Compositions and methods for detecting microorganisms
阅读说明：本技术 用于检测微生物的组合物和方法 (Compositions and methods for detecting microorganisms ) 是由 N.科托夫 J.S.瓦内普斯 K.沃德 于 2018-11-06 设计创作，主要内容包括：本文提供用于检测微生物的组合物、系统和方法。具体而言,本文提供用于快速、多重检测未经纯化的生物样品中的微生物的组合物、系统和方法。(Provided herein are compositions, systems, and methods for detecting microorganisms. In particular, provided herein are compositions, systems, and methods for rapid, multiplexed detection of microorganisms in an unpurified biological sample.)
1. A method of detecting a plurality of microorganisms in a biological sample, comprising:
a) performing nucleic acid amplification on a biological sample to produce a plurality of amplification products of different lengths comprising nanoparticles;
b) depositing a metal shell on the nanoparticles incorporated into the amplification product to produce an amplification product comprising metal-coated nanoparticles; and
c) detecting the amplification product comprising the metal-coated nanoparticle using Circular Dichroism (CD).
2. The method of claim 1, wherein the amplification is PCR.
3. The method of claim 2, wherein the PCR utilizes a plurality of forward and reverse primers, wherein the primers are attached to nanoparticles.
4. The method of any one of claims 1-3, wherein the nanoparticle is a gold nanoparticle.
5. The method of claim 3 or 4, wherein the forward and the reverse primers comprise nanoparticles of different sizes.
6. The method of any one of claims 1-5, wherein the nanoparticles are nanorods.
7. The method of any one of claims 1-6, wherein the amplification products comprising metal nanoparticles are assembled into side-by-side ladders comprising interstitial distances between the nanoparticles.
8. The method of any one of claims 1-6, wherein the amplification products of different lengths exhibit CD peaks at different wavelengths.
9. The method of any one of claims 1-7, wherein the metal shell is gold, copper, or silver.
10. The method of any one of claims 1-9, wherein the microorganism is a bacterium.
11. The method of claim 10, wherein the bacteria are pathogenic bacteria.
12. The method of claim 10 or 11, wherein the bacteria are antibiotic resistant bacteria.
13. The method of claim 3, wherein the primers amplify a plurality of organism-specific and/or antibiotic resistance genes.
14. The method of any one of claims 1-13, wherein the biological sample is whole blood.
15. The method of claim 14, wherein the sample is not cultured or purified prior to performing the method.
16. The process of any one of claims 1-15, wherein the process is carried out in 3 hours or less.
17. The process of any one of claims 1-16, wherein the process is carried out in 2.5 hours or less.
18. The method of any one of claims 1-17, further comprising the step of contacting the sample with an antibiotic prior to performing the method.
19. A method of detecting a plurality of microorganisms in a biological sample, comprising:
a) contacting a biological sample comprising pathogenic bacteria with an antibiotic;
b) amplifying the biological sample to produce a plurality of different lengths of amplification products comprising nanoparticles;
c) depositing a metal shell on the nanoparticles incorporated into the amplification product to produce an amplification product comprising metal-coated nanoparticles; and
d) detecting the amplification product comprising the metal-coated nanoparticle using Circular Dichroism (CD).
20. The method of claim 19, wherein the amplification is PCR.
21. The method of claim 20, wherein the amplification utilizes a plurality of forward and reverse primers, wherein the primers are attached to nanoparticles.
22. The method of any one of claims 19-21, wherein the biological sample is incubated with the antibiotic.
23. The method of any one of claims 19-22, wherein the nanoparticle is a gold nanoparticle.
24. The method of any one of claims 19-23, wherein the forward and the reverse primers comprise nanoparticles of different sizes.
25. The method of any one of claims 19-24, wherein the nanoparticles are nanorods.
26. The method of any one of claims 19-25, wherein the amplification products comprising metal nanoparticles are assembled into side-by-side ladders comprising interstitial distances between the nanoparticles.
27. The method of any one of claims 19-26, wherein the amplification products of different lengths exhibit CD peaks at different wavelengths.
28. The method of any one of claims 19-27, wherein the metal shell is gold, copper, or silver.
29. The method of any one of claims 19-28, wherein the bacteria are antibiotic resistant bacteria.
30. The method of claim 21, wherein the primers amplify a plurality of organism-specific and/or antibiotic resistance genes.
31. The method of any one of claims 19-30, wherein the biological sample is whole blood.
32. The process of any one of claims 19-31, wherein steps b) through d) of the process are performed in 3 hours or less.
33. The process of any one of claims 19-32, wherein the process is carried out in 2.5 hours or less.
34. A composition or kit comprising at least one pair of amplification primers comprising a plurality of forward and reverse primers, wherein the primers are attached to nanoparticles.
35. The composition or kit of claim 34, wherein the nanoparticle is a gold nanoparticle.
36. The composition or kit of claim 34 or 35, wherein the forward and the reverse primers comprise nanoparticles of different sizes.
37. The composition or kit of any one of claims 34-36, wherein the nanoparticle is a nanorod.
38. The composition or kit of any one of claims 34-37, wherein the composition or kit further comprises one or more components selected from the group consisting of: a metal, an antibiotic, a control nucleic acid, one or more buffers, and one or more enzymes.
39. The composition or kit of claim 38, wherein the metal shell is gold, copper, or silver.
40. A reaction mixture, comprising:
a plurality of forward and reverse primers, wherein the primers are attached to nanoparticles that bind to a target nucleic acid or amplification product thereof.
41. The reaction mixture of claim 40, wherein said nanoparticles are gold nanoparticles.
42. The reaction mixture of claim 40 or 41, wherein the forward and the reverse primers comprise nanoparticles of different sizes.
43. The reaction mixture of any one of claims 40-42, wherein said nanoparticles are nanorods.
44. The reaction mixture of any one of claims 40 to 43, wherein the reaction mixture is coated with a metal.
45. The reaction mixture of claim 44, wherein said metal shell is gold, copper or silver.
Description of the drawings
FIG. 1 shows (A) a PCR replication procedure in which a DNA strand is amplified using a primer, a template DNA, taq plus polymerase and 4 different DNA bases. (B) PCR-based gold Nanorods (NR) end-to-end (ETE) assemblies. (C) PCR-based gold NR side-by-side (SBS) modules with inter-NR gaps d; for clarity, the DNA strands are removed at the bottom of the figure. Representative electron microscopy images of ETE (D-F) and SBS (G-I) modules obtained after different numbers of PCR cycles, n =2 (D & G), n =10 (E & H), n =15 (F & I). Cryo-electron tomography (J) and CD spectroscopy of SBS components.
Figure 2 shows a schematic representation of (a) PCR assembly of nanoparticle composites and post-assembly deposition of Ag and Au shells. (B) Shell-mediated spectral modulation of chiral plasmon bands. (C) A calibration curve was analyzed which correlated the intensity of the CD band with the Au shell to the concentration of DNA.
FIG. 3 shows doping with 0-104Gel electrophoresis of mecA PCR of whole blood in serial dilutions of live MRSA in the CFU/ml blood range.
FIG. 4 shows UV-vis spectra of (A) whole blood. (B) CD spectra of whole blood with and without bacterial DNA spiked at 0-104Serial dilutions of live MRSA in the CFU/ml blood range.
Fig. 5 shows the calculated CD spectra of NR complexes with gaps of 2, 5, 10, 20, 30 and 40 nm.
FIG. 6 shows a hypothetical readout of AST from NR-PCR.
Definition of
To facilitate an understanding of the present technology, a number of terms and phrases are defined below. Additional definitions are set forth throughout the detailed description.
The term "nucleic acid molecule" as used herein refers to any nucleic acid-containing molecule, including but not limited to DNA or RNA. The sequences encompassed by this term include any known base analog of DNA and RNA, including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseuduracil, 1-methylguanine, 1-methylinosine, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylbraided glycoside (beta D mannosylqueosine), 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl uracil-5-oxoacetate, oxybutoxythymidine (oxybutoxosine), pseudouracil, braided glycoside (queosine), 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, methyl N-uracil-5-oxoacetate, N-methylacryloyl-5-oxoacetate, N-methoxyuracil-2-oxoacetate, N-methoxyuracil, 5-methoxyuracil, 2-methylthioadenine, 2-methylthiouracil, 2-methyluracil, 5-oxoacetate, and mixtures thereof, Pseudouracil, stevioside, 2-thiocytosine and 2, 6-diaminopurine.
The term "gene" refers to a nucleic acid (e.g., DNA) sequence comprising coding sequences necessary for the production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA). A polypeptide can be encoded by a full-length coding sequence or any portion of a coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, immunogenicity, etc.) of the full-length or fragment are retained. The term also encompasses the coding region of the structural gene and sequences located adjacent to the coding region on both the 5 'and 3' ends (about 1kb or longer on either end) such that the gene corresponds to the length of the full-length mRNA. Sequences located 5 'to the coding region and present on the mRNA are referred to as 5' untranslated sequences. Sequences located 3 'or downstream of the coding region and present on the mRNA are referred to as 3' untranslated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene.
The term "primer" refers to an oligonucleotide (whether naturally occurring (as in a purified restriction digest) or synthetically produced) that is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product complementary to a nucleic acid strand is induced (i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH). For maximum amplification efficiency, the primer is preferably single-stranded, but may alternatively be double-stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare an extension product. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be long enough to prime the synthesis of extension products in the presence of an inducing agent. The exact length of the primer will depend on many factors, including temperature, source of primer, and use of the method.
The term "target" when used in reference to the polymerase chain reaction refers to a region of nucleic acid to which primers used in the polymerase chain reaction bind. Therefore, it is sought to select "targets" from other nucleic acid sequences. A "segment" is defined as a region of nucleic acid within a target sequence.
The term "amplicon" as used herein refers to the nucleic acid produced by the amplification reaction. Amplicons are generally double-stranded DNA; however, it may be RNA and/or DNA RNA. The amplicon comprises DNA complementary to the sample nucleic acid. In some embodiments, the primer pair is configured to generate an amplicon from the sample nucleic acid. Thus, the base composition of any given amplicon may include the primer pair, the complementary sequence of the primer pair, and the region of the sample nucleic acid that is amplified to produce the amplicon. It is understood by those skilled in the art that the designed primer pair sequences are incorporated into the amplicon in place of the native sequence at the primer binding site and its complement. In certain embodiments, after amplification of the target region using the primers, the resulting amplicons with the primer sequences are used for subsequent analysis. In some embodiments, the amplicon further comprises a length that is compatible with subsequent analysis.
In the context of nucleic acids, the term "amplification" or "amplification" refers to the generation of multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting with a small amount of the polynucleotide (e.g., as few as a single polynucleotide molecule) in which amplification products or amplicons are typically detectable. Amplification of polynucleotides encompasses a variety of chemical and enzymatic processes. During Polymerase Chain Reaction (PCR) or Ligase Chain Reaction (LCR), multiple copies of DNA are generated from one or several copies of a target or template DNA molecule in amplified form. Amplification is not limited to the strict replication of the starting molecule. For example, Reverse Transcription (RT) -PCR is used to generate multiple cDNA molecules from a limited amount of RNA in a sample as an amplified version. In addition, the production of multiple RNA molecules from a single DNA molecule during the transcription process is also an amplified form.
The terms "subject" and "patient" as used herein refer to any animal, such as a dog, cat, bird, livestock, and particularly a mammal, and preferably a human.
The term "sample" as used herein is used in its broadest sense. In a sense, this is meant to include representative fractions or cultures obtained from any source, including biological and environmental sources. Biological samples can be obtained from animals, including humans, and include fluids, solids, tissues, and gases. Biological samples include blood and blood products such as plasma, serum, and the like. However, such examples should not be construed as limiting the type of sample that is suitable for use in the present disclosure.
Detailed description of the invention
While the disclosure herein refers to certain illustrated embodiments, it is to be understood that these embodiments are presented by way of example and not by way of limitation.
Provided herein are compositions, systems, and methods for detecting microorganisms. In particular, provided herein are compositions, systems, and methods for rapid, multiplexed detection of microorganisms in an unpurified biological sample.
The cardiovascular device market, particularly those in direct contact with the blood stream (e.g., pacemakers, defibrillators, prosthetic heart valves, left ventricular assist devices, stents, vascular access devices, and venous filters) is one of the fastest growing medical device industries. This increase is due to the increasing prevalence of Cardiovascular diseases (Mozaffarian, D. et al A Report from the American Heart Association, doi:10.1161/cir.0000000000000350 (2015)), enlarged indications for implantation (Praz, F., et al JACC: Cardiovasular Intervision 8, 1777-1796, doi:10.1016/j. jcin.2015.08.015 (2015)), Zhan, C., et al Journal of general internal Medicine 23, 13-19, doi:10.1007/s11606-007-0392-0 (2008)) and improvements in basic bioengineering in materials and electronics (Khan, W., Muntimadugu, E., Jaffe, M. & Dombin J. Control. J. Paul [ 13 ] 14, J. (2014, Sphij. 19)). While these devices provide significant benefits, any foreign object placed within the bloodstream can still constitute a significant threat of infection. Although The rate of implantation has increased by as much as 65% over The past few decades (Zhan et al supra; Isaacs, A. J., et al The Journal of clinical and cardiovascular surgery delivery 149, 1262-1269.e1263, doi:10.1016/j. jtcvs.2015.01.052 (2015)), The rate of infection has increased disproportionately (i.e. >200%) (Nielsen et al supra; Greenspon et al supra; Wang, A. et al Jama 297, 1354-. As a result, implanted medical devices are a leading cause of blood stream infections in the united states. As the implanted indications continue to expand (Praz et al, supra; Zhan et al, supra), patients with increased mesocomplications receive these devices. For example, transcatheter aortic valve replacement procedures allow patients who may not otherwise be good candidates for open surgery to receive valve replacement (Alsara, O., et al Journal of Geriatric Cardiology: JGC 11, 163-170, doi:10.3969/j. issn.1671-5411.2014.02.004 (2014)). Thus, it is expected to have greater co-morbidity and immunosuppression, and therefore greater risk of infection. While new antimicrobial device strategies are being investigated, the time span for their development, regulatory approval, and clinical adoption is long.
The main challenge associated with device-related infections is that they are caused by biofilms. Biofilms are sessile bacterial communities encapsulated in extracellular matrix. They begin with colonization of non-biological materials and may progress to disseminated infection 16. Thus, in many cases, the initial performance is inert and diagnosis is generally not made until the infection has disseminated. In this regard, the device is coated in a mature biofilm that is recalcitrant to antibiotic or host immune responses. Finally, the device must be surgically removed and replaced, followed by long-term systemic antibiotic therapy. Thus, early diagnosis of intravascular device contamination would reduce morbidity and mortality, and even save precious devices.
Current gold standards for diagnosing intravascular device-related infections require bacterial detection, Identification (ID) and AST via traditional blood culture. This is a very poor diagnosis with profound clinical implications. First, the time to obtain blood culture results is generally in the range of 1-5 days (Cleven, B.E. E. et al Journal of clinical microbiology 44, 2389-2397, doi:10.1128/jcm.02291-05 (2006)). Once the patient exhibits systemic symptoms, broad spectrum antibiotic treatment is initiated while waiting for the culture results. This empirical use of antibiotics can lead to opportunistic infections, drug-related toxicity, and antibiotic resistance. Second, the sensitivity of blood culture is low. When the number of bacteria in the blood is low (such as when the device is colonized but before dissemination), growth is slow enough to produce a negative result (Neut, D., et al Acta orthopathica 78, 299-. Still worse, some bacteria do not grow at all under standard culture conditions (Peters, R.P. H., et al, The Lancet Infections Diseases 4, 751-760, doi: http:// dx. doi. org/10.1016/S1473-3099 (04) 01205-8 (2004)). For example, up to 30% of prosthetic valve endocarditis is initially culture negative (Habib, G., et al European heart journal 36, 3075-3128, doi:10.1093/eurheartj/ehv319 (2015)). Thus, infection is not detected until the biofilm matures and is less susceptible to antibiotics and host responses. Third, cultures are often contaminated with normal skin flora (Weinstein, M.P. Journal of clinical microbiology 41, 2275-2278, doi:10.1128/jcm.41.6.2275-2278.2003 (2003)) that can grow rapidly and compete for certain pathogens in the culture medium. Repeated incubations to confirm contamination and infection further extend the diagnostic time.
There is a need for a rapid, ultrasensitive, culture-free diagnostic for bacteremia detection, ID and AST. Non-cultured molecular diagnostic methods are rapidly being incorporated into standard medical microbiology laboratories. Most techniques are based on nucleic acid detection and/or amplification (e.g., PCR). However, direct detection of bacterial nucleic acids in whole blood remains a challenge. This is related to the fact that: at high background concentrations of human cells, the concentration of bacteria in blood can be very low (e.g., <1 CFU/ml) (Valencia-Shelton, F. & Loeffellholz, M. Future microbiology 9, 543-. To date, there is no approved diagnostic for the direct detection of bacteria without culture pre-enrichment. Although current molecular techniques utilizing initial culture enrichment improve time to AST and demonstrate clinical benefits including hospital stays and health costs (Sango, A. et al Journal of clinical microbiology 51, 4008-. The only viable whole blood nucleic acid detection system that does not require culture enrichment is the Septifast technology (Roche Diagnostics, Mannheim, Germany). Based on the manufacturer's instructions, it could identify bacteria within <6 hours. However, it does not have a full AST. In addition, it can only detect as low as 300 CFU/ml. Although this may be useful in patients with severe infections or pre-treatment with antibiotics (Yanagihara, K. et al Critical Care 14, R159-R159, doi:10.1186/cc9234(2010)), it is not sensitive enough for many cases of indolent bacteremia caused by medical device infections, which may have single digit CFU/ml concentrations.
Accordingly, provided herein is a detection method that addresses an unmet clinical need for detecting microorganisms (e.g., in whole blood). In some embodiments, the PCR reactions described herein generate composite nanorod "ladders" by coupling standard PCR primers to nanoparticles (e.g., gold nanorods (NR-PCR)). These assemblies have unique chiral plasma properties that allow for the use of Circular Dichroism (CD) spectrophotometry for the
The preferred NR-PCR method (1) can be applied to whole blood without prior culturing; (2) can be performed in sufficient multiplex to detect most pathogens; (3) providing an antibiotic susceptibility profile; and (4) providing a time to result of less than 3 hours.
By coupling standard PCR primers to gold nanorods (NR-PCR; FIGS. 1A-C), the PCR reaction produces a composite nanorod assembly, the length of which depends on the number of PCR cycles (FIGS. 1D-I). The side-by-side (SBS) 'ladder' has unique chiral plasma properties (fig. 1J), which allows detection of as low as
The use of this highly sensitive and specific PCR technique in combination with primers that target the identification of species-specific genes greatly affects the detection and ID of bacteria in whole blood. The sensitivity is improved, and the bacteria which are difficult to be cultured and have ultralow concentration can be detected. It also allows DNA detection without prior culture growth enrichment. High specificity increases the number of gene targets that can be assayed in a multiplex system. It also allows detection of bacterial DNA without sample treatment to remove excess human DNA.
In some embodiments, the technique utilizes metal (e.g., Au, Ag, Cu) deposition around the NP module — a post-PCR reaction (fig. 2A). These shell coatings provide a means to modulate the CD spectral peaks (fig. 2B) (Zhao et al, supra). In addition, it increases the detection limit down to
The present disclosure is not limited to a particular microorganism. In some embodiments, bacteria (e.g., pathogenic bacteria or antibiotic resistant bacteria) are detected. Examples of pathogenic bacteria include, but are not limited to, methicillin-resistant Staphylococcus aureus (methicillin resistant)S. Aureus) (MRSA), Mycobacterium tuberculosis: (M. tuberculosis) (antibiotic sensitive or resistant), Streptococcus (Streptococcus sp.) Staphylococcus genus (1)Staphylococcus spSalmonella genus (II)Salmonella sp.) Listeria genus (a), (b), (c), (d), (Listeria sp.) Escherichia coli (E.coli)E. coli) Shigella (A), (B), (C)Shigella sp.) Campylobacter (I), (II)Campylobacter sp.) Pseudomonas (a)Pseudomonas sp.) Bacillus (B) and (C)Bacillus sp.) The whole body of Ba (a) ((B))Bartonella sp.) Bordetella (Bordetella), (Bordetella) and (Bordetella)Bordetella sp.) Borrelia genus (Borrelia sp.) Brucella (Brucella) (Brucella)Brucella sp.) The genus of Chlamydia (A)Chlamydia sp.) Clostridium (f) <Clostridium sp.) Corynebacterium genus (A), (B), (C)Corynebacterium sp.) Enterococcus genus (A)Enterococcus sp.) Francisella (Francisella)Francisella sp.) Haemophilus genus (A), (B), (CHaemophilus sp.) Helicobacter genus (A)Helicobacter sp.) Legionella (Legionella)Legionella sp.) Leptospira genus (Leptospira sp.) Mycobacterium genus (A), (B), (CMycobacterium sp.) Mycoplasma genus (A), (B), (CMycoplasma sp.) Rickettsia belongs to (Rickettsia sp.) Treponema (A) and (B)Treponema sp.) Ureaplasma genus (A), (B), (CUreaplasma sp.) Genus Vibrio: (Vibrio sp.) And Yersinia genus (Yersinia sp.) And Neisseria species (Neisseria sp.)。
In some embodiments, a whole blood sample or other biological sample from a subject is used for analysis with or without further purification. In some embodiments, the subject has an implanted medical device (e.g., including but not limited to a defibrillator, an artificial bone or joint, a cardiac pacemaker, a cosmetic implant, a screw, a rod, a disc, etc., an IUD, and a coronary stent).
In some embodiments, the NR-PCR comprises the following steps. In some embodiments, a biological sample is first subjected to PCR with a plurality of forward and reverse primers attached to nanoparticles. In some embodiments, primers for 1 or more (e.g., 2, 3, 4, 5, or more) targets are used. In some embodiments, each pair of primers is designed to produce PCR products of different lengths, such that a plurality of PCR products of different lengths are produced that comprise nanoparticles.
The present disclosure is not limited to particular microparticles or nanoparticles. Examples include, but are not limited to, nanorods. In some embodiments, the nanoparticles or nanorods are gold or other suitable materials. In some embodiments, the forward and reverse primers comprise nanoparticles of different sizes. In some embodiments, PCR products comprising metal nanoparticles are assembled into side-by-side ladders comprising interstitial distances between the nanoparticles. In some embodiments, the difference in gap size is expected to result in different CD profiles for different length PCR products.
In some embodiments, after PCR, a metal shell is deposited on the PCR product comprising the nanoparticles. In some embodiments, the metal shell is gold, copper, or silver, or another metal, and may be the same or different from the nanoparticle material.
In some embodiments, after deposition, the metal-coated PCR products comprising the nanoparticles are detected using Circular Dichroism (CD). In some embodiments, PCR products of different lengths exhibit different peak CD shift wavelengths, allowing multiplexing to occur. Circular Dichroism (CD) is dichroism involving circularly polarized light, i.e. differential absorption of left and right handed light. Left-handed circular (LHC) and right-handed circular (RHC) polarized light represent two possible spin angular momentum states of a photon, and thus circular dichroism is also referred to as dichroism of spin angular momentum. Which is present in the absorption band of the optically active chiral molecule. CD spectrophotometry has a wide range of applications in many different fields. UV CD was used to study the secondary structure of proteins. UV/Vis CD was used to study the charge transfer transitions. Near infrared CD is used to study geometry and electronic structure by detecting metallic d → d transitions. Vibrational circular dichroism using light from an infrared energy region is used for structural studies of small organic molecules, and recently, for structural studies of proteins and DNA. In some embodiments, UV/Vis CD is used to detect NR-PCR products.
In some embodiments, the NR-PCR method from sample collection to data collection and analysis (including CD detection) is completed in 3 hours or less (e.g., 3.0, 2.75, 2.5, 2.25, 2.0 hours or less).
In some embodiments, the NR-PCR method comprises the step of determining the antibiotic resistance status and/or specificity of bacteria (e.g., pathogenic bacteria) in the sample. For example, in some embodiments, the sample is contacted with an antibiotic prior to performing the NR-PCR detection method. In some embodiments, each of the plurality of samples is contacted with a different antibiotic (e.g., of a different class or of the same class). In some embodiments, the sample is incubated with the antibiotic for a period of several hours. In some embodiments, the NR-PCR method is performed at different time points and the growth of the bacteria is determined. The sensitivity of the sample to each antibiotic was determined by comparing the levels of the detected NR-PCR products reflecting bacterial growth. In some embodiments, a genetic profile of the microorganism is determined to reveal antibiotic susceptibility.
In some embodiments, the antibiotic sensitivity of the sample is used to determine and optionally administer a course of therapeutic action (e.g., selecting one or more antibiotics to administer to the subject).
In some embodiments, a computer-based analysis program is used to translate the raw data (e.g., the presence, absence, or amount of a given microorganism) generated by a detection assay into data that is of predictive value to a clinician. The clinician may access the predictive data using any suitable means. Thus, in some preferred embodiments, the present disclosure provides the further benefit that clinicians who are unlikely to receive genetic or molecular biological training do not need to understand the raw data. The data is presented directly to the clinician in its most useful form. The clinician can then immediately utilize this information to optimize the care of the subject.
The present disclosure contemplates any method capable of receiving, processing, and transmitting information to and from laboratories, information providers, medical personnel, and subjects conducting assays. For example, in some embodiments of the present disclosure, a sample (e.g., a blood sample) is obtained from a subject and submitted to a profiling service (e.g., a clinical laboratory of a medical facility, a genomic profiling service, etc.) to generate raw data. If the sample comprises a tissue or other biological sample, the subject may access a medical center to obtain the sample and send it to a profiling center, or the subject may collect the sample (e.g., a blood sample) by itself and send it directly to the profiling center. If the sample contains previously determined biological information, this information can be sent by the subject directly to the profiling service (e.g., an information card containing the information can be scanned by a computer and the data transmitted to the computer of the profiling center using an electronic communication system). Once received by the profiling service, the sample is processed and a profile (e.g., the presence of bacteria and/or antibiotic resistance status in the sample) is generated that is specific to the diagnostic or prognostic information desired by the subject.
The profile data is then prepared in a format suitable for interpretation by the attending physician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment of the subject (e.g., the presence or absence of a microorganism or antibiotic-resistant microorganism) and a recommendation for a particular treatment option. The data may be displayed to the clinician by any suitable method. For example, in some embodiments, the profiling service generates a report that can be printed for the clinician (e.g., at a point of care) or displayed to the clinician on a computer monitor.
In some embodiments, the information is first analyzed at the point-of-care or regional facility. The raw data is then sent to a central processing facility for further analysis and/or conversion into information useful to a clinician or patient. The central processing facility provides the advantages of privacy (all data stored in the central facility with a uniform security protocol), speed, and consistency of data analysis. The central processing facility may then control the fate of the data after the subject is treated. For example, using an electronic communication system, the central facility may provide data to a clinician, subject, or researcher.
In some embodiments, the subject is able to access the data directly using an electronic communication system. The subject may select further intervention or counseling based on the results. In some embodiments, the data is for research use. For example, the data can be used to further optimize the inclusion or elimination of markers, as a useful indicator of a particular condition or stage of disease or as a companion diagnostic to determine the course of therapeutic action (e.g., selection of antibiotics).
In some embodiments, the present disclosure provides kits and systems for isolating and analyzing nucleic acids (e.g., microorganism-specific nucleic acids). In some embodiments, the kit comprises reagents (e.g., primers, nanoparticles, metals, enzymes, buffers, controls, instructions, instruments, processing devices, etc.) necessary, sufficient, or useful for detecting nucleic acids.
In some embodiments, the kit comprises one or more containers comprising primers, nanoparticles, metals, enzymes, buffers, controls, instructions, and the like. In some embodiments, each component of the kit is packaged in a separate container. In some embodiments, the containers are packaged and/or shipped in the same kit or box for use together. In some embodiments, one or more components of the kit are shipped and/or packaged separately.
In some embodiments, the system includes an automated sample and reagent processing device (e.g., a robot).
Experiment of
The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present disclosure, and should not be construed as limiting the scope thereof.
Example 1
Direct detection of bacteria in whole blood by NR-PCR
Many components of whole blood (e.g., hemoglobin) produce chiral plasma signals that can interfere with CD spectroscopic detection. Whole blood is a complex medium containing intact cells, proteins/peptides, lipids, and many small molecules that may interfere with nucleic acid detection. There are several known PCR inhibitors in whole blood, including hemoglobin and immunoglobulins, as well as proteases (Kermekchiev, M.B., et al, Nucleic Acids Research, doi:10.1093/nar/gkn1055 (2009)). To solve this problem, a specific polymerase kit designed for whole blood was used. Using this kit, it was possible to detect the presence of the methicillin resistance gene (mecA) in whole blood spiked with live methicillin-resistant Staphylococcus aureus (MRSA) at concentrations as low as-300 CFU/ml by simple gel electrophoresis (FIG. 3).
Whole blood NR-PCR assay
The data show that the NR 'ladder' after PCR can be shell-coated to modulate the CD band (fig. 2). Specifically, the Ag shell produces a blue shift in the peak CD band, while the Au shell produces a red shift. Copper shells were also tested, which produced a large red shift. These spectral shifts also depend on the shell thickness 8. The shell thickness is controlled by the amount of metal added in the shell coating reaction. This control of the CD spectral band allows shifting the peak to the less noisy portion of the whole blood spectrum. It has been found that increasing concentrations of NH2Hc to additionally reduce Cu and Au shells, additional control over spectral peak positions and their intensities can be added.
The peak CD signal intensity of the NR-PCR reaction in whole blood was evaluated as a function of the shell thickness of the 3 metals (i.e., Au, Ag and Cu). Genomic DNA from Staphylococcus aureus (Staphylococcus aureus) COL was used as a template for the PCR reaction. The strain is a prototype MRSA strain. Genomic DNA was isolated from mid-log cultures of Staphylococcus aureus using the enzyme Staphylococcus aureus (Sigma-Aldrich, St. Louis, MO), Trizol (Life technologies, Carlsbad, Calif.) and the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). Au NR was synthesized by growth from Au seeds and modified as previously described (Ma et al, supra) for conjugation in SBS configuration (fig. 1C) to PCR primers for staphylococcus aureus (table 1). PCR reactions were performed using a 5 XPhusion Blood Direct PCR kit (Thermo Fisher scientific, Waltham, Mass.) with template genomic DNA and Au-NR conjugated primers. The thermocycling protocol starts with a 5 minute pre-denaturation step at 95 ℃ followed by 20 cycles of a 95 ℃ denaturation (5 s), 50-65 ℃ annealing (5 s) and 72 ℃ extension (15-30 s) step, followed by a further extension at 72 ℃ for 10 minutes. Finally, the PCR system will be maintained at 4 ℃ for 10 minutes. The shell coating is applied based on the previously described protocol (Zhao et al, supra). Briefly, the PCR product was centrifuged at 7000 rpm for 5 minutes and the pellet was resuspended in PBS-CTAB (20 mM CTAB). Then, AgNO is added3(0.1 mM-10 mM) plus 0.1M ascorbic acid; HAuCl4(0.3-10 mM) plus 1 mM NH2OH, HCl; or CuCl2(0.5-5 mM) plus 1 mM NH2Hcl was mixed into the PCR product. After 5 minutes of reaction, the mixture was centrifuged at 6000 rpm for 5 minutes. Then precipitatingThe material was dissolved in deionized water and UV-vis and CD spectra were obtained via a JASCO J-815 spectrophotometer.
It is expected that the shift to the near red portion of the spectrum will provide the best signal-to-noise ratio and the highest assay sensitivity.
Comparison with Standard blood culture
The sensitivity and specificity of the assay was confirmed by comparison with current clinical gold standards (i.e. blood culture).
Human whole blood (commercially available from Valley Biomedical, Virginia) obtained from healthy donors was spiked with defined concentrations of methicillin-sensitive staphylococcus aureus (MSSA) and methicillin-resistant staphylococcus aureus (MRSA) and wild-type e. All bacterial strains were available as frozen glycerol stocks stored at-80 ℃. Single colonies of a given species were inoculated into Tryptic Soy Broth (TSBG) containing 1% glucose and grown to mid-log phase. The bacteria were then serially diluted in increments of 10 x. One aliquot of each dilution was used for spiking the whole blood sample. The other was used for plating and colony counting. Blood without bacteria is known to be used as a negative control. The samples were treated simultaneously for NR-PCR (primer sequences see Table 1) and standard blood culture. Test samples were prepared by laboratory personnel other than those performing the platform assay. A plot of CD intensity versus bacterial concentration was used to determine the limit of detection. Standard 2x2 tables were constructed to determine sensitivity and specificity.
If untreated blood is used and single molecule detection is achieved, the maximum theoretical detection limit is 50 CFU/ml. In some cases, 2 ml of whole blood was initially denatured at 98 ℃ prior to NR-PCR, and then centrifuged to concentrate large macromolecules (including DNA). In some cases, multiple (~100) simultaneous NR-PCR reactions are also performed on the same sample, similar to digital PCR, but without the complexity of generating picoliter size reactions.
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