MNP marker site, primer group and kit for identifying multiple pathogens in sewage 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 multiple pathogens in sewage and application of the MNP marker site, the primer group and the kit. The MNP marker site comprises at least one of MNP-1 to MNP-19, the primer group comprises at least one of a first primer pair to a nineteenth primer pair, and a forward primer of the first primer pair, a reverse primer of the first primer pair to a forward primer of the nineteenth primer pair and a reverse primer of the nineteenth primer pair are sequentially shown as SEQ ID NO: 1 to SEQ ID NO: 38 in a sequence table. The invention provides MNP marker sites for identifying multiple pathogens in sewage, a primer group, a kit and application, the primer group aims at 19 MNP marker sites, 1-3 marker sites are detected for each pathogen, the pathogens in the multiple types of sewage can be identified, the comprehensive, efficient and accurate detection effect is achieved, and the primer group is suitable for identifying the multiple pathogens in the sewage.

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 pathogens in sewage. Background Art

[0002] In current healthcare and public safety prevention measures, detecting pathogens in sewage offers the advantage of early warning, making accurate identification of pathogens in sewage crucial. The wide variety of pathogens in sewage, including viruses and bacteria, poses significant challenges to disease diagnosis and prevention.

[0003] Currently, pathogen detection in wastewater primarily relies on PCR-based nucleic acid detection and metagenomic sequencing. However, PCR only detects a single marker per pathogen, resulting in low detection efficiency and an inability to detect sequence variations. Metagenomic sequencing also tends to miss viral pathogens with small, easily degradable genomes, limiting the development of pathogen detection in wastewater. Therefore, a method that can simultaneously detect multiple pathogens in wastewater is urgently needed.

[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] On the one hand, the present disclosure provides an MNP labeling site for identifying multiple pathogens in sewage, wherein the MNP labeling site is used to identify multiple pathogens in sewage, and 1 to 3 MNP markers are designed for each pathogen. The MNP labeling site includes at least one of MNP-1 to MNP-19. The positions of MNP-1 to MNP-19 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 pathogens in sewage, the primer set comprising: at least one pair of primer pairs from the first to the nineteenth primer pairs, each of the primer pairs comprising a forward primer and a reverse primer, the 19 primer pairs being used to detect the 19 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 nineteenth primer pair, and the reverse primer of the nineteenth primer pair being respectively as shown in SEQ ID NO: 1 to SEQ ID NO: 38 in the sequence listing.

[0008] In yet another aspect, the present disclosure provides a kit for identifying multiple pathogens in sewage, the kit comprising the above primer set.

[0009] In 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 pathogens in sewage, wherein the pathogens in sewage include: viruses, bacteria, fungi and special pathogens.

[0010] Specifically, the viruses include: adenovirus F, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus, monkeypox 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, Salmonella enterica 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 pathogens in sewage.

[0016] The beneficial effects brought about by 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 pathogens in sewage, wherein the MNP marker site refers to a marker site that is species-specific and has multiple nucleotide polymorphisms within the species screened on the genome of the target pathogen, and has high polymorphism and strong species differentiation ability. The MNP marker site provided by the present disclosure can be screened from the genome of pathogens in sewage, and 1 to 3 markers are screened for each pathogen, which can specifically distinguish multiple pathogens in sewage. The primer set provided by the present disclosure has amplification compatibility, and can be amplified in one reaction system using super-multiplex PCR, realizing the amplification of multiple MNP marker sites in one reaction system, integrating the second-generation sequencing platform to perform sequence analysis of the amplified products, realizing the identification of multiple pathogens in sewage in one reaction, and having 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 efficient identification of multiple pathogens in sewage, 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] Example 1

[0019] The embodiments of the present disclosure provide an MNP labeling site for identifying multiple pathogens in sewage. The MNP labeling site is used to identify multiple pathogens in sewage. The MNP labeling site includes at least one of MNP-1 to MNP-19. The positions of MNP-1 to MNP-19 on the reference sequence are shown in Table 1.

[0020] The present disclosure also provides a primer set for identifying multiple pathogens in sewage, comprising: at least one pair from the first to the nineteenth primer pairs, the nineteen primer pairs being used to detect nineteen 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 nineteenth primer pair, and the reverse primer of the nineteenth primer pair being, respectively, as shown in the sequence listing as SEQ ID NO: 1 to SEQ ID NO: 38. 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 pathogens in sewage, which includes the primer set provided in Example 1.

[0026] Example 3

[0027] An embodiment of the present disclosure provides an application of the above-mentioned primer set or kit, which includes using the primer set to identify multiple pathogens in sewage, and the multiple pathogens in sewage include: viruses, bacteria, fungi and special pathogens.

[0028] Specifically, the viruses include: adenovirus F, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus, monkeypox 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, Salmonella enterica, 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 is used to detect positive samples of 15 target pathogens, including adenovirus F, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackie virus, monkeypox virus, human rhinovirus, Haemophilus influenzae, Salmonella enterica, Pseudomonas aeruginosa, capsular histoplasma, Cryptococcus neoformans and Chlamydia pneumoniae. Specifically, the 15 target pathogen positive samples are nucleic acids of pathogen isolation cultures that have been identified as positive by digital PCR and whose copy numbers have been quantified. The nucleic acids of the above 15 target pathogens are mixed as a simulated mixed sample of multiple pathogens in sewage (hereinafter referred to as the simulated mixed sample). The simulated mixed sample is made into three different copy gradients. Specifically, the three different copy gradients have a copy number of 1 copy / reaction, 10 copies / reaction and 100 copies / reaction for each pathogen, respectively, and are named 1 copy / reaction sample, 10 copies / reaction sample and 100 copies / reaction sample, respectively. Three simulated mixed samples were tested using the kit provided in Example 2. The MNP-labeled detection method in this example is as follows: The total nucleic acid in the simulated mixed sample was reverse transcribed using a commercial reverse transcription kit. For the procedure, refer to the kit's instructions. In this example, a 20 μL reverse transcription system consisted of: 10 μL total nucleic acid, 5 μL 4× HiScript IV RT Mix, and 5 μL Nuclease-free ddHO (sterile, enzyme-free water). The cDNA synthesis reaction protocol was: 55°C for 15 min, 85°C for 5 sec.

[0034] The total nucleic acid of the simulated mixed sample includes DNA and RNA. The reverse transcription is mainly targeted at the RNA portion to obtain a mixed template of cDNA and DNA of the simulated mixed sample. The mixed template is subjected to a first multiplex PCR amplification using the primer set provided in Example 1 to obtain a first multiplex amplification product. The amplification system for each 30 μL of the first multiplex PCR amplification includes: 4 μL of cDNA, 4 μL of the primer set, 4 μL of E1 Mix, and 6 μL of Nuclease-free ddH2O. The amplification program for the first multiplex PCR amplification is shown in Table 2.

[0035] Table 2 shows the amplification program for the first multiplex PCR amplification

[0036]

[0037] The first multiplex amplification product was subjected to a second multiplex PCR amplification using the primer set provided in Example 1 to obtain a second multiplex amplification product. The amplification system for each 30 μL of the second multiplex PCR amplification included: 4 μL of the first multiplex amplification product, 4 μL of the primer set, 10 μL of E1 Mix, and 16 μL of Nuclease-free ddH2O. The amplification program for the first multiplex PCR amplification is shown in Table 3.

[0038] Table 3 shows the amplification program for the second multiplex PCR amplification

[0039]

[0040]

[0041] After purification, the secondary multiplex amplification products were used to construct libraries. The libraries were sequenced to generate sequencing data, and an equal volume of sterile water was added as a blank control, designated as 0 copies / reaction. Three replicate libraries were constructed daily for each sample, and testing continued for three consecutive days, resulting in nine sets of sequencing data for each sample. The results are shown in Table 4.

[0042] Table 4 evaluates the results for each simulated mixed sample

[0043]

[0044] Table 4 summarizes the number of MNP marker sites mapped to the detected sequences, the number of pathogens covered, and the normalized number of sequences mapped to the pathogen's MNP marker sites in samples with different copy numbers. The normalized number of sequences refers to the number of sequences mapped to the pathogen's MNP marker sites per 100,000 sequences. In the data in Table 4, there are cases where the number of detected markers exceeds the number of covered pathogens. This is because this example 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 exceed the number of covered pathogens.

[0045] Evaluation of the stability and sensitivity of this primer set for detecting multiple pathogens in sewage

[0046] As shown in Table 4, when the copy number reached 10 copies / reaction, all 15 pathogens were detected in 9 repetitions, while when the copy number was as low as 1 copy / reaction, not all of them were detected. This shows that the detection sensitivity of this primer set for pathogens is 10 copies / reaction.

[0047] Evaluation of the reproducibility and accuracy of this primer set for multiple pathogens in sewage

[0048] The reproducibility and accuracy of the MNP-labeled detection method for detecting pathogens in wastewater were evaluated based on the reproducibility of the genotypes at the common detected loci between replicates. Specifically, pairwise comparisons were performed for each of the nine data sets at 100 copies / reaction in Table 4, with the results shown in Table 5.

[0049] Table 5 shows the reproducibility and accuracy evaluation of the primer set for detecting pathogen genotypes in sewage

[0050] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r Accuracy S-1 S-2 19 19 100％ 100％ S-1 S-3 19 19 100％ 100％ S-1 S-4 19 19 100％ 100％ S-1 S-5 19 19 100％ 100％ S-1 S-6 19 19 100％ 100％ S-1 S-7 19 19 100％ 100％ S-1 S-8 19 19 100％ 100％ S-1 S-9 19 19 100％ 100％

[0051] As shown in Table 5, the number of MNP sites with discrepancies in the primary genotype 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 reproducible sites in the primary genotype 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%.

[0052] Evaluation of the specificity of this primer set for pathogens in sewage

[0053] As shown in Table 4, 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, through sequence alignment, that the MNP marker site sequences of the 15 pathogens were all covered by the primer set sequences provided in this example. This indicates that the MNP marker sites of the 15 pathogens have high specificity for detecting target microorganisms in complex templates using the primer set.

[0054] The threshold value of this primer set for detecting multiple pathogens in sewage

[0055] As shown in Table 4, most samples with 1 copy / reaction were able to detect sequences that matched to pathogens in sewage. Sequences of pathogens in sewage were also detected in some blank controls. Due to the extreme sensitivity of the MNP-labeled detection method, false positives can easily occur due to data contamination during the detection process. Therefore, in this example, quality control was performed on the test results of simulated samples with different copy numbers for three consecutive days. The quality control plan developed is as follows:

[0056] 1) Sequencing data volume of at least 30 megabases. This is based on the assumption that each sample can detect a maximum of 19 MNP sites and a single sequencing fragment is 300 bases long. Therefore, for most samples with data volume greater than 30 megabases, a single run can guarantee coverage of each MNP marker site by at least 5,000-fold, ensuring accurate analysis of the base sequence at each MNP marker site.

[0057] 2) Calculate the signal-to-noise ratio (SNR=S / N) of each pathogen in the sample based on the sequence number S of the pathogen in each sewage in the test sample and the sequence number N of the pathogen in the sewage in the blank control.

[0058] Based on the sequencing data of the samples tested in Table 4, 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 standard for the primer set provided in this embodiment to determine whether it is positive for pathogens in sewage is: when the signal-to-noise ratio of the pathogen in the sewage in the sample is greater than 10, it is determined that the nucleic acid of the pathogen in the sewage is detected in the sample.

[0059] Application of this primer set in the detection of multiple pathogens in sewage

[0060] This primer set was used to detect pathogens in 25 sewage samples. The sample numbers are shown in Table 6. Three negative controls using sterile water as a template were also constructed. A commercial pathogen total nucleic acid extraction kit was used to prepare nucleic acid for each sample. The prepared pathogen total nucleic acid of the sample was tested using traditional qPCR methods. All 25 sewage samples were positive for adenovirus type F. Adenovirus type F is a common adenovirus that causes diarrhea. Simultaneously, a commercial reverse transcription kit was used to reverse transcribe the pathogen total nucleic acid of the prepared sewage samples to obtain a mixed template of cDNA and DNA from the sewage samples. The primer set provided in Example 1 was used to perform multiplex PCR amplification on the mixed template of cDNA and DNA from the sewage samples to be tested. Multiplex amplification products were obtained, which were used to construct a library. The library was sequenced to obtain sequencing data. The results of the sequencing data were statistically analyzed. Adenovirus type F pathogens were also detected in all 25 sewage samples. The test results of the above 25 positive samples are shown in Table 6 in this example.

[0061] Table 6 shows the normalized number of sequences detected by the primer set for 25 positive samples (unit: sequence)

[0062]

[0063]

[0064] According to the aforementioned threshold, the primer set provided in Example 1 detected all 25 adenovirus F-positive samples detected by qPCR, demonstrating that the sensitivity of this primer set for detecting adenovirus F is no less than that of qPCR. Table 7 summarizes the pathogens detected in the 25 real sewage samples shown in Table 6.

[0065] Table 7 shows the statistics of pathogens detected in 25 real sewage samples

[0066]

[0067]

[0068] Combining Tables 6 and 7, it can be seen that in addition to adenovirus type F, in 25 samples, the primer set provided in Example 1 also detected coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, monkeypox virus, Haemophilus influenzae, human rhinovirus and Pseudomonas aeruginosa, which shows the advantage of the primer set in comprehensive detection.

[0069] Combining Tables 6 and 7, it can be seen that using the primer set provided in Example 1 of the present invention, three MNP markers of the enterovirus genus were detected in three samples, specifically 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 the primer set can achieve the advantage of pathogen type differentiation by detecting multiple markers for each pathogen.

[0070] Example 4

[0071] The application of any primer pair or a combination of multiple primer pairs in the primer set in the detection of pathogens in sewage.

[0072] The primer set provided by the present invention includes 19 pairs of primer pairs, which can realize the detection of 15 target pathogens in the largest range. For applications that only focus on some of the 15 pathogens, a primer combination for some pathogens can be used. For example, the 1st primer pair to the 13th primer pair in the 19 pairs of primer pairs can realize the identification of 9 viral types of pathogens in 15 sewage. This embodiment adopts the primer set of the 1st primer pair to the 13th primer pair to re-test the pathogens in the 25 sewage samples of Example 3, and at the same time constructs 3 negative controls with sterile water as templates. The target pathogens were detected in 25 samples to be tested, which are positive samples. The test results of the positive samples are shown in Table 8.

[0073] Table 8 shows the normalized number of sequences detected by the primer set in real sewage samples (unit: sequence)

[0074]

[0075]

[0076] According to the above-mentioned determination threshold, the test results are shown in Table 8. It can be seen from Table 8 that the viral pathogens in all the samples to be tested can be detected by the 1st primer pair to the 13th primer pair, while the test results of the samples in which the viral pathogens were not detected in Example 3 were all negative. This proves that any pair of primers or a combination of multiple primer pairs in the primer set has high effectiveness in the detection of pathogens in sewage.

[0077] Example 5

[0078] The application includes: using the MNP labeling site, primer set or kit to monitor genetic variation of pathogen strains in sewage.

[0079] As shown in Table 6, adenovirus F was detected in all 25 positive samples. Comparison of the primary genotypes at the MNP marker loci for adenovirus F in each positive sample revealed that multiple genotypes were present in most of the 25 positive samples. Since sewage samples are collected from communities, hospitals, and other institutions, pathogens within them come from multiple sources. This mixed population manifests as multiple alleles at a single marker locus when tested using sequencing-based techniques. For example, the 53,133 normalized sequences detected in sample S240208, when mapped to the adenovirus F MNP marker, represent two distinct genotypes, with 30,243 and 22,890 sequences, respectively. This indicates that adenovirus F can vary within the same sample. This demonstrates that this primer set is suitable for detecting genetic variation within a population of sewage pathogen strains.

[0080] Comparing the genotypes of each sample reveals genetic differences between strains. For example, the six community sewage samples starting with "S"—S240208, S240212, S240215, S240224, and S240304—each had two identical genotypes. Sample S240219, however, had three genotypes: two shared with the previous five samples, and one unique to that sample, suggesting a wider range of pathogen sources. These differences in sample genotypes indicate that this primer set can be used to detect genetic variation in pathogens.

[0081] Example 6

[0082] Specifically, the application includes: using the MNP labeling site, primer set or kit to construct a database of pathogens in sewage.

[0083] As shown in Table 7, after testing the samples using this primer set, the main genotype of the MNP marker site for each pathogen in each sample was obtained. The main genotypes of the same pathogen obtained in all samples were compared, and the main genotypes with differences were entered into the database file to form an MNP fingerprint database for pathogens in sewage. After each comparison of the main genotype of the MNP marker site of the sample or strain to be tested with the constructed MNP fingerprint database, the MNP fingerprint patterns of the strains with different main 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 strain being tested, it is compatible with all high-throughput sequencing data and has the characteristics of being fully co-constructed, shared, and updatable at any time.

[0084] Example 7

[0085] Specifically, the applications include: using MNP marker sites, primer sets or kits to construct fine typing of pathogens in sewage.

[0086] Viral pathogens among the pathogens in sewage are classified taxonomically into the following categories: 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 6, 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 to distinguish the types of pathogens in sewage, and thus can be used to monitor the prevalence of pathogens in sewage.

[0087] 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. An MNP-labeled site for identifying multiple pathogens in sewage, characterized in that: The MNP marker site is used to identify multiple pathogens in sewage. The MNP marker site includes at least one of MNP-1 to MNP-19. The positions of MNP-1 to MNP-19 on the reference sequence are shown in the following table:

2. A primer set for identifying multiple pathogens in sewage, characterized in that: The primer set includes: at least one pair of the first primer pair to the nineteenth 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 nineteenth primer pair, and the reverse primer of the nineteenth primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 38 in the sequence listing.

3. A kit for identifying multiple pathogens in sewage, characterized in that: The kit comprises the primer set according to 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 pathogens in sewage. The types of pathogens in sewage include viruses, bacteria, fungi and special pathogens.

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

6. The use according to claim 4, characterized in that The bacteria include: Haemophilus influenzae, Salmonella enterica 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 database construction, genetic variation monitoring and type differentiation of multiple pathogens in sewage.

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