An MNP labeling site, primer set, kit and application for identifying multiple human respiratory pathogens

Abstract

The present disclosure provides an MNP labeling site, primer set, kit and application for identifying multiple human respiratory pathogens, which belongs to the field of biotechnology. The MNP labeling site includes: at least one of MNP-1 to MNP-17, the primer set includes: at least one of the first primer pair to the seventeenth primer pair, each of the primer pairs includes a forward primer and a reverse primer, 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 present disclosure provides an MNP labeling site, primer set, kit and application for identifying multiple human respiratory pathogens. The primer set targets 17 MNP labeling sites, detects 1 to 3 labeling sites for each pathogen, can identify 13 human respiratory pathogens, 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 medical, health and public safety fields, accurate identification of human respiratory pathogens is crucial. Human respiratory pathogens are diverse, including viruses and bacteria, posing a great challenge to disease diagnosis and prevention.

[0003] Currently, the primary technology for detecting human respiratory pathogens is PCR-based nucleic acid detection. However, PCR detection only detects a single marker per pathogen, resulting in low detection efficiency and an inability to detect sequence variations. This has limited the development of human respiratory pathogen detection, leading to an urgent need for a method that can detect multiple human respiratory pathogens simultaneously.

[0004] Public content

[0005] To address the problems of the prior art, the present disclosure provides a method for identifying multiple MNP marker sites, primer sets, kits, and applications. The technical solution is as follows:

[0006] In one aspect, the present disclosure provides an MNP labeling site for identifying multiple human respiratory pathogens. The MNP labeling site is used to identify multiple human respiratory pathogens. 1-3 MNP labels are designed for each pathogen. 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 found 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 to the seventeenth primer pairs, each primer pair comprising a forward primer and a reverse primer, the 17 primer pairs being used to detect the 17 MNP marker sites, the forward primer of the first primer pair, the reverse primer of the first primer pair to the forward primers of the seventeenth primer pair, and the reverse primer of the seventeenth primer pair being 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 multiple human respiratory pathogens, the kit comprising the above primer set.

[0009] In another aspect, the present disclosure provides an application of the above-mentioned MNP-labeled site, the above-mentioned primer set, or the above-mentioned kit, wherein the application includes using the MNP-labeled 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 beneficial effects of the technical solutions provided by the embodiments of the present disclosure are as follows: the present disclosure provides an MNP marker site, primer set, kit and application for identifying multiple human respiratory pathogens. The MNP marker site refers to a species-specific marker site screened on the genome of the target pathogen and having multiple nucleotide polymorphisms within the species, with high polymorphism and strong species differentiation ability. The MNP marker site provided by the present disclosure can be screened from the genome of human respiratory pathogens, with 1 to 3 markers screened for each pathogen, which can specifically distinguish multiple human respiratory pathogens. The primer set provided by the present disclosure has amplification compatibility and can be amplified using super-multiplex PCR in one reaction system to achieve amplification of multiple MNP marker sites in one reaction system, and integrated with a second-generation sequencing platform for sequence analysis of the amplified products to achieve identification of multiple human respiratory pathogens in one reaction, with comprehensive, efficient, sensitive and accurate detection effects and type differentiation. Compared with the fluorescence PCR-based method, one reaction usually uses only one pair of primers to detect one marker site of a pathogen, and the detection is a fluorescent signal. The primer set and kit provided by the present invention detect up to three marker sites for each pathogen, making the detection more efficient and accurate. It can also detect sequence variations and distinguish pathogen types. It can be used for the efficient identification, genetic variation monitoring, database construction and type differentiation of various human respiratory pathogens. 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] Example 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 of the first through seventeenth primer pairs. The seventeen primer pairs are used to detect seventeen MNP marker sites, each primer pair comprising a forward primer and a reverse primer. The forward primer of the first primer pair, the reverse primer of the first primer pair through the forward primers of the seventeenth primer pair, and the reverse primer of the seventeenth primer pair are, respectively, as shown in the sequence listing as SEQ ID NO: 1 to SEQ ID NO: 34. 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] Example 2

[0025] The present disclosure provides a kit for identifying multiple human respiratory pathogens, which includes the primer set provided in Example 1.

[0026] Example 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 included: a commercial nucleic acid standard with a known copy number of a human respiratory pathogen and nucleic acids from clinical samples of 12 human respiratory pathogens identified as positive by fluorescent quantitative PCR (hereinafter referred to as qPCR) (provided by the Hubei Provincial Center for Disease Control and Prevention, and the sample type was respiratory samples). The pathogen of one commercial nucleic acid standard was the new coronavirus. The nucleic acid standards containing the above 13 target pathogens and the positive sample nucleic acid were mixed to prepare three simulated mixed samples of multiple human respiratory pathogens (hereinafter referred to as simulated mixed samples). In the three simulated mixed samples, the copy numbers of the pathogens of the new coronavirus nucleic acid standard 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 standard 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. Three simulated mixed samples were tested using the kit provided in Example 2. The MNP-labeled detection method in this example was as follows: total nucleic acid (including DNA and RNA) in the simulated mixed sample was reverse transcribed to obtain a mixed template containing sample cDNA and DNA. The sample mixed template was amplified using the primer set provided in Example 1 using multiplex PCR to obtain multiple amplification products, which were used to construct a library. The library was sequenced to obtain sequencing data, and an equal volume of sterile water was set as a blank control, designated as 0 copies / reaction. Three replicate libraries were constructed for each sample daily, and testing was continued for three consecutive days, resulting in nine sets of sequencing data for each sample. The specific results are shown in Table 2.

[0034] Table 2 evaluates the results for each simulated mixed sample

[0035]

[0036] Table 2 summarizes the number of MNP marker sites that the detected sequences aligned to, the number of pathogens covered, and the normalized number of sequences aligned to the pathogen's MNP marker sites in samples with different copy numbers. The normalized number of sequences refers to the number of sequences aligned to the pathogen's MNP marker sites per 100,000 sequences. In the data in Table 2, there are cases where the number of detected markers exceeds the number of covered pathogens. This is because this embodiment detects multiple markers for some pathogens to ensure true positive detection. When multiple markers are detected for a pathogen, 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 shown in Table 2, when the copy number reached 10 copies / reaction, all 13 pathogens were detected in 9 repetitions, indicating that the primer set can stably detect the novel coronavirus nucleic acid standard when the copy number is as low as 10 copies / reaction; however, when the copy number is as low as 1 copy / reaction, not all of them can be 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] The reproducibility and accuracy of the MNP-labeled assay for detecting human respiratory pathogens were evaluated based on the reproducibility of the genotypes at the common detected loci across two replicates. Specifically, pairwise comparisons were performed for each of the nine data sets at 100 copies / reaction in Table 2, with the results shown in Table 3.

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

[0042] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r 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 shown in Table 3, the number of MNP sites where the primary genotype differed was 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy rate a = 1-(1-r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of the number of sites with reproducible primary genotypes to the number of shared sites. In the reproducibility test of this example, the logarithm of the difference in the primary genotype of each sample between different libraries and different library construction batches was 0, the reproducibility rate r = 100%, and the accuracy rate a = 100%.

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

[0045] As shown in Table 2, in the three simulated mixed samples, when the copy number of the pathogen was ≥10 copies, the sequences obtained by sequencing in the simulated samples showed sequence coverage of the MNP markers for all 13 pathogens provided in this example after sequence alignment. This indicates that the MNP marker and primer set have high specificity for 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, sequences of human respiratory pathogens were detected in most samples with 1 copy / reaction. Sequences of human respiratory pathogens were also detected in some blank controls. Due to the extreme sensitivity of the MNP-labeled detection method, data contamination during the test process can easily lead to false positives. Therefore, in this example, based on the test results of simulated samples with different copy numbers over three consecutive days, the following quality control plan was developed:

[0048] 1) The sequencing data volume must be no less than 26 megabases. This calculation is based on the assumption that the maximum number of MNP sites detected per sample is 17, and a single sequencing fragment is 300 bases long. Therefore, when the data volume exceeds 26 megabases, a single experiment can ensure that the number of sequencing fragments covering each site reaches more than 5000 times, ensuring accurate analysis of the base sequence of each MNP site.

[0049] 2) Calculate the signal-to-noise ratio (SNR) of each pathogen in the sample based on 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: SNR = S / N.

[0050] Based on the sequencing data of the samples tested 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 into account sensitivity, the criterion for determining whether the primer set provided in this example is 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 determined 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 pathogens in 30 human throat swab test samples provided by the Hubei Provincial Center for Disease Control and Prevention. The test sample numbers are shown in Table 4. At the same time, three negative controls using sterile water as a template were constructed. Each test sample was prepared using a commercial pathogen total nucleic acid extraction kit. The prepared pathogen total nucleic acid of the test sample was tested for the new coronavirus by qPCR. After qPCR identification, 10 of the 30 human throat swab test samples were positive for the new coronavirus. At the same time, a commercial reverse transcription kit was used to reverse transcribe the pathogen total nucleic acid of the prepared test sample to obtain a mixed template of cDNA and DNA of the test sample. The primer set provided in this Example 1 was used to perform multiple PCR amplification on the mixed template of cDNA and DNA of the test sample to obtain multiple amplification products, which were used to construct a library. The library was sequenced to obtain sequencing data. The results of the statistical sequencing data showed that among the 30 human throat swab test samples, 25 of the test samples detected the target pathogen and were positive samples, while the other 5 test samples did not detect the target pathogen and were negative samples. 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: sequence)

[0054]

[0055]

[0056] According to the threshold values ​​specified above, the primer set provided in Example 1 detected all 10 COVID-19 positive samples detected by qPCR, and also detected one additional COVID-19 positive sample (S2542). This indicates that the sensitivity of this primer set for detecting COVID-19 is no less than that of qPCR. The pathogens detected in 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 in 30 real human throat swab samples

[0058] Serial number Target pathogens Number of detected samples 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 seven other pathogens, including Pseudomonas aeruginosa, Haemophilus influenzae, human rhinovirus, coronavirus NL63, coronavirus HKU1, coronavirus 229E, and coronavirus OC43, which shows the advantage of this primer set in comprehensive detection.

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

[0061] Example 4

[0062] Any one primer pair or a combination of multiple primer pairs 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 primer pair to the 12th primer pair in the 17 pairs of primer pairs can realize the identification of 8 viral types of 13 human respiratory pathogens. This example uses the primer set of the 1st primer pair to the 12th primer pair to re-test the pathogens in the 30 human throat swab samples of Example 3, and at the same time constructs 3 negative controls with sterile water as templates. The target pathogens were detected in 15 samples to be tested, which are 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 in real human throat swab samples (unit: sequence)

[0065]

[0066]

[0067] According to the above-mentioned determination threshold, the test results are shown in Table 6. As can be seen from Table 6, the first primer pair to the twelfth primer pair can detect all viral pathogens in the tested samples, while the test results of the samples in which no viral pathogens were detected in Example 3 were all negative. This proves the effectiveness of any primer pair or combination of multiple primer pairs in the primer set in detecting human respiratory pathogens.

[0068] Example 5

[0069] The application includes: using the MNP labeling 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. Comparison of the primary genotypes of the novel coronavirus at the MNP marker site in each sample revealed that the genotypes in the 10 samples were not completely identical and could be divided into two categories: the primary genotypes of the novel coronavirus at the MNP marker site in samples S1712 and S2016 were identical, while the primary genotypes of the novel coronavirus at the MNP marker site in the other eight samples were identical. The presence of genotypic differences between strains indicates that this primer set can be used to detect 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 manifests 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 manifest as a low-frequency allele type. Low-frequency allele types are often mixed with technical errors, making it difficult to distinguish with existing technology. This embodiment detects highly polymorphic MNP markers. 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 minor allele type in this example was 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 certain 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 the thresholds for the number of sequencing reads for the minor allele at each MNP marker were 1.03% and 0.0994% (n ≥ 2), respectively, a true minor allele was determined only when the number of sequencing reads for the minor allele exceeded the threshold. When multiple candidate minor alleles were present, the P value for each candidate allele was corrected for multiple alleles, and a candidate allele with an FDR < 0.5% was determined to be a true minor allele.

[0078] Parameters involved in Table 6 max (n=1) and e max(n≥2) refers to the highest proportion of the number of sequencing reads carrying the wrong allele of n SNPs to the total number of sequencing reads at that 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] Using the above parameters, we analyzed the genotypes of the novel coronavirus in the 10 test samples shown in Table 4. The 10,612 sequences detected in the S1712 test sample that mapped to the novel coronavirus MNP marker represented two different genotypes, with 2,744 and 7,868 sequences, respectively. This indicates that the novel coronavirus within the same test sample exhibits internal variation. This demonstrates that this primer set is suitable for detecting genetic variation within a population of human respiratory pathogen strains.

[0080] Example 6

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

[0082] As shown in Table 5, after testing the test samples using this primer set, the primary genotype of the MNP marker site for each pathogen in each test sample is obtained. The primary genotypes of the same pathogen obtained in all samples are compared, and the main genotypes with differences are entered into a database file to form an MNP fingerprint database for human respiratory pathogens. After each comparison of the primary genotype of the MNP marker site of the test sample or strain with the constructed MNP fingerprint database, the MNP fingerprint patterns of strains with different primary genotypes 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 genetic sequence of the tested strains, it is compatible with all high-throughput sequencing data, making it fully co-constructed, shared, and updatable at any time.

[0083] Example 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 among human respiratory pathogens are classified taxonomically into genera, species, subspecies and subtypes. For example, the enterovirus genus provided in this embodiment can be specifically divided 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). Based on the detection of conservative markers and specific markers, the pathogens can be accurately identified and finely typed. As shown in Table 4, 3 enterovirus markers were detected in all 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 that the primer set provided in this embodiment can be used for type differentiation of human respiratory pathogens, and thus can be used for monitoring the prevalence of human respiratory pathogens.

[0086] The above description is merely an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.

Claims

1. A primer set for identifying multiple human respiratory pathogens, characterized in that: The primer set includes: the first primer pair to the seventeenth 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 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 listing.

2. A kit for identifying multiple human respiratory pathogens, characterized in that: The kit comprises the primer set according to claim 1.

3. Use of the primer set according to claim 1 or the kit according to claim 2 in preparing a reagent for identifying human respiratory pathogens, characterized in that: The types of human respiratory pathogens are: new coronavirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackievirus, human rhinovirus, Haemophilus influenzae, Pseudomonas aeruginosa, capsular histoplasma, Cryptococcus neoformans and Chlamydia pneumoniae.

4. Use of the primer set according to claim 1 or the kit according to claim 2 in preparing reagents for constructing a database of pathogens of the human respiratory tract, monitoring genetic variations, and distinguishing types, characterized in that: The types of human respiratory pathogens are: new coronavirus, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackievirus, human rhinovirus, Haemophilus influenzae, Pseudomonas aeruginosa, capsular histoplasma, Cryptococcus neoformans and Chlamydia pneumoniae.

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