Rapid identification of bacterial pathogens

文档序号：1835919 发布日期：2021-11-12 浏览：6次中文

阅读说明：本技术 细菌致病菌的快速鉴定 (Rapid identification of bacterial pathogens ) 是由 P·洛克哈特 R·温克沃斯 P·麦克莱纳肯 N·格伦海特 R·方于 2019-11-08 设计创作，主要内容包括：本文公开用于特异性检测样品中的Mycobacterium spp.和用在与抗生素抗性有关或直接参与的Mycobacterium spp.内对多个基因位点建立图谱的方法和组合物。特别地,该方法采用能够进行本文公开的基于全基因组序列方案的一组独特的核酸扩增引物,从而允许对Mycobacterium spp.的抗生素抗性谱进行全面表征。(Disclosed herein are methods and compositions for specifically detecting and mapping multiple gene sites within a Mycobacterium spp. In particular, the method employs a unique set of nucleic acid amplification primers capable of performing the genome-wide sequence-based protocol disclosed herein, thereby allowing for the comprehensive characterization of the antibiotic resistance profile of Mycobacterium spp.)

1. A composition comprising 7 to 12 unique oligonucleotide primers, each primer consisting of 11 or 12 nucleotides, wherein each of the oligonucleotide primers specifically binds to a nucleic acid sequence in the mycobacterium tuberculosis genome.

2. The composition of claim 1, comprising 7 to 15 unique oligonucleotide primers.

3. The composition of claim 1 or 2, comprising at least 7 unique oligonucleotide primers selected from the group consisting of: p1(SEQ ID NO:1), P2(SEQ ID NO:2), P3(SEQ ID NO:3), P4(SEQ ID NO:4), P5(SEQ ID NO:5), P6(SEQ ID NO:6), P7(SEQ ID NO:7), P8(SEQ ID NO:8), P9(SEQ ID NO:9), P10(SEQ ID NO:10), P11(SEQ ID NO:11), P12(SEQ ID NO:12), P13(SEQ ID NO:13), P14(SEQ ID NO:14) and P15(SEQ ID NO: 15).

4. The composition of claim 3, wherein the oligonucleotide primers comprise P1-P6 and P12.

5. The composition of claim 3 or 4, wherein the oligonucleotide primer consists essentially of P1-P6 and P12.

6. The composition of any one of claims 1 to 5, further comprising at least one enzyme that catalyzes nucleic acid amplification.

7. The composition of claim 3, wherein the enzyme is Φ 29 polymerase or Bst polymerase.

8. A method of selectively amplifying genomic DNA of at least one bacterial species or strain from a sample, the method comprising:

contacting the sample with a composition comprising 7-12 unique oligonucleotide primers, each primer consisting of 11 or 12 nucleotides, wherein each of these oligonucleotide primers specifically binds to a nucleic acid sequence in the genome of the bacterial species or strain,

selectively amplifying DNA from a bacterial species or strain of interest in a Multiple Displacement Amplification (MDA) reaction,

identifying from the selectively amplified DNA a DNA sequence that assigns with high confidence to the genome of at least one bacterial species or strain.

9. The method of claim 8, wherein the unique oligonucleotide introductions are defined as in the composition of any one of claims 1-5.

10. The method of claim 9, wherein the bacterial species or strain is a Mycobacterium spp.

11. The method of claim 10, wherein the Mycobacterium spp.

12. The method of any one of claims 8 to 11, wherein the sample is a sample containing or suspected of containing DNA from: mycobacterium tuberculosis or m.bovis; and at least one other organism, preferably at least 2, preferably at least 5, preferably at least 10, preferably at least 15 other organisms.

13. The method of any one of claims 8 to 12, wherein the sample is a sputum or saliva sample.

14. The method of any one of claims 8 to 13, wherein the sample is from a human or a bovine.

15. A method of determining antibiotic resistance of a Mycobacterium strain, the method comprising:

contacting a sample containing or suspected of containing at least one Mycobacterium spp. with a composition comprising 7 to 12 unique oligonucleotide primers selected from the group consisting of P1-P14 and P15;

selectively amplifying DNA from the at least one Mycobacterium spp in a Multiple Displacement Amplification (MDA) reaction, and

identifying in a selectively amplified DNA pool a DNA sequence encoding at least one Mycobacterium spp.

16. The method of claim 15, wherein identifying in the selectively amplified DNA pool a DNA sequence encoding a bacterial gene product associated with or directly involved in conferring antibiotic resistance in the at least one Mycobacterium spp comprises: generating an antibiotic resistance profile by Whole Genome Sequencing (WGS) and bioinformatic analysis of the amplified DNA to determine the nucleotide sequence of at least one genetic locus associated with or directly involved in antibiotic resistance in the at least one Mycobacterium spp.

17. The method of claim 16, wherein the genetic locus is selected from the group consisting of: alkyl hydroperoxide reductase subunit c (ahpc), arabinosyltransferase b (embb), 7-methylguanosine methyltransferase (gidB), DNA gyrase (gyrA), DNA gyrase (gyrB), NADH-dependent enoyl-acyl carrier protein reductase (inhA), catalase/peroxidase (katG), pyrazinamidase/nicotinamidase (pncA), RNA polymerase beta subunit (rpoB), ribosomal protein S12(rpsL), 16S rRNA (rrs), thymidylate synthase (thyA), and rRNA methyltransferase (tlyA).

18. The method of any one of claims 15 to 17, wherein the unique oligonucleotide introductions are as defined by the composition of any one of claims 1 to 5.

19. The method of claim 18, wherein the Mycobacterium spp.

Technical Field

The present invention relates generally to methods and compositions for detecting mycobacterium tuberculosis in a sample and mapping multiple genetic loci within mycobacterium tuberculosis that are associated with or directly involved in antibiotic resistance in the mycobacterium tuberculosis.

Background

Tuberculosis (TB) is an infectious disease, usually caused by members of the mycobacterium tuberculosis species complex. The complex includes four species-mycobacterium tuberculosis, m.bovis, m.africanum, and m.microti. These species differ significantly epidemiologically but are genetically very similar, having 85-100% identity at the DNA level.

TB is curable in humans, but this disease causes 180 million deaths each year (WHO global tuberculosis report 2016). In addition, there are 17 billion potential TBs. These individuals are usually asymptomatic, but can develop this disease if immune function is low (Houben & double 2016). Despite the large financial investment, current methods of diagnosing TB have failed to reduce the infection rate, which hampers the current efforts of the World Health Organization (WHO) to eradicate TB (Klopper et al.2013; Callaway 2017).

Bovine tuberculosis (bTB) is an infectious disease caused by m. This chronic disease affects cattle, other domestic and wild animals, and can also lead to human disease. bTB is considered one of the seven most frequently overlooked endemic zoonotic diseases worldwide. Once the disease occurs, the disease has great influence on social economy and public health, and the trade of animals and products thereof is severely restricted.

The emergence of multi-drug resistant (MDR) and broadly drug resistant (XDR) strains of tuberculosis is an important obstacle to the control of tuberculosis (Nguyen 2017). The former is resistant to one or more first-line drugs, such as Isoniazid (INH) and Rifampicin (RIF), while the latter is also resistant to Fluoroquinolone (FLQ) and one or more second-line drugs, such as kappacin (CPR), Kanamycin (KAN), Ofloxacin (OFX) and Amikacin (AMK) (Migliori et al 2008). Perhaps the most worry is that a fully drug resistant strain has recently emerged in south Africa (Klopper et al, 2013).

The subsequent culture of conventional light microscopes has remained the "gold standard" for the diagnosis of active tuberculosis for decades. The identification of Ziehl-Neelsen stained Mycobacterium tuberculosis under optical microscopy is both rapid and inexpensive. However, once bacteria are identified, they must be cultured in culture for 4-8 weeks and then subjected to phenotypic drug susceptibility testing, which may take an additional 6 weeks. Only at the end of this process can the resistance and therefore the susceptibility of the strain be determined. Thus, the patient may need to wait up to 14 weeks to receive appropriate treatment.

Recently, molecular methods for evaluating resistance curves have been developed. The commercially available Hain line probe assay utilizes a combination of PCR and DNA-DNA hybridization to simultaneously identify mycobacterium tuberculosis and detect mutations associated with resistance to several antibiotics (Dookie et al 2018). The open format of the line probe assay is a disadvantage and requires specialized infrastructure. In 2010, the WHO approved the use of the cartridge-based Xpert MTB/RIF test for the detection of mycobacterium tuberculosis. Although Xpert MTB/RIF was considered a major breakthrough, TB incidence did not decrease significantly since its introduction (Callaway 2017). This may reflect the limitations of the Xpert MTB/RIF test. These include the high cost of the equipment and test cartridge, and the limited shelf life of the test cartridge. Perot MTB/RIF also requires air-conditioned facilities, maintains a constant power supply, and must be maintained regularly (Kane et al, 2016; Evans, 2011), perhaps more importantly. In countries where tuberculosis is prevalent, these other requirements are often difficult to meet, except in central authorities. The inability of Xpert MTB/RIF to deploy to low resource settings limits its utility.

All existing molecular diagnostic methods for drug-resistant mycobacterium tuberculosis suffer from a limitation in that they do not fully characterize the antibiotic resistance spectrum of any given mycobacterium tuberculosis strain.

An alternative approach to DNA amplification-based testing involves analysis of Whole Genome Sequencing (WGS) data. This approach allows for genome-wide evaluation of genetic mutations, and the use of WGS allows identification of known drug-resistance-inducing sequence variations and identification of new variations. As a result, WGS has become the first choice for diagnosis of tuberculosis in research laboratory settings, especially where multi-drug resistant and broadly drug resistant tuberculosis are expected (Gilpin et al, 2016). The increased affordability and speed of DNA sequencing and the advent of personal DNA sequencing equipment (e.g., Oxford Nanopore MinION) make WGS an increasingly attractive option for rapid tuberculosis diagnosis. However, obstacles still exist. For example, WGS analysis of patient sputum DNA may not contain enough sequence reads from Mycobacterium tuberculosis to identify mutations that induce antibiotic resistance and determine the antibiotic resistance profile (Doherty 2014; Brown et al 2015).

Therefore, there is a need in the art for alternative methods of detecting mycobacterium tuberculosis and m.bovis and mapping antibiotic resistance of various strains of mycobacterium tuberculosis and m.bovis that can be operated more quickly at lower cost and with lower infrastructure.

It is an object of the present invention to at least somehow meet this need and/or to provide a fast, low cost method to selectively amplify mycobacterium tuberculosis DNA using Multiple Displacement Amplification (MDA) and/or to provide methods and compositions for detecting mycobacterium tuberculosis and for mapping multiple genetic loci that are associated or directly related to antibiotic resistance in mycobacterium tuberculosis and/or to provide methods and compositions for detecting m.bovis and for mapping multiple genetic loci that are associated or directly related to antibiotic resistance in m.bovis and/or to at least provide the public with a useful choice.

Summary of The Invention

In one aspect, the invention relates to a composition comprising 7 to 12 unique oligonucleotide primers, each primer consisting of 11 or 12 nucleotides, wherein each of these oligonucleotide primers specifically binds to a nucleic acid sequence in the mycobacterium tuberculosis genome. In one embodiment, the composition comprises 7 to 15 unique oligonucleotide primers.

In another aspect, the invention relates to a composition comprising at least 7 unique oligonucleotide primers selected from the group consisting of: p1(SEQ ID NO:1), P2(SEQ ID NO:2), P3(SEQ ID NO:3), P4(SEQ ID NO:4), P5(SEQ ID NO:5), P6(SEQ ID NO:6), P7(SEQ ID NO:7), P8(SEQ ID NO:8), P9(SEQ ID NO:9), P10(SEQ ID NO:10), P11(SEQ ID NO:11), P12(SEQ ID NO:12), P13(SEQ ID NO:13), P14(SEQ ID NO:14) and P15(SEQ ID NO: 15).

In a further aspect, the invention relates to a kit comprising at least 7 unique oligonucleotide primers selected from the group consisting of: P1-P14 and P15, and at least one enzyme that catalyzes nucleic acid replication.

In a further aspect, the present invention relates to a method of selectively amplifying genomic DNA of at least one bacterial species or strain from a sample, the method comprising:

selectively amplifying DNA from a bacterial species or strain of interest in a Multiple Displacement Amplification (MDA) reaction,

from the selectively amplified DNA, a DNA sequence is identified which assigns with high confidence to the genome of the bacterial species or strain of interest.

In a further aspect, the present invention relates to a method for selectively amplifying Mycobacterium tuberculosis genomic DNA from a sample, the method comprising:

contacting the sample with a composition comprising 7-12 unique oligonucleotide primers, each primer selected from the group consisting of P1-P14 and P15;

selectively amplifying DNA from mycobacterium tuberculosis in a Multiple Displacement Amplification (MDA) reaction; and

the DNA sequence assigned with high confidence to the genome of Mycobacterium tuberculosis is identified from the selectively amplified DNA.

In a further aspect, the present invention relates to a method of selectively amplifying Mycobacterium bovis genomic DNA from a sample, the method comprising:

contacting the sample with a composition comprising 7-12 unique oligonucleotide primers, each primer selected from the group consisting of P1-P14 and P15;

selectively amplifying DNA from m.bovis in a Multiple Displacement Amplification (MDA) reaction; and

identifying from the selectively amplified DNA a DNA sequence of the genome assigned to m.bovis with high confidence.

In a further aspect, the invention relates to a method of determining the antibiotic resistance profile of a mycobacterium tuberculosis strain, the method comprising:

contacting a sample containing or suspected of containing mycobacterium tuberculosis with a composition comprising 7 to 12 unique oligonucleotide primers selected from the group consisting of P1-P14 and P15;

selectively amplifying DNA from Mycobacterium tuberculosis in a Multiple Displacement Amplification (MDA) reaction, and

identifying in the selectively amplified DNA pool a DNA sequence encoding a Mycobacterium tuberculosis gene product that is associated with or directly involved in antibiotic resistance in Mycobacterium tuberculosis.

In a further aspect, the present invention relates to a method of determining the antibiotic resistance profile of a m.bovis strain, the method comprising:

selectively amplifying DNA from m.bovis in a Multiple Displacement Amplification (MDA) reaction; and

identifying in a selectively amplified DNA pool a DNA sequence encoding a m.bovis gene product that is associated with or directly involved in antibiotic resistance in m.bovis.

Various embodiments of the different aspects of the present invention as described above are also set forth below in the detailed description of the invention, but the present invention is not limited thereto. Other aspects of the invention will become apparent from the following description, given by way of example only and with reference to the accompanying drawings.

The invention may be said to broadly consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.

Brief Description of Drawings

The invention will now be described with reference to the drawings of the accompanying drawings.

FIG. 1 is a simplified map of the MTB H37Rv reference genome, labeled with 13 genetic loci commonly associated with antibiotic resistance. The binding site for each of the 15 MDA primers is shown below the genomic map.

FIG. 2.PerkinElmerGX Touch HT three targeted MDA reactions were analyzed using 10-20ng of DNA from sample 5734 as starting template and unamplified samples. From left to right, 40kb ladder, 16h MDA for Illumina MiSeq analysis, 6h MDA for Illumina MiSeq analysis, 16h MDA for oxford nanopore MinION analysis, and unamplified DNA from the original sample. There were significant differences in the size and number of the most common DNA fragments in each sample. These results indicate the amplification and concatenation of DNA during the MDA reaction. The largest difference appears to be between unamplified and 16hr MDA; the intensity of the 6h MDA band was similar to that of the 16hr sample, indicating strong amplification, but its smaller size indicates that the tandem was more restricted.

FIG. 3 quality scores for Illumina MiSeq sequencing of target MDA reactions in raw unamplified samples 5734(A, B read 1 and read 2, respectively) and 6hr (C, D read 1 and read 2, respectively) and 16hr (E, F read 1 and read 2, respectively) using 10-20ng DNA as starting template. The graph is from fastQC showing Illumina mass scores of 1-151 bases of sequence reads. In all cases, sequencing was of high quality; the mass fraction of the vast majority of reads is Q30 and above. However, the quality score for read 2 was higher when the MDA template was sequenced.

Definition of

The following definitions are provided to better define the invention and to serve as guidelines for the practice of the invention by those of ordinary skill in the art.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the relevant art to which this disclosure belongs. Examples of definitions of terms commonly used in Microbiology, Molecular biology and biochemistry can be found in Methods for General and Molecular Microbiology,3rd Edition, c.a. reddy et al (eds.), ASM Press (2008); encyclopedia of Microbiology,2nd ed., Joshua Lederburg, (ed.), Academic Press, (2000); microbiology By Cliffs Notes, i.e. edward Alcamo, Wiley, (1996); dictionary of Microbiology and Molecular Biology, Singleton et al (2d ed.) (1994); biology of Microorganisms 11^th ed.,Brock et al.,Pearson Prentice Hall,(2006)；Genes IX,Benjamin Lewin,Jones&Bartlett Publishing, (2007); the Encyclopedia of Molecular Biology, Kendrew et al, (eds.), Blackwell Science Ltd. (1994) and Molecular Biology and Biotechnology, aCompressent Desk Reference, Robert A. Meyers (ed.), VCH Publishers, Inc. (1995).

The term "comprising" as used in the present specification and claims means "consisting at least in part of … …"; that is, in interpreting statements in this specification and claims which include "comprising," the features prefaced by that term in each statement all need to be present but other features can also be present. Related terms such as "include" and "comprise" are to be interpreted in a similar manner.

As used herein, the term "consisting essentially of … …" refers to particular materials or steps, as well as materials or steps, that do not materially affect the basic and novel characteristics of the claimed invention.

The term "consisting of … …" as used herein refers to a particular material or step of the claimed invention, excluding any element, step, or ingredient not specified in the claims.

The term "specifically binds" as used herein in relation to an oligonucleotide primer that binds to nucleic acid, in particular DNA, refers to annealing of a specific primer to a portion of the nucleic acid that contains a nucleotide sequence complementary to the nucleotide sequence of the primer. The degree of complementarity between the nucleic acid and the oligonucleotide primer is generally determined by the conditions under which they are contacted; that is, the oligonucleotide primer may bind to a portion of the nucleic acid that is at least partially complementary, preferably fully complementary, over the entire length of the primer or a portion thereof, thereby allowing amplification to begin.

One skilled in the art will appreciate that, in the context of the present invention, partially complementary oligonucleotide primers can specifically bind to a target nucleic acid to initiate replication under suitable conditions. Thus, in some embodiments, the oligonucleotide primer of the invention is partially complementary to the target nucleic acid in that it contains 1, 2, or 3 mismatches, but still specifically binds to a nucleic acid sequence in the mycobacterium tuberculosis genome.

The phrase "selectively amplifying" with respect to nucleic acids, particularly DNA, as used herein refers to the use of a DNA polymerase to preferentially replicate nucleic acid of one species or strain of bacteria from a sample containing nucleic acid from two or more species or strains of bacteria. Related terms such as "selective amplification" and "selective amplification" should be interpreted in a similar manner.

As used herein, the term "identifying from amplified DNA" refers to a bioinformatic analysis of DNA sequences from the amplified DNA that allows a skilled person to determine the likely source of any given DNA sequence in the amplified DNA given a suitable set of reference sequences.

As used herein, the term "assigning amplified DNA to a particular bacterial species or strain with high confidence" refers to the fact that a given DNA sequence is more likely to represent a given genome than any other sequence in the reference set in view of the criteria employed for bioinformatic analysis to identify DNA sequences from the amplified DNA.

As used herein, the terms "associated or directly associated with antibiotic resistance of a species or strain" and "associated or directly associated with antibiotic resistance of mycobacterium tuberculosis" and grammatical variants thereof refer to genes that have been reported to be mutated, which are presumed or have been shown to be responsible for the ability of a bacterial species or strain to survive application of the antibiotic.

As used herein, the term "antibiotic resistance profile" refers to a descriptive list of antibiotics to which a bacterial species or strain has acquired resistance.

Reference to a range of numbers disclosed herein (e.g., 1 to 10) is also intended to include all relevant numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) and any rational number range within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7), and thus, all subranges of all ranges explicitly disclosed herein are explicitly disclosed. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest value and the highest value enumerated, are to be considered to be expressly stated in this application in a similar manner.

Detailed Description

The present invention relates generally to a set of unique oligonucleotide primers that specifically bind to genomic DNA of certain mycobacteria and allow for the selective amplification of mycobacteria. DNA from a mixed sample comprising DNA from multiple microbial species. The present invention also relates generally to methods for selectively amplifying the DNA of at least one mycobacterium tuberculosis or mycobacterium bovis strain from a sample using a set of 7 to 12 unique oligonucleotide primers.

Multiple Displacement Amplification (MDA) is an isothermal method of enzymatic DNA amplification. Rather than using high temperatures to make nucleic acids single stranded prior to replication, the enzyme used in the MDA reaction has innate strand displacement activity. These enzymes include phage Φ 29DNA polymerase (Pan et al 2008) and Bst DNA polymerase (Gadkar et al 2014). In an MDA reaction, oligonucleotide primers that bind to multiple sites on a nucleic acid template are used to initiate amplification. As strand synthesis proceeds, the polymerase replaces the newly generated DNA strand, which itself becomes the template for further amplification. This process continues and produces a hyperbranched network of double-stranded DNA fragments.

The inventors designed the 15 M.tuberculosis specific oligonucleotide primers described herein. These oligonucleotide primers 11 were selected in part because the possible binding sites on each M.tuberculosis genome, either in the upstream or downstream direction, included at least one site within 5kb of one or more of the 13 genes that are associated with or directly involved in M.tuberculosis antibiotic resistance. More than 30 genetic loci are associated with antibiotic resistance in mycobacterium tuberculosis (Dookie et al 2018), but in more than 95% of clinical cases resistance is associated with one of the 13 targeted genes (Feuerriegel et al 2015). The use of these oligonucleotide primers, or possibly a subset of 6 of them, ensures that in the subsequent WTS the amplification product of the MDA reaction covers 13 genes associated with or directly involved in the antibiotic resistance of mycobacterium tuberculosis. The remaining four primers were chosen primarily because they bind to the M.tuberculosis genome more frequently than to other test genomes. These latter primers have significantly more binding sites on M.tuberculosis and, when used alone or in combination, increase the overall efficiency of the MDA reaction. The inventors have surprisingly found that the amplification products produced using their inventive primers provide sufficient DNA for a robust WGS of Mycobacterium tuberculosis. The resulting nucleic acids can be analyzed to assess the DNA sequence of the genome as a whole or of genetic loci associated with antibiotic resistance in mycobacterium tuberculosis. These data can then be used to provide an antibiotic resistance profile for the sequenced strain.

As is commonly employed in the art, the goal of MDA is to increase the total concentration of DNA in a sample. In this case, the oligonucleotide primer usually consists of six random nucleotides (so-called "random hexamers"). It is expected that these hexamers will bind to approximately the same extent to all nucleic acids in the sample, and thus the enrichment of DNA in the sample will be unbiased. More recently, researchers have begun to use longer oligonucleotide primers that have greater specificity for the genome of interest (Leichty & Brisson 2014; Clarke et al.2017). In the context of the present invention, the second method is used for the selective amplification of M.tuberculosis genomic DNA in the presence (e.g.sputum samples) or absence (e.g.young culture medium) of DNA from other organisms. The use of MDA for the selective amplification of mycobacterium tuberculosis genomic DNA from a sample comprising multiple genomes requires that the binding site of the oligonucleotide primer used in the reaction occur more frequently in the genomic sequence of the target organism than in the genomic sequence of any other organism that may be present in the sample (Leichty & Brisson 2014).

To identify suitable oligonucleotide primers, the inventors performed a whole genome "k-mer" (i.e., a nucleotide string of "k" length) search for mycobacterium tuberculosis, 15 other organisms common in human and human respiratory tracts (table 2). For these analyses, all k-mers from 6 to 15 nucleotides in length were used to compare the genomes. From these analyses, the inventors determined that some oligonucleotide primers, 11 and 12 nucleotides in length, contain sufficient complexity to selectively amplify M.tuberculosis DNA. In this case, both oligonucleotide primer length and base composition are important considerations; other workers have recently developed MDA primers that allow whole genome amplification of mycobacterium tuberculosis DNA (Clarke et al.2017), but these primers are not suitable for enriching mycobacterium tuberculosis from sputum samples because of their shorter length and lower melting temperature.

The frequency and distribution of specific oligonucleotide primers was then assessed using the mycobacterium tuberculosis H37Rv reference genome. The 11 12-mers were selected in part because for each of these primers, in either upstream or downstream direction, the possible binding sites on the mycobacterium tuberculosis genome include at least one site within 5kb of one or more of the 13 genes commonly associated with or directly involved in antibiotic resistance in mycobacterium tuberculosis. The remaining four primers were chosen primarily because they bind to the M.tuberculosis genome more frequently than to other test genomes. In some embodiments, a primer set comprising less than 15 oligonucleotide primers may be used.

The skilled artisan using the primers and methods described herein has a number of unexpected advantages over other cell-free methods of detecting Mycobacterium tuberculosis and analyzing the antibiotic resistance capability of bacteria currently known in the art. For example, it is very important and highly unexpected that the inventors have found that conducting MDA at a higher temperature than is commonly used increases the specificity of selective amplification of mycobacterium tuberculosis when present in a complex mixture of microorganisms, such as that present in sputum.

Thus, in one aspect, the invention relates to a composition comprising 7 to 12 unique oligonucleotide primers, each primer consisting of 11 or 12 nucleotides, wherein each of these oligonucleotide primers specifically binds to a nucleic acid sequence in the mycobacterium tuberculosis genome.

In one embodiment, the composition comprises 7 to 15 unique oligonucleotide primers.

In one embodiment, the composition consists essentially of unique oligonucleotide primers.

In one embodiment, each oligonucleotide primer selectively binds to a genetic site in the genome of Mycobacterium tuberculosis associated with or directly involved in antibiotic resistance no more than 12kb, preferably no more than 10kb, preferably no more than 8kb, preferably no more than 5kb, preferably no more than 1 kb.

In one embodiment, each oligonucleotide primer selectively binds to a gene site in the genome of Mycobacterium tuberculosis associated with or directly involved in antibiotic resistance at a distance of about 12kb or less, preferably about 10kb or less, preferably about 8kb or less, preferably about 5kb or less, preferably about 1kb or less.

In one embodiment, the genetic locus is selected from the group consisting of: alkyl hydroperoxide reductase subunit c (ahpc), arabinosyltransferase b (embb), 7-methylguanosine methyltransferase (gidB), DNA gyrase (gyrA), DNA gyrase (gyrB), NADH-dependent enoyl-acyl carrier protein reductase (inhA), catalase/peroxidase (katG), pyrazinamidase/nicotinamidase (pncA), RNA polymerase beta subunit (rpoB), ribosomal protein S12(rpsL), 16S rRNA (rrs), thymidylate synthase (thyA), and rRNA methyltransferase (tlyA).

In one embodiment, each oligonucleotide primer selectively binds within 12kb, preferably within 10kb, preferably within 8kb, preferably within 5kb of each of alkyl hydroperoxide reductase subunit c (ahpc), arabinotransferase b (embb), 7-methylguanosinyl methyltransferase (gidB), DNA gyrase (gyrA), DNA gyrase (gyrB), NADH-dependent enoyl-acyl carrier protein reductase (inhA), catalase/peroxidase (katG), pyrazinamidase/nicotinamidase (pncA), RNA polymerase beta subunit (rpoB), ribosomal protein S12(rpsL), 16S rRNA (rrs), thymidylate synthase (thyA), and rRNA methyltransferase (tlyA).

In one embodiment, the oligonucleotide primer is selected from the group consisting of: p1(SEQ ID NO:1), P2(SEQ ID NO:2), P3(SEQ ID NO:3), P4(SEQ ID NO:4), P5(SEQ ID NO:5), P6(SEQ ID NO:6), P7(SEQ ID NO:7), P8(SEQ ID NO:8), P9(SEQ ID NO:9), P10(SEQ ID NO:10), P11(SEQ ID NO:11), P12(SEQ ID NO:12), P13(SEQ ID NO:13), P14(SEQ ID NO:14) and P15(SEQ ID NO: 15).

In one embodiment, the composition comprises P1-P6 and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition consists essentially of P1-P6 and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition comprises P1-P9 and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition consists essentially of P1-P9 and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition comprises P1-P11 and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition consists essentially of P1-P11 and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition comprises P1-P6 and P12. In one embodiment, the composition comprises P1-P6 and P13. In one embodiment, the composition comprises P1-P6 and P14. In one embodiment, the composition comprises P1-P6 and P15. In one embodiment, the composition comprises P1-P6, P12, and P13. In one embodiment, the composition comprises P1-P6, P12, and P14. In one embodiment, the composition comprises P1-P6, P12, and P15. In one embodiment, the composition comprises P1-P6, P13, and P14. In one embodiment, the composition comprises P1-P6, P13, and P15. In one embodiment, the composition comprises P1-P6, P14, and P15. In one embodiment, the composition comprises P1-P6, P12, P13, and P14. In one embodiment, the composition comprises P1-P6, P12, P13, and P15. In one embodiment, the composition comprises P1-P6, P12, P14, and P15. In one embodiment, the composition comprises P1-P6, P13, P14, and P15. In one embodiment, the composition comprises P1-P6, P12, P13, P14, and P15. In one embodiment, the composition comprises P1-P9 and P12. In one embodiment, the composition comprises P1-P9 and P13. In one embodiment, the composition comprises P1-P9 and P14. In one embodiment, the composition comprises P1-P9 and P15. In one embodiment, the composition comprises P1-P9, P12, and P13. In one embodiment, the composition comprises P1-P9, P12, and P14. In one embodiment, the composition comprises P1-P9, P12, and P15. In one embodiment, the composition comprises P1-P9, P13, and P14. In one embodiment, the composition comprises P1-P9, P13, and P15. In one embodiment, the composition comprises P1-P9, P14, and P15. In one embodiment, the composition comprises P1-P9, P12, P13, and P14. In one embodiment, the composition comprises P1-P9, P12, P13, and P15. In one embodiment, the composition comprises P1-P9, P12, P14, and P15. In one embodiment, the composition comprises P1-P9, P13, P14, and P15. In one embodiment, the composition comprises P1-P9, P12, P13, P14, and P15. In one embodiment, the composition comprises P1-P12. In one embodiment, the composition comprises P1-P11 and P13. In one embodiment, the composition comprises P1-P11 and P14. In one embodiment, the composition comprises P1-P11 and P15. In one embodiment, the composition comprises P1-P12 and P13. In one embodiment, the composition comprises P1-P12 and P14.

In one embodiment, the composition comprises P1-P12 and P15. In one embodiment, the composition comprises P1-P11, P13, and P14. In one embodiment, the composition comprises P1-P11, P13, and P15. In one embodiment, the composition comprises P1-P11, P14, and P15. In one embodiment, the composition comprises P1-P12, P13, and P14. In one embodiment, the composition comprises P1-P12, P13, and P15. In one embodiment, the composition comprises P1-P12, P14, and P15. In one embodiment, the composition comprises P1-P11, P13, P14, and P15. In one embodiment, the composition comprises P1-P15.

In one embodiment, the composition consists essentially of P1-P6 and P12. In one embodiment, the composition consists essentially of P1-P6 and P13. In one embodiment, the composition consists essentially of P1-P6 and P14. In one embodiment, the composition consists essentially of P1-P6 and P15. In one embodiment, the composition consists essentially of P1-P6, P12, and P13. In one embodiment, the composition consists essentially of P1-P6, P12, and P14. In one embodiment, the composition consists essentially of P1-P6, P12, and P15. In one embodiment, the composition consists essentially of P1-P6, P13, and P14. In one embodiment, the composition consists essentially of P1-P6, P13, and P15. In one embodiment, the composition consists essentially of P1-P6, P14, and P15. In one embodiment, the composition consists essentially of P1-P6, P12, P13, and P14. In one embodiment, the composition consists essentially of P1-P6, P12, P13, and P15. In one embodiment, the composition consists essentially of P1-P6, P12, P14, and P15. In one embodiment, the composition consists essentially of P1-P6, P13, P14, and P15. In one embodiment, the composition consists essentially of P1-P6, P12, P13, P14, and P15. In one embodiment, the composition consists essentially of P1-P9 and P12. In one embodiment, the composition consists essentially of P1-P9 and P13. In one embodiment, the composition consists essentially of P1-P9 and P14. In one embodiment, the composition consists essentially of P1-P9 and P15. In one embodiment, the composition consists essentially of P1-P9, P12, and P13. In one embodiment, the composition consists essentially of P1-P9, P12, and P14. In one embodiment, the composition consists essentially of P1-P9, P12, and P15. In one embodiment, the composition consists essentially of P1-P9, P13, and P14. In one embodiment, the composition consists essentially of P1-P9, P13, and P15. In one embodiment, the composition consists essentially of P1-P9, P14, and P15. In one embodiment, the composition consists essentially of P1-P9, P12, P13, and P14. In one embodiment, the composition consists essentially of P1-P9, P12, P13, and P15. In one embodiment, the composition consists essentially of P1-P9, P12, P14, and P15. In one embodiment, the composition consists essentially of P1-P9, P13, P14, and P15. In one embodiment, the composition consists essentially of P1-P9, P12, P13, P14, and P15. In one embodiment, the composition consists essentially of P1-P12. In one embodiment, the composition consists essentially of P1-P11 and P13. In one embodiment, the composition consists essentially of P1-P11 and P14. In one embodiment, the composition consists essentially of P1-P11 and P15. In one embodiment, the composition consists essentially of P1-P12 and P13. In one embodiment, the composition consists essentially of P1-P12 and P14. In one embodiment, the composition consists essentially of P1-P12 and P15. In one embodiment, the composition consists essentially of P1-P11, P13, and P14. In one embodiment, the composition consists essentially of P1-P11, P13, and P15. In one embodiment, the composition consists essentially of P1-P11, P14, and P15. In one embodiment, the composition consists essentially of P1-P12, P13, and P14. In one embodiment, the composition consists essentially of P1-P12, P13, and P15. In one embodiment, the composition consists essentially of P1-P12, P14, and P15. In one embodiment, the composition consists essentially of P1-P11, P13, P14, and P15. In one embodiment, the composition consists essentially of P1-P15.

The nucleotide sequences of P1-P15 are shown in Table 1

TABLE 1 Mycobacterium tuberculosis oligonucleotide primers

In one embodiment, the composition comprises P1-P6, and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition consists essentially of P1-P6 and at least one, preferably at least two, preferably at least three, preferably all four of P1-P6, P12, P13, P14 or P15.

In one embodiment, the composition comprises P1-P9 and at least one, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition consists essentially of P1-P9 and at least one, preferably at least two, preferably at least three, preferably all four of P1-P9, P12, P13, P14 or P15.

In one embodiment, the composition comprises P1-P11, and at least four, preferably at least two, preferably at least three, preferably all four of P12, P13, P14 or P15.

In one embodiment, the composition consists essentially of P1-P11 and at least one, preferably at least two, preferably at least three, preferably all four of P1-P11, P12, P13, P14 or P15.

In one embodiment, the composition further comprises at least one enzyme that catalyzes nucleic acid replication. In one embodiment, the enzyme is Φ 29 polymerase. In one embodiment, the enzyme is Bst polymerase.

In a further aspect, the invention relates to a kit comprising at least 7 unique oligonucleotide primers selected from the group consisting of: P1-P14 and P15; and at least one enzyme that catalyzes the replication of nucleic acids.

In one embodiment, the enzyme is Φ 29 polymerase.

In one embodiment, the enzyme is Bst polymerase.

In one embodiment, the kit comprises P1-P6 and P12. In one embodiment, the kit comprises P1-P6 and P13. In one embodiment, the kit comprises P1-P6 and P14. In one embodiment, the kit comprises P1-P6 and P15. In one embodiment, the kit comprises P1-P6, P12, and P13. In one embodiment, the kit comprises P1-P6, P12, and P14. In one embodiment, the kit comprises P1-P6, P12, and P15. In one embodiment, the kit comprises P1-P6, P13, and P14. In one embodiment, the kit comprises P1-P6, P13, and P15. In one embodiment, the kit comprises P1-P6, P14, and P15. In one embodiment, the kit comprises P1-P6, P12, P13, and P14. In one embodiment, the kit comprises P1-P6, P12, P13, and P15. In one embodiment, the kit comprises P1-P6, P12, P14, and P15. In one embodiment, the kit comprises P1-P6, P13, P14, and P15. In one embodiment, the kit comprises P1-P6, P12, P13, P14, and P15. In one embodiment, the kit comprises P1-P9 and P12. In one embodiment, the kit comprises P1-P9 and P13. In one embodiment, the kit comprises P1-P9 and P14. In one embodiment, the kit comprises P1-P9 and P15. In one embodiment, the kit comprises P1-P9, P12, and P13. In one embodiment, the kit comprises P1-P9, P12, and P14. In one embodiment, the kit comprises P1-P9, P12, and P15. In one embodiment, the kit comprises P1-P9, P13, and P14. In one embodiment, the kit comprises P1-P9, P13, and P15. In one embodiment, the kit comprises P1-P9, P14, and P15. In one embodiment, the kit comprises P1-P9, P12, P13, and P14. In one embodiment, the kit comprises P1-P9, P12, P13, and P15. In one embodiment, the kit comprises P1-P9, P12, P14, and P15. In one embodiment, the kit comprises P1-P9, P13, P14, and P15. In one embodiment, the kit comprises P1-P9, P12, P13, P14, and P15. In one embodiment, the kit comprises P1-P12. In one embodiment, the kit comprises P1-P11 and P13. In one embodiment, the kit comprises P1-P11 and P14. In one embodiment, the kit comprises P1-P11 and P15. In one embodiment, the kit comprises P1-P12 and P13. In one embodiment, the kit comprises P1-P12 and P14. In one embodiment, the kit comprises P1-P12 and P15. In one embodiment, the kit comprises P1-P11, P13, and P14. In one embodiment, the kit comprises P1-P11, P13, and P15. In one embodiment, the kit comprises P1-P11, P14, and P15. In one embodiment, the kit comprises P1-P12, P13, and P14. In one embodiment, the kit comprises P1-P12, P13, and P15. In one embodiment, the kit comprises P1-P12, P14, and P15. In one embodiment, the kit comprises P1-P11, P13, P14, and P15. In one embodiment, the kit comprises P1-P15.

In one embodiment, the kit consists essentially of P1-P6 and P12. In one embodiment, the kit consists essentially of P1-P6 and P13. In one embodiment, the kit consists essentially of P1-P6 and P14. In one embodiment, the kit consists essentially of P1-P6 and P15. In one embodiment, the kit consists essentially of P1-P6, P12, and P13. In one embodiment, the kit consists essentially of P1-P6, P12, and P14. In one embodiment, the kit consists essentially of P1-P6, P12, and P15. In one embodiment, the kit consists essentially of P1-P6, P13, and P14. In one embodiment, the kit consists essentially of P1-P6, P13, and P15. In one embodiment, the kit consists essentially of P1-P6, P14, and P15. In one embodiment, the kit consists essentially of P1-P6, P12, P13, and P14. In one embodiment, the kit consists essentially of P1-P6, P12, P13, and P15. In one embodiment, the kit consists essentially of P1-P6, P12, P14, and P15. In one embodiment, the kit consists essentially of P1-P6, P13, P14, and P15. In one embodiment, the kit consists essentially of P1-P6, P12, P13, P14, and P15. In one embodiment, the kit consists essentially of P1-P9 and P12. In one embodiment, the kit consists essentially of P1-P9 and P13. In one embodiment, the kit consists essentially of P1-P9 and P14. In one embodiment, the kit consists essentially of P1-P9 and P15. In one embodiment, the kit consists essentially of P1-P9, P12, and P13. In one embodiment, the kit consists essentially of P1-P9, P12, and P14. In one embodiment, the kit consists essentially of P1-P9, P12, and P15. In one embodiment, the kit consists essentially of P1-P9, P13, and P14. In one embodiment, the kit consists essentially of P1-P9, P13, and P15. In one embodiment, the kit consists essentially of P1-P9, P14, and P15. In one embodiment, the kit consists essentially of P1-P9, P12, P13, and P14. In one embodiment, the kit consists essentially of P1-P9, P12, P13, and P15. In one embodiment, the kit consists essentially of P1-P9, P12, P14, and P15. In one embodiment, the kit consists essentially of P1-P9, P13, P14, and P15. In one embodiment, the kit consists essentially of P1-P9, P12, P13, P14, and P15. In one embodiment, the kit consists essentially of P1-P12. In one embodiment, the kit consists essentially of P1-P11 and P13. In one embodiment, the kit consists essentially of P1-P11 and P14. In one embodiment, the kit consists essentially of P1-P11 and P15. In one embodiment, the kit consists essentially of P1-P12 and P13. In one embodiment, the kit consists essentially of P1-P12 and P14. In one embodiment, the kit consists essentially of P1-P12 and P15. In one embodiment, the kit consists essentially of P1-P11, P13, and P14. In one embodiment, the kit consists essentially of P1-P11, P13, and P15. In one embodiment, the kit consists essentially of P1-P11, P14, and P15. In one embodiment, the kit consists essentially of P1-P12, P13, and P14. In one embodiment, the kit consists essentially of P1-P12, P13, and P15. In one embodiment, the kit consists essentially of P1-P12, P14, and P15. In one embodiment, the kit consists essentially of P1-P11, P13, P14, and P15. In one embodiment, the kit consists essentially of P1-P15.

In a further aspect, the present invention relates to a method of selectively amplifying genomic DNA of at least one bacterial species or strain from a sample, the method comprising:

selectively amplifying DNA from a bacterial species or strain of interest in a Multiple Displacement Amplification (MDA) reaction;

from the selectively amplified DNA, a DNA sequence is identified which assigns with high confidence to the genome of the bacterial species or strain of interest.

In one embodiment, the composition comprises 7 to 15 unique oligonucleotide primers.

Methods of selectively amplifying genomic DNA of at least one bacterial species or strain that are particularly contemplated as embodiments of this aspect of the invention are all embodiments of the invention set forth in the compositional aspects of the invention, including the use of and combinations of unique oligonucleotide primers described in the compositional aspects and embodiments of the invention.

In one embodiment, the identification comprises sequencing and bioinformatic analysis of the amplified DNA product.

In one embodiment, the DNA sequence is identified as encoding a bacterial gene product that is associated with or directly involved in conferring antibiotic resistance in at least one bacterial species or strain.

In one embodiment, the DNA sequence is identified as encoding a protein or portion thereof, or an RNA or portion thereof, associated with or directly involved in conferring antibiotic resistance in at least one bacterial species or strain.

In one embodiment, identifying from the selectively amplified DNA a DNA sequence that assigns with high confidence to the genome of the bacterial species or strain of interest comprises performing Whole Genome Sequencing (WGS) on the amplified DNA and performing bioinformatic analysis on the obtained nucleotide sequence to determine the nucleotide sequence of at least one bacterial species or strain.

In one embodiment, the method further comprises: identifying a DNA sequence from the selectively amplified DNA as encoding a bacterial gene product associated with or directly involved in conferring antibiotic resistance in at least one bacterial species or strain comprises generating an antibiotic resistance profile by: whole Genome Sequencing (WGS) and bioinformatic analysis of the amplified DNA is performed to determine the nucleotide sequence of at least one genetic locus associated with or directly involved in antibiotic resistance in a bacterial species or strain.

In one embodiment, generating the antibiotic resistance profile comprises subjecting the amplified DNA to WGS and bioinformatic analysis to determine the nucleotide sequence of at least one, preferably at least two, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably 13, genetic locus associated with or directly involved in antibiotic resistance in at least one bacterial species or strain.

In some embodiments, the method comprises identifying an antibiotic that is effective against at least one bacterial species or strain based on an antibiotic resistance profile.

In one embodiment, at least one bacterial species or strain is Mycobacterium. In one embodiment, the Mycobacterium is Mycobacterium tuberculosis or Mycobacterium bovis.

In one embodiment, the sample is a culture of mycobacterium tuberculosis or m.bovis.

In one embodiment, the culture is for less than 5 days, preferably for less than 4 days, preferably for less than 3 days.

In one embodiment, the MDA reaction is performed using Φ 29 polymerase.

In one embodiment, the MDA reaction is conducted at a first temperature of about 25 ℃ to about 40 ℃, preferably about 26 ℃ to about 38 ℃, preferably about 27 ℃ to about 36 ℃, preferably about 28 ℃ to about 34 ℃, preferably about 29 ℃ to about 32 ℃, preferably about 30 ℃.

In one embodiment, the MDA reaction is conducted at a first temperature of about 25 ℃ to about 45 ℃, preferably about 30 ℃ to about 44 ℃, preferably about 32 ℃ to about 43 ℃, preferably about 33 ℃ to about 42 ℃, preferably about 34 ℃ to about 41 ℃, preferably about 35 ℃ to about 40 ℃, preferably about 36 ℃ to about 39 ℃, preferably about 36 ℃ to about 38 ℃, preferably about 37 ℃.

In one embodiment, the MDA reaction is carried out at a first temperature of from 25 ℃ to 40 ℃, preferably from 26 ℃ to 38 ℃, preferably from 27 ℃ to 36 ℃, preferably from 28 ℃ to 34 ℃, preferably from 29 ℃ to 32 ℃, preferably 30 ℃.

In one embodiment, the MDA reaction is carried out at a first temperature of 25 ℃ to 45 ℃, preferably 30 ℃ to 44 ℃, preferably 32 ℃ to 43 ℃, preferably 33 ℃ to 42 ℃, preferably 34 ℃ to 41 ℃, preferably 35 ℃ to 40 ℃, preferably 36 ℃ to 39 ℃, preferably 36 ℃ to 38 ℃, preferably 37 ℃.

In one embodiment, the MDA reaction is incubated at the first temperature for at least 1 hour, preferably at least 2 hours, preferably at least 3 hours, preferably at least 4 hours, preferably at least 5 hours, preferably at least 6 hours, preferably at least 7 hours, preferably at least 8 hours, preferably at least 9 hours, preferably at least 10 hours, preferably at least 11 hours, preferably at least 12 hours, preferably at least 13 hours, preferably at least 14 hours, preferably at least 15 hours, preferably at least 16 hours.

In one embodiment, the MDA reaction is carried out at the first temperature for up to 1 hour, preferably up to 2 hours, preferably up to 3 hours, preferably up to 4 hours, preferably up to 5 hours, preferably up to 6 hours, preferably up to 7 hours, preferably up to 8 hours, preferably up to 9 hours, preferably up to 10 hours, preferably up to 11 hours, preferably up to 12 hours, preferably up to 13 hours, preferably up to 14 hours, preferably up to 15 hours, preferably up to 16 hours.

In one embodiment, the MDA reaction is carried out at the first temperature for about 1 hour, preferably about 2 hours, preferably about 3 hours, preferably about 4 hours, preferably about 5 hours, preferably about 6 hours, preferably about 7 hours, preferably about 8 hours, preferably about 9 hours, preferably about 10 hours, preferably about 11 hours, preferably about 12 hours, preferably about 13 hours, preferably about 14 hours, preferably about 15 hours, preferably about 16 hours.

In one embodiment, the MDA reaction is conducted at a first temperature of about 25 ℃ to about 45 ℃, preferably about 30 ℃ to about 44 ℃, preferably about 32 ℃ to about 43 ℃, preferably about 33 ℃ to about 42 ℃, preferably about 34 ℃ to about 25 ℃ 41 ℃, preferably about 35 ℃ to about 40 ℃, preferably about 36 ℃ to about 39 ℃, preferably about 36 ℃ to about 38 ℃, preferably about 37 ℃.

In one embodiment, the MDA reaction is carried out at a first temperature of 25 ℃ to 40 ℃, preferably 26 ℃ to 38 ℃, preferably 27 ℃ to 36 ℃, preferably 28 ℃ to 34 ℃, preferably 29 ℃ to 32 ℃, preferably 30 ℃.

In one embodiment, the MDA reaction is further incubated at the second temperature for about 10 minutes, preferably 10 minutes.

In one embodiment, the second temperature is about 65 ℃, preferably 65 ℃.

In one embodiment, the MDA reaction comprises a buffer, deoxyribonucleotide triphosphates, bovine serum albumin, and trehalose dihydrate. In one embodiment, the MDA reaction includes yeast inorganic pyrophosphatase and/or potassium chloride.

In one embodiment, the MDA reaction contains less than 30ng, preferably less than 15ng, preferably less than 10ng, preferably less than 5ng, preferably less than 2ng, preferably less than 0.2ng, preferably less than 0.02ng, preferably less than 0.002ng of mycobacterium tuberculosis or m.bovis DNA.

In one embodiment, the sample is a sample containing or suspected of containing DNA from: mycobacterium tuberculosis or m.bovis; and at least one other organism, preferably at least 2, preferably at least 5, preferably at least 10, preferably at least 15 other organisms.

In one embodiment, the at least one other organism is selected from the group consisting of prokaryotes and eukaryotes. In one embodiment, the prokaryote is a bacterium. In one embodiment, the bacterium is a gram-negative or gram-positive bacterium or both.

In one embodiment, the eukaryote is a protist or an animal.

In one embodiment, the animal is a mammal.

In one embodiment, the mammal is selected from the group consisting of humans, cows, sheep, deer, dogs, cats, pigs and camelids.

In one embodiment, the sample comprises or further comprises DNA or RNA of a virus.

In one embodiment, the sample is from a human.

In one embodiment, the sample is from a bovine.

In one embodiment, the sample is a sputum sample.

In one embodiment, the sample is a saliva sample.

In this embodiment, the MDA reaction is performed using Bst polymerase.

In one embodiment, the MDA reaction is conducted at a first temperature of about 38 ℃ to about 60 ℃, preferably about 40 ℃ to about 56 ℃, preferably about 42 ℃ to about 52 ℃, preferably about 44 ℃ to about 48 ℃, preferably about 45 ℃, preferably about 46 ℃, preferably about 47 ℃.

In one embodiment, the MDA reaction is carried out at a first temperature of 38 ℃ to 60 ℃, preferably 40 ℃ to 56 ℃, preferably 42 ℃ to 52 ℃, preferably 44 ℃ to 48 ℃, preferably 45 ℃, preferably 46 ℃, preferably 47 ℃.

In one embodiment, the MDA reaction is carried out at the first temperature for 1 hour, preferably 2h, preferably 3h, preferably 4h, preferably 5h, preferably 6 h.

In one embodiment, the MDA reaction is conducted at a first temperature of 38 ℃ to 60 ℃, preferably 40 ℃ to 56 ℃, preferably 42 ℃ to 52 ℃, preferably 44 ℃ to about 48 ℃, preferably 45 ℃, preferably 46 ℃, preferably 47 ℃.

In one embodiment, the MDA reaction is further incubated at the second temperature for about 10 minutes, preferably 10 minutes.

In one embodiment, the second temperature is about 80 ℃, preferably 80 ℃.

In one embodiment, the MDA reaction comprises a buffer, deoxyribonucleotide triphosphates, dimethyl sulfoxide, and T4Gene32 protein.

In a further aspect, the present invention relates to a method for selectively amplifying Mycobacterium tuberculosis genomic DNA from a sample, the method comprising:

contacting the sample with a composition comprising 7-12 unique oligonucleotide primers, each primer selected from the group consisting of P1-P14 and P15,

selectively amplifying DNA from Mycobacterium tuberculosis in a Multiple Displacement Amplification (MDA) reaction, and

the DNA sequence assigned with high confidence to the genome of Mycobacterium tuberculosis is identified from the selectively amplified DNA.

In one embodiment, the composition comprises 7 to 15 unique oligonucleotide primers.

In one embodiment, the identification comprises sequencing and bioinformatic analysis of the amplified DNA product.

In one embodiment, the DNA sequence is identified as encoding a bacterial gene product that is associated with or directly involved in conferring antibiotic resistance in mycobacterium tuberculosis.

In one embodiment, identifying a DNA sequence assigned with high confidence to the genome of Mycobacterium tuberculosis from the selectively amplified DNA comprises performing Whole Genome Sequencing (WGS) on the amplified DNA and performing bioinformatic analysis on the obtained nucleotide sequence to determine the nucleotide sequence of the Mycobacterium tuberculosis genome.

In one embodiment, the method further comprises: identifying a DNA sequence from the selectively amplified DNA as encoding a bacterial gene product associated with or directly involved in conferring antibiotic resistance in the species or strain comprises generating an antibiotic resistance profile by: the amplified DNA is subjected to Whole Genome Sequencing (WGS) and bioinformatics analysis to determine the nucleotide sequence of at least one genetic locus associated with or directly involved in antibiotic resistance in Mycobacterium tuberculosis.

In one embodiment, generating the antibiotic resistance profile comprises subjecting the amplified DNA to WGS and bioinformatic analysis to determine the nucleotide sequence of at least one, preferably at least two, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably 13, genetic locus(s) associated with or directly involved in antibiotic resistance in mycobacterium tuberculosis.

In some embodiments, the method comprises identifying an antibiotic effective against mycobacterium tuberculosis based on the antibiotic resistance profile.

In one embodiment, the sample is a culture of mycobacterium tuberculosis.

In one embodiment, the culture is for less than 5 days, preferably for less than 4 days, preferably for less than 3 days.

In one embodiment, the MDA reaction is performed using Φ 29 polymerase.

In one embodiment, the MDA reaction is conducted at a first temperature of about 25 ℃ to about 45 ℃, preferably about 30 ℃ to about 44 ℃, preferably about 32 ℃ to about 43 ℃, preferably about 33 ℃ to about 42 ℃, preferably about 34 ℃ to about 25 ℃ 41 ℃, preferably about 35 ℃ to about 40 ℃, preferably about 36 ℃ to about 39 ℃, preferably about 36 ℃ to about 38 ℃, preferably about 37 ℃.

In one embodiment, the MDA reaction is further incubated at the second temperature for about 10 minutes, preferably 10 minutes.

In one embodiment, the second temperature is sufficient to inactivate the polymerase.

In one embodiment, the second temperature is about 65 ℃, preferably 65 ℃.

In one embodiment, the sample is a sample containing or suspected of containing DNA from: mycobacterium tuberculosis; and at least one other organism, preferably at least 2, preferably at least 5, preferably at least 10, preferably at least 15 other organisms.

In one embodiment, the eukaryote is a protist or an animal.

In one embodiment, the animal is a mammal.

In one embodiment, the mammal is selected from the group consisting of humans, cows, sheep, deer, dogs, cats, pigs and camelids.

In one embodiment, the sample comprises or further comprises DNA or RNA of a virus.

In one embodiment, the sample is from a human.

In one embodiment, the sample is from a bovine.

In one embodiment, the sample is a sputum sample.

In one embodiment, the sample is a saliva sample.

In this embodiment, the MDA reaction is performed using Bst polymerase.

In one embodiment, the MDA reaction is carried out at the first temperature for 1 hour, preferably 2h, preferably 3h, preferably 4h, preferably 5h, preferably 6 h.

In one embodiment, the MDA reaction is further incubated at the second temperature for about 10 minutes, preferably 10 minutes.

In one embodiment, the second temperature is sufficient to inactivate the polymerase.

In one embodiment, the second temperature is about 80 ℃, preferably 80 ℃.

In one embodiment, the MDA reaction comprises a buffer, deoxyribonucleotide triphosphates, dimethyl sulfoxide, and T4Gene32 protein.

In addition, the method of selectively amplifying genomic DNA of Mycobacterium tuberculosis specifically contemplated as an embodiment of this aspect of the present invention is all embodiments of the present invention set forth in the compositional aspect of the present invention, and is a method of selectively amplifying genomic DNA of at least one species or strain of bacteria, comprising the use of unique oligonucleotide primers and a combination of unique oligonucleotide primers as described in the compositional aspect and embodiments of the present invention.

In a further aspect, the present invention relates to a method of selectively amplifying Mycobacterium bovis genomic DNA from a sample, the method comprising:

contacting the sample with a composition comprising 7-12 unique oligonucleotide primers, each primer selected from the group consisting of P1-P14 and P15;

selectively amplifying DNA from m.bovis in a Multiple Displacement Amplification (MDA) reaction; and

identifying from the selectively amplified DNA a DNA sequence of the genome assigned to m.bovis with high confidence.

In one embodiment, the composition comprises 7 to 15 unique oligonucleotide primers.

Bovis's genomic DNA selective amplification methods are specifically contemplated as embodiments of this aspect of the invention are all embodiments of the invention set forth in the compositional aspects of the invention, are methods of selectively amplifying genomic DNA of mycobacterium tuberculosis, include the use of unique oligonucleotide primers and combinations of unique oligonucleotide primers as described in the compositional aspects and embodiments of the invention.

In a further aspect, the invention relates to a method of determining the antibiotic resistance profile of a mycobacterium tuberculosis strain, the method comprising:

selectively amplifying DNA from mycobacterium tuberculosis in a Multiple Displacement Amplification (MDA) reaction; and

In one embodiment, the composition comprises 7 to 15 unique oligonucleotide primers.

In one embodiment, the identification comprises sequencing and bioinformatic analysis of the amplified DNA product.

In one embodiment, the DNA sequence is identified as encoding a bacterial gene product that is associated with or directly involved in conferring antibiotic resistance in mycobacterium tuberculosis.

In one embodiment, identifying a DNA sequence within the pool of selectively amplified DNA that is assigned with high confidence to the genome of mycobacterium tuberculosis includes performing Whole Genome Sequencing (WGS) on the amplified DNA and performing bioinformatic analysis on the obtained nucleotide sequence to determine the nucleotide sequence of the mycobacterium tuberculosis genome.

In one embodiment, identifying a DNA sequence within a selectively amplified pool of DNA as encoding a bacterial gene product associated with or directly involved in conferring antibiotic resistance in mycobacterium tuberculosis comprises generating an antibiotic resistance profile by: the amplified DNA is subjected to Whole Genome Sequencing (WGS) and bioinformatics analysis to determine the nucleotide sequence of at least one genetic locus associated with or directly involved in antibiotic resistance in Mycobacterium tuberculosis.

In one embodiment, generating the antibiotic resistance profile comprises subjecting the amplified DNA to WGS and bioinformatic analysis to determine the nucleotide sequence of at least one, preferably at least two, preferably at least 3, preferably at least 4, preferably at least 5, preferably at least 6, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12, preferably 13, genetic locus(s) associated with or directly involved in antibiotic resistance in mycobacterium tuberculosis.

In some embodiments, the method comprises identifying an antibiotic effective against mycobacterium tuberculosis based on the antibiotic resistance profile.

The method of determining the antibiotic resistance profile of a mycobacterium tuberculosis strain specifically contemplated as an embodiment of this aspect of the invention is all embodiments of the invention set forth in the compositional aspect of the invention, being a method of selectively amplifying genomic DNA of mycobacterium tuberculosis comprising the use of and a combination of unique oligonucleotide primers as described in the compositional aspect and embodiments of the invention.

In a further aspect, the present invention relates to a method of determining the antibiotic resistance profile of a m.bovis strain, the method comprising:

selectively amplifying DNA from m.bovis in a Multiple Displacement Amplification (MDA) reaction; and

identifying in a selectively amplified DNA pool a DNA sequence encoding a m.bovis gene product that is associated with or directly involved in antibiotic resistance in m.bovis.

In one embodiment, the composition comprises 7 to 15 unique oligonucleotide primers.

In one embodiment, the identification comprises sequencing and bioinformatic analysis of the amplified DNA product.

In one embodiment, the DNA sequence is identified as encoding a bacterial gene product associated with or directly involved in conferring antibiotic resistance in m.bovis.

In one embodiment, identifying DNA sequences from the selectively amplified DNA that assign with high confidence to the genome of m.bovis comprises: whole Genome Sequencing (WGS) was performed on the amplified DNA, and bioinformatics analysis was performed on the obtained nucleotide sequence to determine the nucleotide sequence of m.bovis.

In one embodiment, identifying DNA sequences from the selectively amplified DNA that encode bacterial gene products that are associated with or directly involved in conferring antibiotic resistance in m.bovis comprises generating an antibiotic resistance profile by: performing Whole Genome Sequencing (WGS) and bioinformatic analysis on the amplified DNA to determine the nucleotide sequence of at least one genetic locus associated with or directly involved in antibiotic resistance in m.bovis.

In one embodiment, the method further comprises identifying an antibiotic that is effective or expected to be effective against m.bovis based on the antibiotic resistance profile.

The method of determining the antibiotic resistance profile of a m.bovis strain, which is specifically contemplated as an embodiment of this aspect of the invention, is all embodiments of the invention set forth in the compositional aspect of the invention, being a method of selectively amplifying genomic DNA of mycobacterium tuberculosis, comprising the use of unique oligonucleotide primers and a combination of unique oligonucleotide primers as described in the compositional aspect and embodiments of the invention.

In this specification where reference has been made to patent specifications, other external documents or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. The citation of such external documents is not to be construed as an admission that such documents are available unless otherwise specifically indicated; or such sources of information in any jurisdiction, is prior art, or forms part of the common general knowledge in the art.

The invention will now be illustrated in a non-limiting manner with reference to the following examples.

Examples

Materials and methods

MDA primer selection

A subset of 12 MDA primers was selected from the full set of 15 MDA primers. 11 of the 12 MDA primers were selected because for these 11 primers, the possible binding sites on the mycobacterium tuberculosis genome, either in the upstream or downstream direction, included within 5kb of one or more of at least one of the 13 genes commonly associated with or directly involved in mycobacterium tuberculosis antibiotic resistance (table 2). The twelfth primer was chosen primarily because it frequently binds to the M.tuberculosis genome. The inclusion of this primer also ensured that there was at least one binding site within 10kb for all 13 genes normally associated with or directly involved in mycobacterium tuberculosis antibiotic resistance, whether in the upstream or downstream direction (table 2).

Genomic DNA

The collaborators at the university of Ottago provide samples of Mycobacterium tuberculosis DNA. This sample (designated 5734) is derived from a Mycobacterium tuberculosis strain cultured on Lowenstein-Jensen medium for 6-8 weeks and usedDNA was extracted using a microbial DNA isolation and purification kit (Qiagen). After purification, the DNA sample was incubated in a boiling water bath for 10 minutes to ensure that no viable bacteria remained.

MDA of Mycobacterium tuberculosis DNA

To provide a template for WGS analysis, Mycobacterium tuberculosis DNA was enriched using 12 selected MDA primers (SEQ ID NO:1-SEQ ID NO:12) and other reaction conditions, as suggested by the manufacturer for Φ 29 polymerase, reaction buffer, DTT and bovine serum albumin (New England Biolabs, USA). The exception was that although the amount of primer (125 pmol each) and template (10-20ng) added was the recommended amount for a 50ul reaction volume, the reaction volume was halved to 25 ul. The amplification reaction was incubated at 30 ℃ for 6 or 16 hours in a thermal cycler and then at 85 ℃ for 15 minutes.

After incubation, the reaction product was used directly for Illumina and Oxford Nanopore sequencing library preparation.

Illumina short read long sequencing

Products from targeted MDA reactions were prepared for WGS using an Illumina Nextera XT DNA library preparation kit following the recommended protocol (e.g., Lamble et al 2013; Tyler et al 2016). Briefly, the input DNA is enzymatically cleaved into fragments of approximately 300bp in length, which are then labeled with specific linkers. After adaptor ligation, the indexes were added using 15-cycle PCR amplification.

High-throughput sequencing was performed on an Illumina MiSeq instrument using Illumina MiSeq Reagent kit v2(300 cycles) to generate 150 nucleotide paired-end sequence reads. The raw sequence reads were processed using the standard workflow in trimmatic v0.35 (Bolger et al 2014); this would delete the sequence corresponding to the Illumina linker as well as the low quality region (i.e. phred score < 33). The processed sequence reads were then mapped to a mycobacterium tuberculosis H37rv reference genome using BWA 0.7 and sequence positions known to be associated with antibiotic resistance (Li & Durbin 2009) to determine the drug resistance profile of the strain.

Oxford Nanopore MinION sequencing

According to the manufacturer's recommended protocol, Oxford nanopore 1D was used²The ligation sequencing kit prepares the product from the targeted MDA reaction for WGS. Briefly, input DNA is blunt-ended repaired prior to addition of the flow cell adapter and hairpin adapter (for reverse complement reading). To obtain the final library, the DNA fragments were purified using standard magnetic bead methods.

After the MinION flow-through cell (FLO-MIN 106R9.4) QC, the DNA library was loaded and a standard 48 hour sequencing run was initiated using MinKNOW ONT software. After the run was completed, sequence reads were drawn using Geneious 9.0 for mycobacterium tuberculosis H37rv reference genome (Kearse et al 2012) and sequence positions known to be associated with antibiotic resistance were evaluated to determine the resistance of the strain.

Results

The results of the above design and validation work are listed in the following tables (tables 2-5).

TABLE 2 number of binding sites for Mycobacterium tuberculosis MDA oligonucleotide primers on the reference genome of Mycobacterium tuberculosis H37rv and on the genomes of 15 other bacteria common to the human upper respiratory tract

^aGenBank accession numbers of the genomes used for comparison. Mycobacterium tuberculosis (NC-000962), Haemophilus influenzae (NC-000907), Chlamydia pneumoniae (NC-000922), Pseudomonas aeruginosa (NC-002516), Escherichia coli (NC-002695), Bordetella pertussis (NC-002929), Neisseria meningitidis (NC-003112), Listeria (NC-003210), Lactobacillus brevis (NC-008497), Leuconostoc mesenteroides (NC-008531), Clostridium difficile (NC-009089), Porphyromonas gingivalis (NC-010729), Veillonellaplavulula (NC-013520), Moraxella catarrhalis (NC-014147), Enterobacter aeogenes (NC-015663), Photococcus aureus (NZ-CP 010295).

TABLE 3 position of 15 MDA oligonucleotide primers relative to 13 putative antibiotic resistance loci when mapped to the M.tuberculosis H37rv reference genome

Table 4 genome coverage statistics and antibiotic resistance profiles when mapped to the mycobacterium tuberculosis H37rv reference genome required the original non-amplified sample 5734 and 6 and 16 hour targeted MDA reactions using 10-20ng DNA as starting template

Table 5 raw non-amplified sample 5734 and gene-by-gene coverage statistics of 6 and 16 hour targeted MDA reactions using 10-20ng DNA as starting template when mapped to the mycobacterium tuberculosis H37rv reference genome.

Conclusion

As provided in tables 1-5 above and the accompanying figures, the inventors developed a set of oligonucleotide primers that when used with Φ 29 or Bst enzyme under standard MDA conditions, resulted in the selective amplification of Mycobacterium tuberculosis genomic DNA. This has potential applications in genotyping mycobacterium tuberculosis from small amounts of starting material and WGS (Illumina and MinION) sequencing of mycobacterium tuberculosis genomes from young cultures and sputum samples.

Sequencing of these templates on Illumina MiSEQ and Oxford Nanopore MinION instruments resulted in a statistically high similarity in percentage of mapped sequence reads and depth of coverage versus non-MDA controls. Specifically, for the non-MDA control, 99.1% of the reads mapped to the reference genome, with an average genome coverage of 23.4-fold, and an average genome coverage per genetic locus currently associated with antibiotic resistance of 17.3-42.5-fold. For MDA products, the percentage of mapped sequence reads is 99.3-99.5%, and when the number of reads collected is normalized, the coverage of the genome as a whole and of the individual genes is 18.8-20.2 and 16.1-37.2, respectively.

Although the overall coverage of MDA-produced templates is somewhat lower, the depth of coverage of these templates is greater for several genetic loci. For example, the average coverage of gid loci is controlled to 24.2 for non-MDA, but 28.7-36.0 for MDA-generated templates. Importantly, the MDA generated template provides the same spectrum of antibiotic resistance as the non-MDA control.

Existing methods for assessing drug susceptibility of mycobacterium tuberculosis isolated from patients have several limitations, including the need for specialized infrastructure, slow diagnosis, and incomplete characterization of antibiotic resistance. However, efficient implementation of the unique oligonucleotide primers and selective amplification methods of the present invention as described herein provides advantages by allowing the rapid establishment of a complete antibiotic resistance profile for individual TB patients.

Whole genome sequencing has become the first choice for diagnosis of MDR-TB and XDR-TB as it allows for whole genome assessment of genetic mutations and thus complete characterization of antibiotic resistance. However, due to infrastructure requirements, this activity is often limited to resource-intensive research laboratories (e.g., large, expensive instruments that require controlled conditions and specialized maintenance). WGS can also be effectively used to support clinical diagnosis of drug sensitivity to mycobacterium tuberculosis isolated from a patient using the selective MDA primers and methods described herein. In particular, the use of these methods would eliminate the need to isolate and culture M.tuberculosis from sputum samples prior to WGS in a central laboratory, thereby reducing diagnosis time by as much as several weeks.

The MDA-based approach described herein also enables WGS-based diagnostics to be performed in a low-infrastructure point-of-care setting. By employing the MDA-based methods described herein, a sufficient amount of DNA can be generated to allow the use of WGS of the Oxford Nanopore MinION platform. Such personal DNA sequencing equipment has little infrastructure requirements and when used to analyze DNA templates generated using selective MDA, can achieve rapid tuberculosis diagnosis-hours rather than weeks-at the point of care.

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Industrial applications

The oligonucleotide primers according to the invention and the methods of using them have industrial applications in molecular biology to provide a rapid method to identify pathogenic strains of bacteria, in particular mycobacterium tuberculosis and mycobacterium bovis.

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