MNP marker site, primer group and kit for identifying multiple human respiratory pathogens and application of MNP marker site, primer group and kit

Abstract

The invention provides an MNP marker site for identifying multiple human respiratory pathogens, a primer group, a kit and application, and belongs to the technical field of biology. The MNP marker site comprises at least one of MNP-1 to MNP-17, the primer group comprises at least one of a first primer pair to a seventeenth primer pair, and each primer pair comprises a forward primer and a reverse primer, a forward primer of the first primer pair, a reverse primer of the first primer pair, a forward primer of the 17th primer pair and a reverse primer of the 17th primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 34 in a sequence table. The invention provides MNP marker sites for identifying multiple human respiratory pathogens, a primer group, a kit and application, the primer group aims at 17 MNP marker sites, 1-3 marker sites are detected for each pathogen, 13 human respiratory pathogens can be identified, and the primer group has a comprehensive, efficient and accurate detection effect and is suitable for identifying multiple human respiratory pathogens.

Description

Technical Field

[0001] The present disclosure relates to the field of biotechnology, and in particular to an MNP labeling site, a primer set, a kit and applications for identifying multiple human respiratory pathogens. Background Art

[0002] In the current field of medical health and public safety, accurate identification of human respiratory pathogens is crucial. There are many types of human respiratory pathogens, including viruses and bacteria, which poses a great challenge to the diagnosis and prevention of diseases.

[0003] At present, the detection technology of human respiratory pathogens is mainly based on PCR nucleic acid detection technology. However, during PCR detection, only one marker is detected for one pathogen, which makes its detection efficiency low and cannot detect the sequence information of mutations. This limits the development of human respiratory pathogen detection. Therefore, a method that can detect multiple human respiratory pathogens at one time is urgently needed.

[0004] Public Content

[0005] In order to solve the problems of the prior art, the embodiments of the present disclosure provide a method for identifying a plurality of MNP marker sites, a primer set, a kit and an application. The technical solution is as follows:

[0006] On the one hand, the present disclosure provides an MNP labeling site for identifying multiple human respiratory pathogens, wherein the MNP labeling site is used to identify multiple human respiratory pathogens, 1-3 MNP labels are designed for each pathogen, and the MNP labeling site includes at least one of MNP-1 to MNP-17. The positions of MNP-1 to MNP-17 on the reference sequence can be seen in Table 1.

[0007] On the other hand, the present disclosure provides a primer set for identifying multiple human respiratory pathogens, the primer set comprising: at least one pair of primer pairs from the first primer pair to the seventeenth primer pair, each of the primer pairs comprising a forward primer and a reverse primer, the 17 primer pairs are used to detect the 17 MNP marker sites, and the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primer of the seventeenth primer pair, and the reverse primer of the seventeenth primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 34 in the sequence listing.

[0008] In yet another aspect, the present disclosure provides a kit for identifying a plurality of human respiratory pathogens, the kit comprising the above primer set.

[0009] In yet another aspect, the present disclosure provides an application of the above-mentioned MNP labeling site, the above-mentioned primer set or the above-mentioned kit, wherein the application includes using the MNP labeling site, the above-mentioned primer set or the above-mentioned kit to identify human respiratory pathogens, wherein the human respiratory pathogens include: viruses, bacteria, fungi and special pathogens.

[0010] Specifically, the viruses include: new coronavirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus and human rhinovirus.

[0011] Specifically, the enterovirus 71, coxsackievirus and human rhinovirus all belong to the genus Enterovirus.

[0012] Specifically, the bacteria include: Haemophilus influenzae and Pseudomonas aeruginosa.

[0013] Specifically, the fungi include: Histoplasma capsulatum and Cryptococcus neoformans.

[0014] Specifically, the special pathogens include Chlamydia pneumoniae.

[0015] Specifically, the application includes: using the MNP marker site, the primer set or the kit for database construction, genetic variation monitoring and type differentiation of multiple human respiratory pathogens.

[0016] The technical solution provided by the embodiments of the present disclosure has the following beneficial effects: the present disclosure provides an MNP marker site, primer set, kit and application for identifying a variety of human respiratory pathogens. The MNP marker site refers to a marker site that is species-specific and has multiple nucleotide polymorphisms within the species screened on the target pathogen genome, and has high polymorphism and strong species differentiation ability. The MNP marker site provided by the present disclosure can be screened from the human respiratory pathogen genome, and 1 to 3 markers are screened for each pathogen, which can specifically distinguish a variety of human respiratory pathogens. The primer set provided by the present disclosure has amplification compatibility, and can be amplified in a reaction system using super-multiplex PCR to achieve amplification of multiple MNP marker sites in a reaction system, and the second-generation sequencing platform is integrated to perform sequence analysis of the amplified products, so as to achieve identification of a variety of human respiratory pathogens in one reaction, with comprehensive, efficient, sensitive and accurate detection effects and type differentiation. Compared with the fluorescence PCR-based method, in which one reaction usually uses only one pair of primers to detect one marker site of a pathogen, and what is detected is the fluorescent signal, the primer set and kit provided in the present invention detect up to 3 marker sites for each pathogen, making the detection more efficient and accurate, and can also detect sequence variations and distinguish pathogen types, and can be used for efficient identification of a variety of human respiratory pathogens, genetic variation monitoring, database construction and type differentiation. DETAILED DESCRIPTION

[0017] 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.

[0018] Embodiment 1

[0019] The embodiments of the present disclosure provide an MNP labeling site for identifying multiple human respiratory pathogens. The MNP labeling site is used to identify multiple human respiratory pathogens. The MNP labeling site includes at least one of MNP-1 to MNP-17. The positions of MNP-1 to MNP-17 on the reference sequence are shown in Table 1.

[0020] The present disclosure also provides a primer set for identifying multiple human respiratory pathogens, including: at least one pair of primer pairs from the first primer pair to the seventeenth primer pair, the seventeen primer pairs are used to detect 17 MNP marker sites, 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 seventeenth primer pair, and the reverse primer of the seventeenth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 34 in the sequence table. The specific sequences are shown in Table 1.

[0021] Table 1 shows the primer set sequences and the locations of their corresponding MNP labeling sites

[0022]

[0023]

[0024] Embodiment 2

[0025] The disclosed embodiments provide a kit for identifying a variety of human respiratory pathogens, the kit comprising the primer set provided in Embodiment 1.

[0026] Embodiment 3

[0027] The disclosed embodiments provide an application of the primer set or kit, which includes using the primer set to identify a variety of human respiratory pathogens, including viruses, bacteria, fungi and special pathogens.

[0028] Specifically, the viruses include: new coronavirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus and human rhinovirus.

[0029] Specifically, enterovirus 71, coxsackievirus, and human rhinovirus all belong to the genus Enterovirus.

[0030] Specifically, the bacteria include: Haemophilus influenzae and Pseudomonas aeruginosa.

[0031] Specifically, fungi include: Histoplasma capsulatum and Cryptococcus neoformans.

[0032] Specifically, special pathogens include Chlamydia pneumoniae.

[0033] In this embodiment, a primer set was used to detect 13 target pathogen positive samples. Specifically, the 13 target pathogen positive samples include: 1 commercial nucleic acid standard with a known copy number of human respiratory pathogens and 12 human respiratory pathogens identified as positive clinical samples by fluorescence quantitative PCR (hereinafter referred to as qPCR) (provided by Hubei Provincial Center for Disease Control and Prevention, and the sample type is respiratory samples). The pathogen of 1 commercial nucleic acid standard is a new coronavirus. The nucleic acid standards containing the above 13 target pathogens and the positive sample nucleic acid were mixed to prepare 3 simulated mixed samples of human respiratory multi-pathogens (hereinafter referred to as simulated mixed samples). In the 3 simulated mixed samples, the copy numbers of the pathogens of the new coronavirus nucleic acid standards were 1 copy / reaction, 10 copies / reaction and 100 copies / reaction, respectively, and the CT values ​​of the other 10 pathogens ranged from 20 to 34. Based on the copy numbers of the new coronavirus nucleic acid standards in the above 3 simulated mixed samples, the above 3 simulated mixed samples were named 1 copy / reaction, 10 copies / reaction and 100 copies / reaction samples, respectively. The kit provided in Example 2 was used to detect three simulated mixed samples. In this example, 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.

[0034] Table 2 Evaluation results for each simulated mixed sample

[0035]

[0036] Table 2 counts the number of MNP marker sites that the detected sequences are aligned to, the number of pathogens covered, and the normalized number of sequences aligned to the MNP marker sites of pathogens in samples with different copy numbers. Among them, the normalized number of sequences refers to the number of sequences aligned to the MNP marker sites of pathogens in every 100,000 sequences. In the data in Table 2, there is a situation where the number of detected markers is greater than the number of covered pathogens. The reason is that this embodiment detects multiple markers for some pathogens to ensure the true positive detection. When a pathogen detects multiple markers, the number of detected markers will be greater than the number of covered pathogens.

[0037] Evaluation of the stability and sensitivity of this primer set for detecting multiple human respiratory pathogens

[0038] As can be seen from Table 2, when the copy number reaches 10 copies / reaction, all 13 pathogens are detected in 9 repetitions, indicating that the primer set can stably detect the new coronavirus nucleic acid standard when the copy number is as low as 10 copies / reaction; but when it is as low as 1 copy / reaction, it cannot be fully detected. This shows that the detection sensitivity of this primer set for pathogens is 10 copies / reaction.

[0039] Evaluation of the reproducibility and accuracy of this primer set for multiple human respiratory pathogens

[0040] 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 with 100 copies / reaction in Table 2 were compared in pairs, and the results are shown in Table 3.

[0041] Table 3 shows the reproducibility and accuracy evaluation of the primer set for detecting genotypes of human respiratory pathogens

[0042] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Reproducibility Accuracy S-1 S-2 17 17 100％ 100％ S-1 S-3 17 17 100％ 100％ S-1 S-4 17 17 100％ 100％ S-1 S-5 17 17 100％ 100％ S-1 S-6 17 17 100％ 100％ S-1 S-7 17 17 100％ 100％ S-1 S-8 17 17 100％ 100％ S-1 S-9 17 17 100％ 100％

[0043] 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 that can be reproducible in the main genotype 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%.

[0044] Evaluation of the specificity of this primer set for human respiratory pathogens

[0045] As can be seen from Table 2, in the three simulated mixed 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 13 pathogens provided in this example all have sequence coverage, which indicates that the MNP marker and primer set have high specificity in detecting target microorganisms in complex templates.

[0046] The threshold value of this primer set for detecting multiple human respiratory pathogens

[0047] 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:

[0048] 1) The amount of sequencing data is no less than 26 megabases. The calculation is based on the fact that the maximum number of MNP sites detected for each sample is 17, and the length of a sequencing fragment is 300 bases. Therefore, when the amount of data is greater than 26 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.

[0049] 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.

[0050] Based on the sequencing data of the samples detected in Table 2, when the copy number of the pathogen is ≥10 copies, the SNR value of each pathogen is ≥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 the human respiratory pathogen in the sample is greater than 10, it is judged that the nucleic acid of the human respiratory pathogen is detected in the sample.

[0051] Application of this primer set in the detection of multiple human respiratory pathogens

[0052] The primer set was used to detect the pathogens of 30 human throat swab samples provided by the Hubei Provincial Center for Disease Control and Prevention. The sample numbers are shown in Table 4, and three negative controls with sterile water as the template were constructed. Each sample to be tested uses a commercial pathogen total nucleic acid extraction kit to prepare nucleic acid, and the prepared pathogen total nucleic acid of the sample to be tested is tested for the new coronavirus by the qPCR method. After qPCR identification, 10 new coronavirus positive samples were found in the 30 human throat swab samples to be tested. At the same time, a commercial reverse transcription kit was used to reverse transcribe the pathogen total nucleic acid of the prepared sample to be tested, and the cDNA and DNA mixed template of the sample to be tested were obtained. The primer set provided in this embodiment was used to perform multiple PCR amplification on the cDNA and DNA mixed template of the sample to be tested, and multiple amplification products were obtained for library construction, and the library was sequenced to obtain sequencing data. The results of the statistical sequencing data showed that among the 30 human throat swab samples to be tested, 25 samples to be tested detected the target pathogen, which was a positive sample, and the other 5 samples to be tested did not detect the target pathogen, which was a negative sample. In this example, the test results of the above 25 positive samples are shown in Table 4.

[0053] Table 4 shows the normalized number of sequences detected by the primer set for 25 positive samples (unit: bar)

[0054]

[0055]

[0056] According to the above-mentioned determination threshold, all 10 novel coronavirus positive samples detected by qPCR were detected by the primer set provided in Example 1, and one additional novel coronavirus positive sample (S2542) was detected, which indicates that the sensitivity of the primer set in detecting novel coronavirus is not lower than that of qPCR. The pathogens detected by the 30 real human throat swab samples shown in Table 4 are shown in Table 5.

[0057] Table 5 shows the statistics of pathogens detected from 30 real human throat swab samples

[0058] Serial number Target pathogens Number of samples detected 1 Pseudomonas aeruginosa 14 2 Novel Coronavirus 10 3 Haemophilus influenzae 5 4 Human rhinovirus 3 5 Coronavirus NL63 2 6 Coronavirus HKU1 1 7 Coronavirus 229E 1 8 Coronavirus OC43 1

[0059] Combining Tables 4 and 5, it can be seen that in addition to the new coronavirus, in 30 samples, the primer set provided in Example 2 also detected 7 other pathogens including Pseudomonas aeruginosa, Haemophilus influenzae, human rhinovirus, coronavirus NL63, coronavirus HKU1, coronavirus 229E and coronavirus OC43, which shows the advantage of the primer set in comprehensive detection.

[0060] Combining Table 4 and Table 5, it can be seen that by using the primer set provided in this embodiment, three MNP markers of the enterovirus genus were detected in three samples, which can be further subdivided into one enterovirus genus marker and two human rhinovirus markers. Therefore, according to the genotypes of the three MNP markers, the enterovirus can be further identified as human rhinovirus of the enterovirus genus, which shows that the primer set can achieve the advantage of pathogen type differentiation by detecting multiple markers for each pathogen.

[0061] Embodiment 4

[0062] Any one pair of primers or a combination of multiple pairs of primers in the primer set is used in the detection of human respiratory pathogens.

[0063] The primer set provided by the present invention includes 17 pairs of primer pairs, which can realize the detection of 13 target pathogens in the largest range. For applications that only focus on some of the 13 pathogens, primer combinations for some pathogens can be used. For example, the 1st to 12th primer pairs in the 17 pairs of primers can realize the identification of 8 viruses for 13 human respiratory pathogens. This embodiment uses the primer set of the 1st to 12th primer pairs to re-test the pathogens in the 30 human throat swab samples of Example 3, and constructs 3 negative controls with sterile water as the template. The target pathogens were detected in 15 samples to be tested, which were positive samples. The test results of the positive samples are shown in Table 6.

[0064] Table 6 shows the normalized number of sequences detected by the primer set for real human throat swab samples (unit: number)

[0065]

[0066]

[0067] According to the above-specified judgment threshold, the test results are shown in Table 6. It can be seen from Table 6 that the viral pathogens in all the samples to be tested can be detected by the 1st primer pair to the 12th primer pair, and the test results of the samples in which the viral pathogens were not detected in Example 3 were all negative, which proves the effectiveness of any pair of primers or a combination of multiple pairs of primers in the primer set in the detection of human respiratory pathogens.

[0068] Embodiment 5

[0069] The application includes: using the MNP marker site, primer set or kit for monitoring genetic variation of human respiratory pathogen strains.

[0070] As shown in Table 4, the novel coronavirus was detected in all 10 samples to be tested. The main genotypes of the MNP marker sites of the novel coronavirus in each sample to be tested were compared, and it was found that the genotypes in the 10 samples to be tested were not exactly the same, and could be divided into two categories. The main genotypes of the novel coronavirus MNP marker sites in the S1712 and S2016 samples to be tested were the same, and the main genotypes of the novel coronavirus MNP marker sites in the other 8 samples to be tested were the same. There are differences in genotypes between strains, indicating that this primer set can be used for the detection of genetic variations in pathogens.

[0071] 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, thereby affecting the stability and consistency of the experimental microbial phenotype. This heterogeneous heterozygous group is manifested as an allele type other than the main genotype of the site when the group is subjected to molecular marker detection. When the mutant individuals have not yet accumulated, they only account for a very small part of the group and are manifested as low-frequency allele types. Low-frequency allele types are often mixed with technical errors, making it difficult to distinguish with existing technologies. This embodiment detects highly polymorphic MNP markers. Based on the probability of multiple errors occurring simultaneously is lower than the probability of one error occurring, and the technical error rate of MNP markers is significantly lower than that of SNP markers.

[0072] The authenticity of the sub-allele type in this example is determined based on the number and ratio of sequenced sequences in Table 7.

[0073] Table 7 shows the critical values ​​for determining minor allele types at some sequencing depths

[0074]

[0075]

[0076]

[0077] Table 7 lists the calculations based on the BINOM.INV function with a probability guarantee of α = 99.9999%, e max (n=1) and e max When (n≥2) is 1.03% and 0.0994% respectively, the critical value of the number of sequencing sequences of the minor allele type in each MNP marker site is that only when the number of sequencing sequences of the minor allele type exceeds the critical value, it is determined to be the true minor allele type. When there are multiple candidate minor alleles, the P value of each candidate allele type is multi-corrected, and the candidate allele with FDR<0.5% is determined to be the true minor allele type.

[0078] Table 6 Parameters involved max (n=1) and e max(n≥2) refers to the highest proportion of the number of sequencing sequences carrying the wrong allele of n SNPs to the total number of sequencing sequences at this site. max (n=1) and e max (n≥2) were 1.03% and 0.0994%, respectively, which were obtained based on the frequencies of all minor alleles detected at 930 homozygous MNP loci.

[0079] According to the above parameters, the genotypes of the new coronavirus in the 10 samples shown in Table 4 were analyzed. The 10,612 sequencing sequences detected in the S1712 sample that were aligned to the new coronavirus MNP marker represented 2 different genotypes, with 2,744 and 7868 sequences, respectively, indicating that there were variations within the new coronavirus in the same sample. It can be seen that this primer set is suitable for detecting genetic variations within the human respiratory pathogen strain population.

[0080] Embodiment 6

[0081] Specifically, the application includes: using the MNP labeling site, primer set or kit to construct a database of human respiratory pathogens.

[0082] 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.

[0083] Embodiment 7

[0084] Specifically, the applications include: using MNP labeling sites, primer sets or kits to construct fine typing of human respiratory pathogens.

[0085] Viral pathogens in human respiratory pathogens are classified into the following categories in taxonomy, and there are also species, subspecies and subtypes. 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 pathogen can be accurately identified and finely typed. As shown in Table 4, 3 enterovirus markers were detected in 3 samples, namely 1 conservative marker of the enterovirus genus and 2 markers specific to human rhinovirus, thereby determining that the enterovirus in the sample is human rhinovirus. It can be seen from this that the primer set provided in this embodiment can be used for the type differentiation of human respiratory pathogens, and thus can be used for the monitoring of the prevalent species (types) of human respiratory pathogens.

[0086] 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 a variety of human respiratory pathogens, characterized in that The MNP marker site is used to identify a variety of human respiratory pathogens. The MNP marker site includes at least one of MNP-1 to MNP-17. The positions of MNP-1 to MNP-17 on the reference sequence are shown in the following table:

2. A primer set for identifying multiple human respiratory pathogens, characterized in that The primer set includes: at least one pair of the first primer pair to the seventeenth primer pair, each of the primer pairs 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 seventeenth primer pair, and the reverse primer of the seventeenth primer pair are respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 34 in the sequence listing.

3. A kit for identifying multiple human respiratory pathogens, 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 labeling site, the primer set or the kit to identify human respiratory pathogens, and the types of 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 and human rhinovirus.

6. The use according to claim 4, characterized in that: The bacteria include: 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 application includes: using the MNP marker site, the primer set or the kit for constructing a database of pathogens of multiple human respiratory tracts, monitoring genetic variations and distinguishing types.

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