MNP marker site, primer group and kit for identifying Xinjiang common human respiratory tract pathogens and drug-resistant genes thereof and application of MNP marker site, primer group and kit

Abstract

The invention provides an MNP marker site, a primer group and a kit for identifying Xinjiang common human respiratory pathogens and drug-resistant genes thereof and application, and belongs to the technical field of biology. The MNP marker sites comprise an MNP-1 to an MNP-25; the primer group comprises a first primer pair to a 25th primer pair, each primer pair comprises a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 25th primer pair and the reverse primer of the 25th primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 50 in a sequence table. The primer group provided by the invention aims at 25 MNP marker sites, 1-5 marker sites are detected for each pathogen, 15 human respiratory pathogens and 5 drug-resistant genes can be identified, a comprehensive, efficient and accurate detection effect is achieved, and the primer group is applicable to detection of multiple respiratory pathogens and drug-resistant genes.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to an MNP marker site, a primer set, a kit and applications for identifying common human respiratory pathogens and drug-resistant genes in Xinjiang. Background Art

[0002] At present, the detection technology of common human respiratory pathogens and their drug resistance genes in Xinjiang is mainly based on PCR (Polymerase Chain Reaction) nucleic acid detection technology. However, PCR detection can only monitor one or a few pathogens at a time, the detection efficiency is low, and it cannot detect the sequence information of the variant, which limits the detection of human respiratory pathogens in Xinjiang and makes it difficult to complete the identification of pathogens and their drug resistance genes at the same time. Therefore, there is an urgent need for a method that can detect multiple common human respiratory pathogens and their drug resistance genes in Xinjiang at one time.

[0003] Public Content

[0004] In order to solve the problems of the prior art, the present disclosure provides an MNP (Multiple Dispersed Nucleotide Polymorphism) marker site, primer set, kit and application for identifying common human respiratory pathogens in Xinjiang and their drug resistance genes. The technical solution is as follows:

[0005] On the one hand, the present disclosure provides an MNP marker site for identifying common human respiratory pathogens in Xinjiang and their drug resistance genes, the MNP marker site is used to identify common human respiratory pathogens in Xinjiang and their drug resistance genes, the MNP marker site is a species-specific MNP marker screened on the genomes of 15 common human respiratory pathogens in Xinjiang, used for the identification of the pathogens and their drug resistance genes, 1-5 MNP markers are screened for each pathogen, the MNP marker site includes MNP-1 to MNP-25, and the positions of MNP-1 to MNP-25 on the reference sequence can be seen in Table 1.

[0006] On the other hand, the present disclosure provides a primer set for identifying common human respiratory pathogens in Xinjiang and their drug resistance genes, the primer set comprising: the 1st primer pair to the 25th primer pair, each of the primer pairs comprising a forward primer and a reverse primer, the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primers of the 25th primer pair, and the reverse primer of the 25th primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 50 in the sequence listing.

[0007] On the other hand, the present disclosure provides a kit for identifying common human respiratory pathogens in Xinjiang and their drug-resistant genes, the kit comprising the above-mentioned primer set.

[0008] On the other hand, the present disclosure provides an application of the above-mentioned MNP marker site, the above-mentioned primer set or the above-mentioned kit, wherein the application includes using the MNP marker site, the above-mentioned primer set or the above-mentioned kit to identify common human respiratory pathogens and their drug resistance genes in Xinjiang, wherein the human respiratory pathogens include: viruses, bacteria, fungi and special pathogens.

[0009] Specifically, the viruses include: novel coronavirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus, human rhinovirus and Xinjiang hemorrhagic fever virus. Among them, enterovirus 71, coxsackie virus and human rhinovirus all belong to the enterovirus genus and can infect the upper respiratory tract.

[0010] Specifically, the bacteria include: Mycobacterium tuberculosis, Haemophilus influenzae and Pseudomonas aeruginosa.

[0011] Specifically, the fungi include: Histoplasma capsulatum and Cryptococcus neoformans.

[0012] Specifically, the special pathogens include Chlamydia pneumoniae.

[0013] Specifically, the drug-resistant genes include rpsA, tlyA, katG, pncA and embB genes of Mycobacterium tuberculosis.

[0014] Specifically, the application includes: using the MNP marker site, the primer set or the kit for identification of pathogens and their drug-resistant genes, database construction, genetic variation monitoring and type differentiation.

[0015] The technical solution provided by the embodiments of the present disclosure has the following beneficial effects: the present disclosure provides an MNP marker site, a primer set, a kit and an application for identifying common human respiratory pathogens and their drug resistance genes in Xinjiang. The MNP marker site can be used to identify common human respiratory pathogens and their drug resistance genes in Xinjiang and their drug resistance genes. The primer set is used for the detection of 25 MNP marker sites. One detection can comprehensively identify 15 human respiratory pathogens and 5 drug resistance genes, thereby realizing the simultaneous identification of human respiratory pathogens and their drug resistance genes, monitoring the genetic variation of strains, building a database of human respiratory pathogens and distinguishing the types of human respiratory pathogens.

[0016] The beneficial effects of the technical solution provided by the embodiment of the present disclosure also include: the second-generation sequencing platform of the present disclosure sequences the amplified products of the primer set provided by the embodiment of the present disclosure, and can realize the sequence analysis of 25 MNP labeling sites in one reaction system. Compared with the fluorescence PCR-based method that only uses a pair of primers to detect one site, and detects the fluorescence signal, the detection of the primer set provided by the embodiment of the present disclosure is more efficient and accurate.

[0017] The beneficial effects of the technical solution provided by the embodiments of the present disclosure also include: the present disclosure detects 1 to 5 markers for each pathogen, for example, a total of 7 markers are detected for 3 viruses of the genus Enterovirus (enterovirus 71, coxsackie virus and human rhinovirus), including 2 specific MNP markers for each virus and 1 marker specific to the genus Enterovirus; 3 markers are detected for Xinjiang hemorrhagic fever virus; 5 markers are detected for Mycobacterium tuberculosis, which can be used for species identification of Mycobacterium tuberculosis and identification of 5 drug resistance genes at the same time. The presence of pathogens and the type of pathogens can be determined and distinguished by the detection of multiple markers, and it has high specificity, sensitivity and accuracy. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be described in further detail below.

[0019] Embodiment 1

[0020] The disclosed embodiment provides an MNP marker site for identifying common human respiratory pathogens in Xinjiang and their drug resistance genes. The MNP marker site is used to identify common human respiratory pathogens in Xinjiang and their drug resistance genes. The MNP marker site includes MNP-1 to MNP-25. The positions of MNP-1 to MNP-25 on the reference sequence are shown in Table 1.

[0021] The present disclosure also provides a primer set for identifying common human respiratory pathogens in Xinjiang, including: the first primer pair to the 25th primer pair, each primer pair includes a forward primer and a reverse primer, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the 25th primer pair, and the reverse primer of the 25th primer pair are shown in the sequence table as SEQ ID NO: 1 to SEQ ID NO: 50. The specific sequence is shown in Table 1.

[0022] 25 primer sets were used to detect 25 MNP marker sites. The positions of the MNP marker sites were determined based on the reference sequence. The specific sequence positions are shown in Table 1.

[0023] Table 1 shows the primer set sequences and their corresponding MNP labeling sites

[0024]

[0025]

[0026] Embodiment 2

[0027] The disclosed embodiments provide a kit for identifying common human respiratory pathogens in Xinjiang and their drug-resistant genes, and the kit includes the primer set provided in Embodiment 1.

[0028] Embodiment 3

[0029] The disclosed embodiments provide an application of the above-mentioned primer set and kit, which includes using the primer set to identify common human respiratory pathogens in Xinjiang and their drug resistance genes, and human respiratory pathogens include: viruses, bacteria, fungi and special pathogens.

[0030] Specifically, the viruses include: new coronavirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus, human rhinovirus and Xinjiang hemorrhagic fever virus.

[0031] Specifically, the enterovirus 71, coxsackievirus and human rhinovirus all belong to the genus Enterovirus.

[0032] Specifically, the bacteria include: Mycobacterium tuberculosis, Haemophilus influenzae and Pseudomonas aeruginosa.

[0033] Specifically, the fungi include: Histoplasma capsulatum and Cryptococcus neoformans.

[0034] Specifically, the special pathogens include Chlamydia pneumoniae.

[0035] Specifically, the drug-resistant genes include rpsA, tlyA, katG, pncA and embB genes of Mycobacterium tuberculosis.

[0036] In this embodiment, a primer set is used to detect 15 target pathogen positive samples. Specifically, the 15 target pathogen positive samples include: 2 commercial nucleic acid standards with known copy numbers of human respiratory pathogens and 13 human respiratory pathogens identified as positive clinical samples by fluorescent quantitative PCR (hereinafter referred to as qPCR) (provided by the Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention, and the sample type is respiratory samples). The pathogens of the two commercial nucleic acid standards are new coronavirus and Mycobacterium tuberculosis. The nucleic acid standards containing the above 15 target pathogens and the positive sample nucleic acid are mixed to prepare three human respiratory multi-pathogen simulated mixed samples (hereinafter referred to as simulated mixed samples). In the three simulated mixed samples, the copy numbers of the pathogens of the new coronavirus and Mycobacterium tuberculosis nucleic acid standards are 1 copy / reaction, 10 copies / reaction and 100 copies / reaction, respectively, and the CT values ​​of the other 10 pathogens range from 20 to 34. Based on the number of copies of the novel coronavirus and Mycobacterium tuberculosis nucleic acid standards in the above three simulated mixed samples, the above three simulated mixed samples were named 1 copy / reaction, 10 copies / reaction and 100 copies / reaction samples, respectively. The three simulated mixed samples were detected using the kit provided in Example 2. In this embodiment, the detection method of MNP labeling is as follows: the total nucleic acid (including DNA and RNA) of the simulated mixed sample is reverse transcribed to obtain a mixed template containing sample cDNA and DNA; the sample mixed template is amplified by multiple PCR using the primer set provided in Example 1 to obtain multiple amplification products for library construction; the library is sequenced to obtain sequencing data, and an equal volume of sterile water is set as a blank control, and the blank control is named 0 copy / reaction. Three replicate libraries are constructed for each sample every day, and the detection is continuous for 3 days, that is, 9 sets of sequencing data are obtained for each sample, and the specific results are shown in Table 2.

[0037] Table 2 evaluates the results for each sample

[0038]

[0039] As can be seen from Table 2, in samples with different copy numbers, the normalized sequence number of the sample detected that is aligned to the MNP marker site of the pathogen, the number of the aligned MNP marker sites, and the number of pathogens covered by these markers are statistically analyzed. Among them, the normalized sequence number refers to the number of sequences aligned to the MNP marker site of the pathogen in every 100,000 sequences. The primer set detects 3 markers for each pathogen for enterovirus 71, human rhinovirus, coxsackievirus A10 and Xinjiang hemorrhagic fever virus, and 5 markers for Mycobacterium tuberculosis, so the number of detected markers is greater than the number of covered pathogens.

[0040] Evaluation of the stability and sensitivity of the detection primer set for human respiratory pathogens

[0041] As can be seen from Table 2, when the copy number is lower than 10 copies / reaction, only a maximum of 8 MNP marker sites can be detected; when the copy number reaches 10 copies / reaction, all 25 MNP marker sites of 15 pathogens are detected in 9 repetitions, indicating that the primer set can stably detect the novel coronavirus and Mycobacterium tuberculosis nucleic acid standards when the copies are as low as 10 copies / reaction; it can be seen that the detection sensitivity of this primer set for pathogens is 10 copies / reaction.

[0042] Evaluation of the reproducibility and accuracy of this primer set for human respiratory pathogens

[0043] Based on whether the genotypes of the common detection sites in two replicates are reproducible, the reproducibility and accuracy of the MNP labeling detection method for detecting human respiratory pathogens were evaluated. Specifically, the 9 groups of data of the 100 copies / reaction samples in Table 2 were compared pairwise, and the results are shown in Table 3.

[0044] Table 3 shows the reproducibility and accuracy evaluation of the primer set for detecting genotypes of human respiratory pathogens

[0045] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Reproducibility Accuracy S-1 S-2 25 25 100％ 100％ S-1 S-3 25 25 100％ 100％ S-1 S-4 25 25 100％ 100％ S-1 S-5 25 25 100％ 100％ S-1 S-6 25 25 100％ 100％ S-1 S-7 25 25 100％ 100％ S-1 S-8 25 25 100％ 100％ S-1 S-9 25 25 100％ 100％

[0046] As can be seen from Table 3, the number of MNP sites with differences in the main genotype is 0; according to the principle that the reproducible genotype between two repeated experiments is considered accurate, the accuracy rate a=1-(1-r) / 2=0.5+0.5r, r represents the reproducibility, that is, the ratio of the number of sites with reproducible main genotypes to the number of common sites. In the reproducibility test of this embodiment, the difference logarithm of the main genotype of MNP markers between different libraries and different library construction batches for each sample is 0, the reproducibility r=100%, and the accuracy rate a=100%.

[0047] Evaluation of the specificity of this primer set for human respiratory pathogens

[0048] As can be seen from Table 2, in the three simulated samples, when the copy number of the pathogen is ≥10 copies, the sequences obtained by sequencing in the simulated samples, after sequence alignment, the MNP markers of the 15 pathogens provided in this example all have sequence coverage, indicating that the MNP marker and primer set have high specificity in detecting target microorganisms in complex templates.

[0049] The threshold value of this primer set for detecting human respiratory pathogens

[0050] As shown in Table 2, most samples with 1 copy / reaction can detect sequences of human respiratory pathogens. Sequences of human respiratory pathogens were also detected in some blank controls. Due to the extreme sensitivity of the MNP labeling detection method, data contamination during the detection process can easily lead to false positives. Therefore, in this embodiment, based on the test results of simulated samples with different copy numbers for 3 consecutive days, the following quality control plan was formulated:

[0051] 1) The amount of sequencing data is no less than 38 megabases. The calculation is based on the fact that the maximum number of MNP sites detected in each sample is 25, and the length of a sequencing fragment is 300 bases. Therefore, when the amount of data is greater than 38 megabases, most samples can ensure that the number of sequencing fragments covering each site reaches more than 5,000 times in one experiment, ensuring accurate analysis of the base sequence of each MNP site.

[0052] 2) According to the sequence number S of each human respiratory pathogen in the test sample and the sequence number N of the human respiratory pathogen in the blank control, the signal-to-noise ratio SNR=S / N of each pathogen in the sample is calculated.

[0053] Based on the data of the samples detected in Table 2, in the simulated mixed samples of 10 copies / reaction and 10 copies / reaction, the SNR value of each pathogen detected each time was ≥10. Therefore, while ensuring accuracy and taking sensitivity into account, the judgment criterion for the primer set provided in this embodiment to be positive for human respiratory pathogens is: when the signal-to-noise ratio of human respiratory pathogens in the sample is greater than 10, it is judged that the nucleic acid of human respiratory pathogens is detected in the sample.

[0054] Application of this primer set in the detection of human respiratory pathogens

[0055] The primer set was used to test 13 human throat swab samples provided by the Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention as test samples. The test sample numbers are shown in Table 4, and 3 negative controls with sterile water as templates were constructed. Each test sample uses a commercial pathogen total nucleic acid extraction kit to prepare nucleic acid, and the prepared nucleic acid is first tested for the new coronavirus by qPCR. Five of the 13 samples are positive for the new coronavirus. Then, the same nucleic acid is also tested using the primer set provided in Example 1 to obtain sequencing data. The results of the statistical sequencing data are shown in Table 4.

[0056] Table 4 shows the normalized number of sequences detected by the primer set for real human throat swab samples (unit: number)

[0057]

[0058]

[0059] According to the above-mentioned determination threshold, all five positive samples of the novel coronavirus detected by the qPCR method were detected by the primer set provided in Example 1, which indicates that the sensitivity of the primer set in detecting the novel coronavirus is not lower than that of qPCR. The pathogens detected by the 13 real human throat swab samples shown in Table 4 are shown in Table 5.

[0060] Table 5 shows the statistics of pathogens detected from 13 real human throat swab samples

[0061] Serial number Target pathogens Number of samples detected 1 Chlamydia pneumoniae 2 2 Coronavirus 229E 1 3 Coronavirus HKU1 2 4 Coronavirus NL63 1 5 Mycobacterium tuberculosis 3 6 Coxsackievirus A10 1 7 Haemophilus influenzae 3 8 Human rhinovirus 2 9 Pseudomonas aeruginosa 3 10 Xinjiang hemorrhagic fever virus 2 11 Novel Coronavirus 5

[0062] Combining Tables 4 and 5, it can be seen that in addition to the new coronavirus, in 13 samples, the primer set provided in Example 1 also detected Chlamydia pneumoniae, coronavirus 229E, coronavirus HKU1, coronavirus NL63, Mycobacterium tuberculosis, Coxsackievirus A10, Haemophilus influenzae, human rhinovirus, Pseudomonas aeruginosa and Xinjiang hemorrhagic fever virus. 10 other pathogens, which shows the advantage of this primer set in comprehensive detection.

[0063] Combining Table 4 and Table 5, it can be seen that by using the primer set provided in Example 1, three MNP markers of enterovirus were detected in three samples (S07, S11 and S12). According to the genotypes of the three MNP markers, the enterovirus was further identified as human rhinovirus and coxsackievirus A10 of the enterovirus genus. It can be seen that the primer set can achieve the advantage of pathogen type differentiation by detecting multiple markers for each pathogen.

[0064] Embodiment 4

[0065] Specifically, the application includes: using the primer set for the detection of Mycobacterium tuberculosis and its drug resistance genes in Xinjiang. The drug resistance genes include: rpsA, tlyA, katG, pncA and embB.

[0066] In this embodiment, a primer set is used to detect nucleic acid samples of 13 known drug-resistant strains of Mycobacterium tuberculosis with drug-resistant genes and mutation types (provided by the Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention, and the sample type is a respiratory sample). The samples are named MTB1-13 in sequence. The 13 samples are detected using the kit provided in Example 2. In this embodiment, the detection method of MNP labeling is as follows: the nucleic acid sample is subjected to multiple PCR amplification using the primer set provided in Example 1 to obtain multiple amplification products for library construction; the library is sequenced to obtain sequencing data, and an equal volume of sterile water is set as a blank control.

[0067] Table 6 shows the drug resistance genes and mutation types detected in Mycobacterium tuberculosis

[0068]

[0069]

[0070] Note: In the mutation type p.Arg133fs, p stands for protein and fs stands for frame shift. p.Arg133fs means that a frame shift mutation has occurred at the amino acid Arg at position 133 of the drug resistance gene. The mutation type p.Gly406Ala means that a mutation from Gly to Ala has occurred at the amino acid at position 406 of the drug resistance gene, and so on.

[0071] As can be seen from Table 6, using the primer set provided in this embodiment, all 5 MNP marker sites of Mycobacterium tuberculosis were identified in 13 samples, and the simultaneous detection of multiple marker sites ensured the accuracy of the primer set for species identification. Mutations of drug-resistant genes within the detection range were detected in 8 cases, and the test results were in line with expectations, indicating that the primer set can be used for durable identification at the same time. In addition, using the primer set provided in this embodiment, pathogens such as Haemophilus influenzae, Chlamydia pneumoniae and new coronavirus were also detected in addition to Mycobacterium tuberculosis, indicating that the primer set can simultaneously identify multiple pathogens and identify drug-resistant gene mutations.

[0072] Embodiment 5

[0073] The application includes: using the MNP marker loci, primer sets or kits for monitoring genetic variation between and within strains of human respiratory pathogens.

[0074] As shown in Table 4, the novel coronavirus was detected in all five samples to be tested. The major genotypes of the novel coronavirus MNP marker sites in each sample to be tested were compared, and it was found that the genotypes in the five samples to be tested were not exactly the same, and could be divided into two categories. The major genotypes of the novel coronavirus MNP marker sites in the samples S01 and S02 to be tested were the same, and the major genotypes of the novel coronavirus MNP marker sites in the other three samples to be tested were the same. There are differences in genotypes between strains, indicating that this primer set can be used to detect genetic variations between pathogen strains and to monitor new variants.

[0075] As a group organism, some individuals within the group of human respiratory pathogens mutate, which will make the group no longer homozygous, forming a heterogeneous heterozygous group. This heterogeneous heterozygous group is detected and analyzed by the primer set or kit of this embodiment, showing that there are secondary alleles other than the main genotype at the same MNP marker site. This embodiment detects highly polymorphic MNP markers. The high polymorphism of MNP markers can fully reflect the multiple genotypes of each marker site. Low-frequency alleles are often mixed with technical errors, making it difficult to distinguish with existing technologies. Based on the probability that multiple errors occur simultaneously is lower than the probability that one error occurs, the technical error rate of MNP markers is significantly lower than that of SNP markers. When the number of sequencing sequences of the secondary allele type of the MNP marker reaches 50% or more of the number of sequences of the main genotype, the secondary allele type cannot be a technical error.

[0076] The genotypes of the novel coronavirus in the five samples shown in Table 4 were analyzed. The 22,329 sequencing sequences detected in the S06 sample that were aligned to the novel coronavirus MNP marker represented two different genotypes, with 12,657 and 9672 sequences, respectively, indicating that there were variations within the novel coronavirus in the same sample. This shows that the primer set is suitable for detecting genetic variations within the human respiratory pathogen strain population and determining the homozygous and heterozygous strains.

[0077] Embodiment 6

[0078] Specifically, the applications include: using the MNP labeling sites, primer sets or kits to construct a database of human respiratory pathogens.

[0079] As shown in Table 5, after the primer set is used to detect the sample to be tested, the main genotype of the MNP marker site of each pathogen in each sample to be tested is obtained, the main genotypes of the same pathogen obtained in all samples are compared, and the main genotypes with differences are entered into the database file to form the MNP fingerprint database of human respiratory pathogens. After the main genotype of the MNP marker site of each sample or strain to be tested is compared with the constructed MNP fingerprint database, the MNP fingerprint map of the strain with a difference in the main genotype can be entered into the constructed MNP fingerprint database. Therefore, in theory, the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequence of the strain detected, it is compatible with all high-throughput sequencing data, and has the characteristics of being fully co-constructed and shared and updatable at any time.

[0080] Embodiment 7

[0081] Specifically, the applications include: using MNP labeling sites, primer sets or kits to construct fine typing of human respiratory pathogens.

[0082] Viral pathogens in human respiratory pathogens are classified into species, subspecies and subtypes in taxonomy. For example, the enterovirus genus provided in this embodiment can be specifically classified into enterovirus type 71, coxsackievirus species and human rhinovirus species. The primer set provided in this embodiment is 7 specific MNP markers for detecting enterovirus. The 7 specific MNP markers include 1 conservative marker of the enterovirus genus and 2 specific markers for each species (type). According to the detection of conservative markers and specific markers, the pathogens can be accurately identified and finely typed. As shown in Table 4, 1 conservative marker of the enterovirus genus and 2 markers specific to human rhinovirus were detected in both samples, thereby determining that the enterovirus in the sample was finely identified as human rhinovirus. In 1 sample, 1 conservative marker of the enterovirus genus and 2 markers specific to coxsackievirus A10 were detected, thereby determining that the enterovirus in the sample was finely identified as coxsackievirus A10. It can be seen that the primer set provided in this embodiment can be used to construct the type differentiation of human respiratory pathogens, and thus can be used to monitor the epidemic species (types) of human respiratory pathogens.

[0083] The above description is only an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the protection scope of the present disclosure.

Claims

1. An MNP marker site for identifying common human respiratory pathogens in Xinjiang and their drug resistance genes, characterized in that: The MNP labeling sites include MNP-1 to MNP-25, and the positions of MNP-1 to MNP-25 on the reference sequence are shown in the following table:

2. A primer set for identifying common human respiratory pathogens in Xinjiang and their drug resistance genes, characterized in that: The primer set includes: the 1st primer pair to the 25th primer pair, each of the primer pairs includes a forward primer and a reverse primer, and the forward primer of the 1st primer pair, the reverse primer of the 1st primer pair to the forward primer of the 25th primer pair and the reverse primer of the 25th primer pair are respectively shown as SEQ ID NO: 1 to SEQ ID NO: 50 in the sequence listing.

3. A kit for identifying common human respiratory pathogens in Xinjiang and their drug resistance genes, characterized in that: The kit comprises the primer set as claimed in claim 2.

4. A use of the MNP marker site according to claim 1, the primer set according to claim 2 or the kit according to claim 3, characterized in that: The application includes using the MNP marker site, the primer set or the kit to identify common human respiratory pathogens and their drug resistance genes in Xinjiang, wherein the human respiratory pathogens include viruses, bacteria, fungi and special pathogens.

5. The use according to claim 4, characterized in that: The viruses include: new coronavirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus, human rhinovirus and Xinjiang hemorrhagic fever virus.

6. The use according to claim 4, characterized in that: The bacteria include: Mycobacterium tuberculosis, Haemophilus influenzae and Pseudomonas aeruginosa.

7. The use according to claim 4, characterized in that: The fungi include: Histoplasma capsulatum and Cryptococcus neoformans.

8. The use according to claim 4, characterized in that: The specific pathogens include Chlamydia pneumoniae.

9. The use according to claim 4, characterized in that: The drug-resistant genes include rpsA, tlyA, katG, pncA and embB genes of Mycobacterium tuberculosis.

10. The use according to claim 4, characterized in that: The application includes: using the MNP marker site, the primer set or the kit for simultaneous identification of pathogens and their drug-resistant genes, database construction, genetic variation monitoring and type differentiation.

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