阅读说明：本技术 一种snp分子标记、pcr引物及应用 (SNP molecular marker, PCR primer and application ) 是由 李卫民 朱晨迪 贾俊楠 于 2021-09-23 设计创作，主要内容包括：本发明提供了一种SNP分子标记、PCR引物及应用,涉及分子标记及其应用技术领域。所述SNP分子标记包括位于rpoB基因上的SNP1～SNP 8和位于gyrB基因上的SNP 9～SNP 15。本发明提供的SNP分子标记在9种分枝杆菌中具有多态性,通过检测分枝杆菌在SNP位点上的碱基类型即可鉴别出待测的分枝杆菌的种类。(The invention provides an SNP molecular marker, a PCR primer and application, and relates to the technical field of molecular markers and application thereof. The SNP molecular markers comprise SNPs 1-SNP 8 positioned on rpoB gene and SNPs 9-SNP 15 positioned on gyrB gene. The SNP molecular marker provided by the invention has polymorphism in 9 kinds of mycobacteria, and the species of the mycobacteria to be detected can be identified by detecting the base type of the mycobacteria at the SNP site.)

1. An SNP molecular marker, which is characterized by comprising SNPs 1 to SNP8 positioned on rpoB gene and SNPs 9 to SNP15 positioned on gyrB gene; wherein

SNP1 is located at 14 th site from 5' end of sequence shown in SEQ ID No.1, and base is A, G;

SNP2 is located at 26 th site from 5' end of sequence shown in SEQ ID No.1, and base is C, G;

SNP3 is located at 65 th site from 5' end of sequence shown in SEQ ID No.1, and base is C, T;

SNP4 is located at 128 bits from the 5' end of the sequence shown in SEQ ID No.1, and the base is C, G;

SNP5 is located at position 170 from the 5' end of the sequence shown in SEQ ID No.1, and the base is G, T or C;

SNP6 is located at 311 bits from the 5' end of the sequence shown in SEQ ID No.1, and the base is C, G or T;

SNP7 is located at 421 position from 5' end of the sequence shown in SEQ ID No.1, and the base is C, T;

SNP8 is located at 498 th from 5' end of sequence shown in SEQ ID No.1, base is G, A;

SNP9 is located at 32 bits from the 5' end of the sequence shown in SEQ ID No.2, and the base is A, G or C;

SNP10 is located at position 38 from the 5' end of the sequence shown in SEQ ID No.2, and the base is A, C or G;

SNP11 is located at 59 th from 5' end of the sequence shown in SEQ ID No.2, and the base is G, T, C or A;

SNP12 is located at 149 th from the 5' end of the sequence shown in SEQ ID No.2, and the base is A, G or C;

SNP13 is located at position 236 of the 5' end of the sequence shown in SEQ ID No.2, and the base is C, T or G;

SNP14 is located at 302 th site from 5' end of the sequence shown in SEQ ID No.2, and the base is C, T;

SNP15 is located at position 437 of the 5' end of the sequence shown in SEQ ID No.2, and the base is G or A.

2.PCR primers for amplifying the SNP molecular marker according to claim 1, comprising primers SEQ ID No.3 and SEQ ID No.4 for amplifying the sequence shown in SEQ ID No. 1; and primers SEQ ID No.5 and SEQ ID No.6 for amplifying the sequence shown in SEQ ID No. 2.

3. A kit for detecting the SNP molecular marker according to claim 1, which comprises the PCR primer according to claim 2; preferably, the kit further comprises dNTPs, Taq DNA polymerase and Mg²⁺PCR reaction buffer solutionAt least one of (1).

4. A gene chip comprising the PCR primer of claim 2.

5. Use of the SNP molecular marker according to claim 1 or the PCR primer according to claim 2 or the kit according to claim 3 for the detection and/or identification of Mycobacterium tuberculosis complex for non-disease diagnostic purposes.

6. A method for identifying Mycobacterium tuberculosis complex for non-disease diagnosis purposes, which is characterized in that the species type of a sample to be tested is judged by detecting the polymorphism of the SNP molecular marker according to claim 1.

7. The method of claim 6, wherein the detection of the polymorphism of the SNP molecular marker comprises one or more of: SNP detection method based on gel electrophoresis, DNA sequencing method, DNA chip method, denaturation high performance liquid chromatography or mass spectrometry detection method.

8. The method of claim 6, wherein the Mycobacterium tuberculosis complex is identified when the polymorphism result shows that the base corresponding to SNP 1-SNP 15 is ACTGGGTAAAGATTA;

when the base corresponding to SNP 1-SNP 15 is GGCCTCCGCCCGGCG, the target is mycobacterium abscessus;

when the base corresponding to SNP 1-SNP 15 is ACTGGGTAAAAATTA, the gene is Mycobacterium carinii;

when the base corresponding to SNP 1-SNP 15 is GGCGCCCACGCGCCG, the nucleotide is M.avium;

when the base corresponding to SNP 1-SNP 15 is GGCCTTCGCCGCGCG, the mycobacterium cheloniae is obtained;

when the base number corresponding to SNP 1-SNP 15 is GGCCTTCACGCCCCG, the mycobacterium fortuitum is determined;

when the base corresponding to SNP 1-SNP 15 is GGCCTCCACGCCCCG, the gene is M.intracellulare;

when the base corresponding to SNP 1-SNP 15 is GGCCTGCAGCGCCCG, the gene is Mycobacterium kansasii;

when the base corresponding to SNP 1-SNP 15 is GGCGTCCACGTCCCG, it is Mycobacterium marinum.

9. The method of claim 6, further comprising obtaining DNA from a sample to be tested; amplifying the DNA as a template by using the PCR primer according to claim 2; sequencing the amplified product to determine the polymorphism of the SNP molecular marker.

10. The method according to claim 9, wherein the concentration of the PCR primers in the amplification procedure is 350-500 nM, preferably 500 nM; preferably, the same annealing temperature is used for the 2 sets of primers during amplification, and the annealing temperature is 58-62 ℃, preferably 60 ℃.

Technical Field

The invention relates to the technical field of molecular markers, in particular to an SNP molecular marker, a PCR primer and application.

Background

Tuberculosis (TB) caused by the mycobacterium Tuberculosis complex is the single most lethal infectious disease. In 2020, the WHO report indicates that 20 hundred million TB-infected people account for 1/3-1/4 of the total number of people, and about 1000 million cases of attack and 140 million cases of death. Correspondingly, the number of TB-infected people is about 3.5 hundred million in China, the number of the sick people is 86.6 ten thousand, the number of the dead people is 3.9 ten thousand, and the people live in the third place of the world.

The Mycobacterium tuberculosis complex belongs to the genus Mycobacterium (Mycobacterium), and mainly comprises several species of Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium bovis (Mycobacterium bovis), Mycobacterium africanum, Mycobacterium microti and Mycobacterium kenneyi. Within the genus Mycobacterium, more than 100 species such as Mycobacterium avium, Mycobacterium intracellulare and the like are included in addition to Mycobacterium tuberculosis complex and Mycobacterium leprae, which are also called non-tuberculosis mycobacteria (NTM).

The traditional method for identifying the mycobacteria is judged according to the phenotypic characteristics of colony morphology, oxygen preference, nicotinic acid accumulation, nitrate reductase activity and the like, and is simple, low in reagent price and easy to popularize and is used up to now. However, it is time-consuming, low in positive rate, highly contaminated and lack of repeatability, and cannot provide accurate and timely reference for clinical diagnosis. With the rapid development of molecular biology technology, based on PCR and other technologies, the identification or differentiation of Mycobacterium tuberculosis strains and other strains in Mycobacterium can be completed by utilizing the specificity of homologous DNA sequences in Mycobacterium, thereby providing objective basis for clinical diagnosis and treatment, and being favored due to the advantages of rapidness, high efficiency, high specificity and the like.

The whole genome sequencing technology is improved along with the rapid progress of molecular biology technology, and becomes the mainstream technology for identifying strains. The mycobacterium is identified to the species or subspecies level according to the specificity of DNA homologous fragment sequences of different species of mycobacteria, the resolution and the accuracy are further greatly improved, and the identification time is obviously shortened. However, because of its high price, it is mostly used in scientific research and is difficult to be applied to clinical diagnosis.

The 16SrDNA is a bacterial DNA sequence, can code a sequence corresponding to the 16SrRNA on a chromosome, is an inherent sequence of all bacterial chromosome genes and can be used for strain identification. The bacillus identification method has the advantages of few species, large content and moderate molecular size, can reflect the difference between different bacteria, can easily detect the sequence by a sequencing technology, and can identify bacteria to a species level as a 'gold standard' for identifying mycobacteria. However, due to the high sequence identity and close affinity of homologous DNA sequences of some mycobacteria, the 16SrDNA sequence composition is completely identical, so that some strains with close affinity, such as Mycobacterium avium and M.intracellulare, Mycobacterium cheloniae and M.abscessus, Kansas and M.gastri, and the like, cannot be distinguished. Therefore, there is an urgent need to develop an improved method for rapidly identifying mycobacteria with high accuracy.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an SNP molecular marker, a PCR primer and application. The SNP molecular marker provided by the invention has polymorphism in 9 kinds of mycobacteria, and the species of the mycobacteria to be detected can be identified by detecting the base type of the mycobacteria at the SNP site.

The technical scheme provided by the invention is as follows:

in a first aspect, the present invention provides an SNP molecular marker for identifying a mycobacterium tuberculosis complex, the SNP molecular marker including SNPs 1-SNP 8 located on rpoB gene and SNPs 9-SNP 15 located on gyrB gene; wherein the content of the first and second substances,

SNP1 is located at 14 th site from 5' end of sequence shown in SEQ ID No.1, and base is A, G;

SNP2 is located at 26 th site from 5' end of sequence shown in SEQ ID No.1, and base is C, G;

SNP3 is located at 65 th site from 5' end of sequence shown in SEQ ID No.1, and base is C, T;

SNP4 is located at 128 bits from the 5' end of the sequence shown in SEQ ID No.1, and the base is C, G;

SNP5 is located at position 170 from the 5' end of the sequence shown in SEQ ID No.1, and the base is G, T or C;

SNP6 is located at 311 bits from the 5' end of the sequence shown in SEQ ID No.1, and the base is C, G or T;

SNP7 is located at 421 position from 5' end of the sequence shown in SEQ ID No.1, and the base is C, T;

SNP8 is located at 498 th from 5' end of sequence shown in SEQ ID No.1, base is G, A;

SNP9 is located at 32 bits from the 5' end of the sequence shown in SEQ ID No.2, and the base is A, G or C;

SNP10 is located at position 38 from the 5' end of the sequence shown in SEQ ID No.2, and the base is G, A or C;

SNP11 is located at 59 th from 5' end of the sequence shown in SEQ ID No.2, and the base is G, T, C or A;

SNP12 is located at 149 th from the 5' end of the sequence shown in SEQ ID No.2, and the base is A, G or C;

SNP13 is located at position 236 of the 5' end of the sequence shown in SEQ ID No.2, and the base is C, T or G;

SNP14 is located at 302 th site from 5' end of the sequence shown in SEQ ID No.2, and the base is C, T;

SNP15 is located at position 437 of the 5' end of the sequence shown in SEQ ID No.2, and the base is G or A.

The SNP molecular markers of the present invention are polymorphic between Mycobacterium tuberculosis complex and nontuberculous mycobacterial NTM (Mycobacterium marinum, Mycobacterium fortuitum, Mycobacterium abscessus, Mycobacterium avium, Mycobacterium cheloniae, Mycobacterium kansasii, Mycobacterium intracellulare, Mycobacterium kansaii).

In a second aspect, the present invention provides PCR primers for amplifying the aforementioned SNP molecular markers, comprising primers SEQ ID No.3 and SEQ ID No.4 for amplifying the sequence shown as SEQ ID No. 1; and primers SEQ ID No.5 and SEQ ID No.6 for amplifying the sequence shown in SEQ ID No. 2.

The primer composition for identifying the mycobacteria provided by the invention can amplify and distribute the SNP marker genes, shortens the gene length through optimization, and can amplify the rpoB gene and the gyrB gene of the mycobacteria in a sample to be detected more quickly, sensitively and specifically.

In a third aspect, the present invention provides a kit for detecting the aforementioned SNP molecular markers, the kit comprising the aforementioned PCR primers; preferably, the kit further comprises dNTPs, Taq DNA polymerase and Mg²⁺And PCR reaction buffer.

In a fourth aspect, the present invention provides a gene chip comprising the PCR primers as described above.

In a fifth aspect, the invention provides the use of said SNP molecular marker or said PCR primer or said kit for the detection and/or identification of Mycobacterium tuberculosis complex, and the invention provides the use of said SNP molecular marker or said PCR primer or said kit for the detection and typing of mycobacteria, said use being for non-disease diagnostic purposes.

In a sixth aspect, the present invention provides a method for identifying Mycobacterium tuberculosis complex for non-disease diagnosis purposes, which determines the species type of a sample to be tested by detecting the polymorphism of the SNP molecular marker as described above.

In one embodiment, the method for detecting the polymorphism of the SNP molecular marker comprises one or more of the following steps: SNP detection method based on gel electrophoresis, DNA sequencing method, DNA chip method, denaturation high performance liquid chromatography or mass spectrometry detection method.

In one embodiment, in the method, when the polymorphism result shows that the base corresponding to SNP 1-SNP 15 is ACTGGGTAAAGATTA, the mycobacterium tuberculosis complex is obtained;

when the base corresponding to SNP 1-SNP 15 is GGCCTCCGCCCGGCG, the target is mycobacterium abscessus;

when the base corresponding to SNP 1-SNP 15 is ACTGGGTAAAAATTA, the gene is Mycobacterium carinii;

when the base corresponding to SNP 1-SNP 15 is GGCGCCCACGCGCCG, the nucleotide is M.avium;

when the base corresponding to SNP 1-SNP 15 is GGCCTTCGCCGCGCG, the mycobacterium cheloniae is obtained;

when the base number corresponding to SNP 1-SNP 15 is GGCCTTCACGCCCCG, the mycobacterium fortuitum is determined;

when the base corresponding to SNP 1-SNP 15 is GGCCTCCACGCCCCG, the gene is M.intracellulare;

when the base corresponding to SNP 1-SNP 15 is GGCCTGCAGCGCCCG, the gene is Mycobacterium kansasii;

when the base corresponding to SNP 1-SNP 15 is GGCGTCCACGTCCCG, it is Mycobacterium marinum.

In one embodiment, the method further comprises obtaining DNA from the sample to be tested; using the DNA as a template and performing amplification by using the PCR primer; sequencing the amplified product to determine the polymorphism of the SNP molecular marker.

In a specific embodiment, the method comprises:

1) extracting the genome DNA of a sample to be detected;

2) respectively amplifying DNA fragments containing core SNP markers by PCR reaction by using the extracted genomic DNA as a template and primers shown in SEQ ID Nos. 3-4 and 5-6;

3) and detecting the PCR amplification product, sequencing the amplification product, analyzing the sequencing result, and judging the polymorphism at the SNP site.

In one embodiment, the concentration of the primer in the system for amplification is 350-500 nM, preferably 500 nM.

In one embodiment, the same annealing temperature is used for the 2 sets of primers during amplification, the annealing temperature being 58 ℃ to 62 ℃, preferably 60 ℃.

Has the advantages that:

(1) the SNP sites contained in the SNP molecular marker provided by the invention are distributed in two different genes, different mycobacteria are distinguished from SNP polymorphism on the two different genes, the polymorphism difference between different mycobacteria SNP can be increased, and the identification accuracy is improved.

(2) The SNP molecular marker provided by the invention has polymorphism in 9 kinds of mycobacteria, and the species of the mycobacteria to be detected can be identified by detecting the base types of the mycobacteria at 15 SNP sites.

(3) The method for identifying the mycobacterium, provided by the invention, does not need complicated steps such as culture, biochemical inspection and the like, and is simple and convenient to operate; the detection result can be quickly obtained without waiting for a long time.

(4) The method can identify clinically common NTM bacteria besides the main mycobacterium tuberculosis infecting human body and causing serious drug resistance, and has comprehensive identification.

(5) The method of the invention does not need large expensive detection instruments, and can carry out experiments only by a common PCR instrument and reagents thereof.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 shows the rpoB and gyrB gene-evolved trees provided in the examples of the present invention (wherein Mycobacterium avium: Mycobacterium avium; Mycobacterium fortuitum: Mycobacterium fortuitum; Mycobacterium chelonae: Mycobacterium cheloniae; Mycobacterium abscessus: Mycobacterium abscessus; Mycobacterium marinum: Mycobacterium marinum; Mycobacterium kansasii: Mycobacterium intracellulare; Mycobacterium intracellulare: Mycobacterium canarii; Mycobacterium canertii: Mycobacterium canarii);

fig. 2 is a validation of clinical results based on rpoB and gyrB genes (a portion of the results are shown in a truncated figure).

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1: SNP molecular marker

The SNP molecular markers selected by the invention are positioned on rpoB genes and gyrB genes. The conservation degree of the selected gene in the mycobacterium is high, and the diversity among different NTMs is strong, the specific method is that the target gene is divided into 10 longer segments through online analysis software MEME, and the segments in the gene are selected as alternative amplification regions according to the principle that the difference between MTBC and NTM is as small as possible and the difference between MTBC and NTM is as large as possible. The selected fragment is introduced into MEGA7.0 software for gene alignment, and a variable site, namely SNP, is marked, and the SNP is screened for later use according to the principle that the diversity of the SNP at the same site is as strong as possible (with two or more base types). The difference is more obvious when the evolutionary tree is constructed according to the target gene, which is shown in figure 1.

As can be seen from the molecules of FIG. 1, there are no differences within the Mycobacterium tuberculosis complex, as shown in the figure by L1-L7 (both of the lines 1-7 belong to the MTB complex, where L1 corresponds to Indo-Oceanic line; L2 East-Asian line; L3 East-African-Indian line; L4 Euro-American line; L5 and L6 are M.africanum West African 1 and M.African West African 2; L7 Ethiopedian line), while there is strong diversity among other NTMs and strong differences among different NTMs, which is an effective combination of molecular markers.

In the provided SNP molecular markers, SNP1, SNP2, SNP3, SNP4, SNP5, SNP6, SNP7 and SNP8 are located in rpoB genes, and SNP9, SNP10, SNP11, SNP12, SNP13, SNP14 and SNP15 are located in gyrB genes.

SNP1 is located at 14 th site from 5' end of sequence shown in SEQ ID No.1, and base is A, G;

SNP2 is located at 26 th site from 5' end of sequence shown in SEQ ID No.1, and base is C, G;

SNP3 is located at 65 th site from 5' end of sequence shown in SEQ ID No.1, and base is C, T;

SNP4 is located at 128 bits from the 5' end of the sequence shown in SEQ ID No.1, and the base is C, G;

SNP5 is located at position 170 from the 5' end of the sequence shown in SEQ ID No.1, and the base is G, T or C;

SNP6 is located at 311 bits from the 5' end of the sequence shown in SEQ ID No.1, and the base is C, G or T;

SNP7 is located at 421 position from 5' end of the sequence shown in SEQ ID No.1, and the base is C, T;

SNP8 is located at 498 th from 5' end of sequence shown in SEQ ID No.1, base is G, A;

SNP9 is located at 32 bits from the 5' end of the sequence shown in SEQ ID No.2, and the base is A, G or C;

SNP10 is located at position 38 from the 5' end of the sequence shown in SEQ ID No.2, and the base is G, A or C;

SNP11 is located at 59 th from 5' end of the sequence shown in SEQ ID No.2, and the base is G, T, C or A;

SNP12 is located at 149 th from the 5' end of the sequence shown in SEQ ID No.2, and the base is A, G or C;

SNP13 is located at position 236 of the 5' end of the sequence shown in SEQ ID No.2, and the base is C, T or G;

SNP14 is located at 302 th site from 5' end of the sequence shown in SEQ ID No.2, and the base is C, T;

SNP15 is located at position 437 of the 5' end of the sequence shown in SEQ ID No.2, and the base is G or A.

The distribution and base types of the SNP sites in the SNP molecular markers are shown in Table 1.

TABLE 1 distribution and base types of individual SNP sites in SNP molecular markers

Example 2: PCR detection of the species of Mycobacterium to be detected

The detection method comprises the following steps:

1) extracting the genome DNA of a sample to be detected;

2) respectively amplifying DNA fragments containing core SNP markers by PCR reaction by using the extracted genomic DNA as a template and primers shown in SEQ ID Nos. 3-4 and 5-6;

3) and detecting the PCR amplification product, sequencing the amplification product, analyzing the sequencing result, and judging the polymorphism at the SNP site.

TABLE 2 PCR primer pairs

SEQ ID No.1：

CGTACGCGGCTCCACTGTTCGTCACCGCCGAGTTCATCAACAACAACACCGGTGAGATCAAGAGTCAGACGGTGTTCATGGGTGACTTCCCGATGATGACCGAGAAGGGCACGTTCATCATCAACGGGACCGAGCGTGTGGTGGTCAGCCAGCTGGTGCGGTCGCCCGGGGTGTACTTCGACGAGACCATTGACAAGTCCACCGACAAGACGCTGCACAGCGTCAAGGTGATCCCGAGCCGCGGCGCGTGGCTCGAGTTTGACGTCGACAAGCGCGACACCGTCGGCGTGCGCATCGACCGCAAACGCCGGCAACCGGTCACCGTGCTGCTCAAGGCGCTGGGCTGGACCAGCGAGCAGATTGTCGAGCGGTTCGGGTTCTCCGAGATCATGCGATCGACGCTGGAGAAGGACAACACCGTCGGCACCGACGAGGCGCTGTTGGACATCTACCGCAAGCTGCGTCCGGGCGAGCCCCCGACCAAAGAGTCAGCGCAGACGCTGTTGGAAAACTTG。

SEQ ID No.2：

ACGCGGTCGACGAGGCGATGGCCGGTTATGCAACCACAGTGAACGTAGTGCTGCTTGAGGATGGCGGTGTCGAGGTCGCCGACGACGGCCGCGGCATTCCGGTCGCCACCCACGCCTCCGGCATACCGACCGTCGACGTGGTGATGACACAACTACATGCCGGCGGCAAGTTCGACTCGGACGCGTATGCGATATCTGGTGGTCTGCACGGCGTCGGCGTGTCGGTGGTTAACGCGCTATCCACCCGGCTCGAAGTCGAGATCAAGCGCGACGGGTACGAGTGGTCTCAGGTTTATGAGAAGTCGGAACCCCTGGGCCTCAAGCAAGGGGCGCCGACCAAGAAGACGGGGTCAACGGTGCGGTTCTGGGCCGACCCCGCTGTTTTCGAAACCACGGAATACGACTTCGAAACCGTCGCCCGCCGGCTGCAAGAGATG。

And (3) PCR reaction system:

using the primer pairs in Table 2, a conventional PCR reaction system was used, as follows:

the total volume was 20. mu.L, including 10. mu.L of 2 XPromix Taq (Code No.: R004A, Takara Co., Premix is a 2-fold concentration Mixture composed of DNA Polymerase, Buffer, dNTP mix), 1. mu.L of each of 10. mu.M upstream and downstream primers (final concentration: 0.5. mu.M);

DNA template 1. mu.L, and double distilled water 7. mu.L to 20. mu.L.

PCR reaction procedure:

94 ℃ for 5 min; 30 cycles: 94 ℃, 45 sec; 60 ℃ for 45 sec; 72 ℃ for 50sec, final extension 72 ℃ for 7 min.

2, the same annealing temperature is adopted for the primers, so that the operation steps are reduced, and the strain identification time is saved.

Judging the polymorphism at the SNP site, and determining the type of the strain:

if the sample to be tested appears at the above 15 sites as: ACTGGGTAAAGATTA, it is Mycobacterium tuberculosis complex;

if the sample to be tested appears at the above 15 sites as: GGCCTCCGCCCGGCG, it is Mycobacterium abscessus;

if the sample to be tested appears at the above 15 sites as: ACTGGGTAAAAATTA, Mycobacterium vaccae;

if the sample to be tested appears at the above 15 sites as: GGCGCCCACGCGCCG, it is Mycobacterium avium;

if the sample to be tested appears at the above 15 sites as: GGCCTTCGCCGCGCG, Mycobacterium cheloniae;

if the sample to be tested appears at the above 15 sites as: GGCCTTCACGCCCCG, it is a fortuitous mycobacterium;

if the sample to be tested appears at the above 15 sites as: GGCCTCCACGCCCCG, it is M.intracellulare;

if the sample to be tested appears at the above 15 sites as: GGCCTGCAGCGCCCG, it is Mycobacterium kansasii;

if the sample to be tested appears at the above 15 sites as: GGCGTCCACGTCCCG, it is Mycobacterium marinum.

According to the verification of PCR experiments, the molecular marker has stronger consistency aiming at clinical tubercle strains and is an effective molecular marker for identifying tubercle bacillus and NTM, and then 200 cases of clinical MTB samples are used for verification, and because SNP differences do not exist among part of clinical strains, only the results of the parts with the differences and part of clinical strains are presented, and the results are shown in figure 2. Results were classified based on clinically confirmed strains, with 100% consistency with clinical results. The SNP classification target has stronger consistency and larger difference with NTM, and is an effective basis for clinical diagnosis.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> the university of capital medical department affiliated to the Beijing thoracic hospital; research institute of tuberculosis and breast tumor in Beijing

<120> SNP molecular marker, PCR primer and application

<130> PA21024390

<160> 6

<170> PatentIn version 3.3

<210> 1

<211> 515

<212> DNA

<213> rpoB

<400> 1

cgtacgcggc tccactgttc gtcaccgccg agttcatcaa caacaacacc ggtgagatca 60

agagtcagac ggtgttcatg ggtgacttcc cgatgatgac cgagaagggc acgttcatca 120

tcaacgggac cgagcgtgtg gtggtcagcc agctggtgcg gtcgcccggg gtgtacttcg 180

acgagaccat tgacaagtcc accgacaaga cgctgcacag cgtcaaggtg atcccgagcc 240

gcggcgcgtg gctcgagttt gacgtcgaca agcgcgacac cgtcggcgtg cgcatcgacc 300

gcaaacgccg gcaaccggtc accgtgctgc tcaaggcgct gggctggacc agcgagcaga 360

ttgtcgagcg gttcgggttc tccgagatca tgcgatcgac gctggagaag gacaacaccg 420

tcggcaccga cgaggcgctg ttggacatct accgcaagct gcgtccgggc gagcccccga 480

ccaaagagtc agcgcagacg ctgttggaaa acttg 515

<210> 2

<211> 437

<212> DNA

<213> gyrB

<400> 2

acgcggtcga cgaggcgatg gccggttatg caaccacagt gaacgtagtg ctgcttgagg 60

atggcggtgt cgaggtcgcc gacgacggcc gcggcattcc ggtcgccacc cacgcctccg 120

gcataccgac cgtcgacgtg gtgatgacac aactacatgc cggcggcaag ttcgactcgg 180

acgcgtatgc gatatctggt ggtctgcacg gcgtcggcgt gtcggtggtt aacgcgctat 240

ccacccggct cgaagtcgag atcaagcgcg acgggtacga gtggtctcag gtttatgaga 300

agtcggaacc cctgggcctc aagcaagggg cgccgaccaa gaagacgggg tcaacggtgc 360

ggttctgggc cgaccccgct gttttcgaaa ccacggaata cgacttcgaa accgtcgccc 420

gccggctgca agagatg 437

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence

<400> 3

gtgcaaagac aaggacatga 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence

<400> 4

tagcgcttct ccttgaagaa 20

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

catttgggag gtggtcgaca 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

agccccttgt tgaggaacgc 20