An MNP marker site, primer set, reagent kit, and application for identifying multiple pathogens in wastewater.

Abstract

This disclosure provides an MNP marker site, primer set, kit, and application for identifying multiple pathogens in wastewater. The MNP marker site includes at least one of MNP-1 to MNP-19. The primer set includes at least one primer pair from the first to the 19th primer pair. The forward primer of the first primer pair, the reverse primer of the first primer pair, the forward primer of the 19th primer pair, and the reverse primer of the 19th primer pair are shown sequentially as SEQ ID NO: 1 to SEQ ID NO: 38 in the sequence listing. This disclosure provides an MNP marker site, primer set, kit, and application for identifying multiple pathogens in wastewater. The primer set targets 19 MNP marker sites, and each pathogen can be detected by 1 to 3 marker sites. It can identify multiple pathogens in wastewater, providing comprehensive, efficient, and accurate detection, and is suitable for identifying multiple pathogens in wastewater.

Description

Technical Field

[0001] This disclosure relates to the field of biotechnology, and in particular to an MNP marker site, primer set, kit, and application for identifying multiple pathogens in wastewater. Background Technology

[0002] In current medical and public safety prevention measures, detecting pathogens in wastewater offers the advantage of early warning; therefore, accurate identification of pathogens in wastewater is crucial. Wastewater contains a wide variety of pathogens, including viruses and bacteria, posing a significant challenge to disease diagnosis and prevention.

[0003] Currently, pathogen detection technologies in wastewater mainly rely on PCR-based nucleic acid detection and metagenomic sequencing. However, PCR detection only detects one marker per pathogen, resulting in low detection efficiency and the inability to detect variant sequence information. Metagenomic sequencing tends to miss small, easily degradable viral pathogens, which limits the development of pathogen detection in wastewater. Therefore, there is an urgent need for a method that can simultaneously detect multiple pathogens in wastewater.

[0004] Public content

[0005] To address the problems of existing technologies, this disclosure provides an embodiment of a primer set, kit, and application for identifying various MNP marker sites. The technical solution is as follows:

[0006] On the one hand, this disclosure provides an MNP marker site for identifying multiple pathogens in wastewater. The MNP marker site is used to identify multiple pathogens in wastewater. One to three MNP markers are designed for each pathogen. 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 can be found in Table 1.

[0007] On the other hand, this disclosure provides a primer set for identifying multiple pathogens in wastewater, the primer set comprising at least one of primer pairs 1 to 19, each primer pair 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 primer of the 19th primer pair and the reverse primer of the 19th primer pair being as shown in SEQ ID NO: 1 to SEQ ID NO: 38 in the sequence listing.

[0008] In another aspect, this disclosure provides a kit for identifying multiple pathogens in wastewater, the kit comprising the aforementioned primer set.

[0009] In another aspect, this disclosure provides an application of the above-mentioned MNP marker site, primer set, or kit, the application of which includes using the MNP marker site, primer set, or kit to identify pathogens in wastewater, the pathogens in the wastewater including viruses, bacteria, fungi, and special pathogens.

[0010] Specifically, the viruses include: adenovirus type F, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus type 71, coxsackievirus, monkeypox virus, and human rhinovirus.

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

[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 specific pathogens include Chlamydia pneumoniae.

[0015] Specifically, the applications include using the MNP marker sites, the primer set, or the kit for database construction, genetic variation monitoring, and type differentiation of multiple pathogens in wastewater.

[0016] The beneficial effects of the technical solution provided in this disclosure are as follows: This disclosure provides an MNP marker site, primer set, kit, and application for identifying multiple pathogens in wastewater. The MNP marker site refers to a species-specific marker site screened on the genome of the target pathogen, exhibiting multiple nucleotide polymorphisms within the species, possessing high polymorphism and strong species discrimination ability. The MNP marker sites provided in this disclosure can be screened from the genomes of pathogens in wastewater, with 1 to 3 markers selected for each pathogen, specifically distinguishing multiple pathogens in wastewater. The primer set provided in this disclosure has amplification compatibility and can be amplified in a single reaction system using ultramultiplex PCR, achieving amplification of multiple MNP marker sites within one reaction system. Combined with next-generation sequencing platform for sequence analysis of the amplified products, this enables the identification of multiple pathogens in wastewater in a single reaction, providing comprehensive, efficient, sensitive, and accurate detection and type differentiation. Compared to fluorescence PCR-based methods, which typically use only one pair of primers to detect one marker site for a pathogen in a single reaction and detect fluorescence signals, the primer set and kit provided in this disclosure can detect up to three marker sites for each pathogen, making the detection more efficient and accurate. Furthermore, it can detect sequence variations and distinguish pathogen types, and can be used for efficient identification, genetic variation monitoring, database construction, and type differentiation of multiple pathogens in wastewater. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below.

[0018] Example 1

[0019] This disclosure provides an MNP marker site for identifying multiple pathogens in wastewater. The MNP marker site is used to identify multiple pathogens in wastewater. 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 Table 1.

[0020] This disclosure also provides a primer set for identifying multiple pathogens in wastewater, comprising at least one of primer pairs 1 to 19, the 19 primer pairs being used to detect 19 MNP marker sites. Each primer pair includes a forward primer and a reverse primer. The forward primer of primer pair 1, the reverse primer of primer pair 1, the forward primer of primer pair 1, and the reverse primer of primer pair 19 are shown sequentially as SEQ ID NO: 1 to SEQ ID NO: 38 in the sequence listing. Specific sequences are shown in Table 1.

[0021] Table 1 shows the primer sequence and the location of the corresponding MNP marker sites.

[0022]

[0023]

[0024] Example 2

[0025] This disclosure provides a kit for identifying multiple pathogens in wastewater, the kit comprising the primer set provided in Example 1.

[0026] Example 3

[0027] This disclosure provides an application of the above-described primer set or kit, which includes using the primer set to identify multiple pathogens in wastewater, including viruses, bacteria, fungi, and specific pathogens.

[0028] Specifically, the viruses include: adenovirus type F, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus type 71, coxsackievirus, monkeypox virus, and human rhinovirus.

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

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

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

[0032] Specifically, special pathogens include Chlamydia pneumoniae.

[0033] In this embodiment, primer sets were used to detect positive samples of 15 target pathogens. These 15 target pathogens include: adenovirus F, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, Coxsackievirus, monkeypoxvirus, human rhinovirus, Haemophilus influenzae, Salmonella enterica, Pseudomonas aeruginosa, Histoplasma capsulatum, Cryptococcus neoformans, and Chlamydia pneumoniae. Specifically, the positive samples of the 15 target pathogens were nucleic acids from pathogen isolates that were identified positive by digital PCR and whose copy numbers were quantified. The nucleic acids of the above 15 target pathogens were mixed to form a simulated multipathogen pooled sample in wastewater (hereinafter referred to as the simulated pooled sample). This simulated pooled sample was prepared into three different copy gradients: 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction for each pathogen, respectively named the 1 copy / reaction sample, the 10 copy / reaction sample, and the 100 copy / reaction sample. The kit provided in Example 2 was used to detect three simulated mixed samples. The MNP marker detection method in this example is as follows: Total nucleic acids from the simulated mixed samples were reverse transcribed. Reverse transcription can be performed using a commercially available reverse transcription kit; the procedure is described in the kit's instruction manual. In this example, the 20 μL reverse transcription system included: 10 μL total nucleic acids, 5 μL 4×HiScript IV RT Mix, and 5 μL Nuclease-free ddH2O (sterile, enzyme-free water). The cDNA synthesis reaction program was: 55℃ for 15 min, 85℃ for 5 sec.

[0034] The total nucleic acids in the simulated mixed sample include DNA and RNA. The reverse transcription mainly targets the RNA portion to obtain a mixed template of cDNA and DNA from the simulated mixed sample. The mixed template is then subjected to a first multiplex PCR amplification using the primer set provided in Example 1 to obtain the first multiplex amplification product. The amplification system for each 30 μL of the first multiplex PCR amplification includes: 4 μL cDNA, 4 μL primer set, 4 μL E1 Mix and 6 μL 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 amplified by a second multiplex PCR using the primer set provided in Example 1 to obtain the second multiplex amplification product. The amplification system of each 30 μL second multiplex PCR amplification included: 4 μL first multiplex amplification product, 4 μL primer set, 10 μL E1 Mix and 16 μL Nuclease-free ddH2O. The amplification program of the first multiplex PCR amplification is shown in Table 3.

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

[0039]

[0040]

[0041] The purified products from the second multiplex amplification were used to construct libraries. Sequencing was performed on these libraries to obtain sequencing data. An equal volume of sterile water was used as a blank control, named 0 copies / reaction. Three replicate libraries were constructed per sample per day for three consecutive days, resulting in nine sets of sequencing data per sample. The specific results are shown in Table 4.

[0042] Table 4. Results evaluation for each simulated mixed sample

[0043]

[0044] Table 4 summarizes the number of MNP marker sites aligned to detected sequences in samples with different copy numbers, the number of pathogens covered, and the normalized number of sequences aligned to MNP marker sites of pathogens. The normalized number of sequences refers to the number of sequences aligned to pathogen MNP marker sites per 100,000 sequences. In the data in Table 4, there are instances where the number of detected markers exceeds the number of pathogens covered. This is because this embodiment detects multiple markers for some pathogens to ensure true positives. When multiple markers are detected for a single pathogen, the number of detected markers will exceed the number of pathogens covered.

[0045] The stability and sensitivity of this primer set for detecting multiple pathogens in wastewater were evaluated.

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

[0047] The primer set was used to assess the reproducibility and accuracy of multiple pathogens in wastewater.

[0048] The reproducibility and accuracy of the MNP marker detection method for detecting pathogens in wastewater were evaluated based on whether the genotypes of common detection sites could be reproduced in two replicates. Specifically, pairwise comparisons were performed on the nine groups of data with 100 copies / reaction in Table 4, and the results are shown in Table 5.

[0049] Table 5 shows the reproducibility and accuracy assessment of primer sets for detecting pathogen genotypes in wastewater.

[0050] Repeat 1 Repeat 2 Number of common sites Number of repeatable sites Recurrence rate r Accuracy a 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 loci with different major genotypes was 0. Based on the principle that reproducible genotypes between two replicates are considered accurate, the accuracy a = 1 - (1 - r) / 2 = 0.5 + 0.5r, where r represents the reproducibility rate, i.e., the ratio of reproducible major genotypes to the total number of shared loci. In the reproducibility test of this embodiment, the logarithm of the difference in major genotypes of MNP markers between different libraries and between different library preparation batches for each sample was 0, the reproducibility rate r = 100%, and the accuracy a = 100%.

[0052] The specificity of this primer set for pathogens in wastewater was assessed.

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

[0054] The primer set has a threshold for detecting multiple pathogens in wastewater.

[0055] Table 4 shows that most samples with one copy / reaction could detect sequences of pathogens that had aligned to wastewater. Pathogen sequences from wastewater were also detected in some blank controls. Because the MNP marker detection method is extremely sensitive, false positives are easily generated due to data contamination during the detection process. Therefore, in this embodiment, quality control was performed on the test results of simulated samples with different copy numbers over three consecutive days. The quality control plan is as follows:

[0056] 1) The sequencing data volume should be no less than 30 megabases. The calculation is based on the fact that the maximum number of MNP sites detected per sample is 19, and the length of a sequencing fragment is 300 bases. Therefore, when the data volume is greater than 30 megabases, most samples can ensure that the number of sequencing fragments covering each MNP marker site reaches more than 5000 times in one experiment, ensuring accurate analysis of the base sequence of each MNP marker site.

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

[0058] Based on the sequencing data of the samples detected 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 criterion for determining the positivity of the primer set for pathogens in sewage provided in this embodiment 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 wastewater

[0060] The primer set was used to detect pathogens in 25 wastewater samples. The sample numbers are shown in Table 6. Three negative controls using sterile water as a template were also constructed. Nucleic acid was prepared from each sample using a commercially available pathogen total nucleic acid extraction kit. The prepared pathogen total nucleic acid was then detected using traditional qPCR. All 25 wastewater samples were positive for adenovirus type F. Adenovirus type F is a diarrhea-causing adenovirus and a common virus found in wastewater. Simultaneously, a commercially available reverse transcription kit was used to reverse transcribe the prepared pathogen total nucleic acid from the wastewater samples, obtaining a mixed template of cDNA and DNA. The primer set provided in Example 1 was used to perform multiplex PCR amplification on the mixed template of cDNA and DNA from the wastewater samples. The multiplex amplification products were used to construct a library, which was then sequenced to obtain sequencing data. The sequencing data showed that adenovirus type F pathogen was detected in all 25 wastewater samples. The specific detection results of the 25 positive samples are shown in Table 6.

[0061] Table 6 shows the normalized sequence counts (unit: sequences) detected in 25 positive samples using the primer set.

[0062]

[0063]

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

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

[0066]

[0067]

[0068] As shown in Tables 6 and 7, in addition to adenovirus type F, 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 in the 25 samples, which demonstrates the advantage of this primer set in terms of comprehensive detection.

[0069] As shown in Tables 6 and 7, using the primer set provided in Example 1 of this invention, three MNP markers of the Enterovirus genus were detected in three samples, specifically one Enterovirus 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 indicates that the primer set has the advantage of differentiating pathogens by detecting multiple markers for each pathogen.

[0070] Example 4

[0071] Application of any one or more primer pairs from this primer set in the detection of pathogens in wastewater.

[0072] The primer set provided by this invention, comprising 19 primer pairs, can achieve the detection of up to 15 target pathogens. For applications focusing only on a subset of these 15 pathogens, primer combinations targeting specific pathogens can be used. For example, primer pairs 1 through 13 of the 19 primer pairs can identify 9 viral classes from 15 pathogens found in wastewater. In this embodiment, primer pairs 1 through 13 were used to re-detect pathogens in 25 wastewater samples from Example 3, while three negative controls were constructed using sterile water as a template. The target pathogens were detected in the 25 samples, which were considered positive samples. The detection 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: sequences).

[0074]

[0075]

[0076] According to the above-mentioned judgment threshold, the detection results are shown in Table 8. As can be seen from Table 8, all viral pathogens in the test samples can be detected by primer pairs 1 to 13. In Example 3, the test results of samples in which no viral pathogens were detected were all negative. This proves that any pair of primers or the combination of multiple pairs of primers in this primer set has high effectiveness in the detection of pathogens in sewage.

[0077] Example 5

[0078] This application includes using MNP marker sites, primer sets, or kits for monitoring genetic variations in pathogen strains in wastewater.

[0079] Table 6 shows that adenovirus type F was detected in all 25 positive samples. Comparison of the major genotypes at the MNP marker sites of adenovirus type F in each positive sample revealed that most of the 25 positive samples contained multiple genotypes. Since the wastewater samples were collected from communities, hospitals, etc., the pathogens within them originated from multiple sources. When this mixed population is detected using sequencing-based techniques, it manifests as multiple alleles at a single marker site. For example, the 53,133 normalized sequences detected in sample S240208, when compared with the sequencing sequences of the adenovirus type F MNP marker, represented two different genotypes, with 30,243 and 22,890 sequences respectively, indicating internal variation within the adenovirus type F in the same sample. Therefore, this primer set is suitable for detecting genetic variation within wastewater pathogen populations.

[0080] Comparing the genotypes of each sample reveals genetic differences in the strains between them. For example, among the six community sewage samples starting with S (S240208, S240212, S240215, S240224, and S240304), each had two identical genotypes. However, sample S240219 had three genotypes: two shared with the previous five samples, and the third unique to that sample, suggesting a broader source of pathogens. These genotypic differences indicate that this primer set can be used for detecting pathogen genetic variation.

[0081] Example 6

[0082] Specifically, applications include using MNP marker sites, primer sets, or kits to construct databases of pathogens in wastewater.

[0083] As shown in Table 7, after using this primer set to detect the test samples, the major genotypes of the MNP marker sites for each pathogen in each test sample were obtained. The major genotypes of the same pathogen obtained from all samples were compared, and the major genotypes with differences were entered into a database file, forming an MNP fingerprint database of pathogens in wastewater. Each time the major genotype of the MNP marker sites of a test sample or strain is compared with the constructed MNP fingerprint database, the MNP fingerprint profiles of strains with different major genotypes can be entered into the constructed MNP fingerprint database. Therefore, theoretically, the constructed MNP fingerprint database can be continuously updated and enriched. Because the constructed database is based on the gene sequences of the detected strains, it is compatible with all high-throughput sequencing data and has the characteristics of being fully collaboratively built and shared, and updated at any time.

[0084] Example 7

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

[0086] Viral pathogens in wastewater are further classified into species, subspecies, and subtypes at the genus level. For example, the enterovirus genus provided in this embodiment can be specifically classified into enterovirus 71, coxsackievirus, and human rhinovirus. The primer set provided in this embodiment contains seven specific MNP markers for detecting enteroviruses. These seven specific MNP markers include one conserved marker for the enterovirus genus and two specific markers for each species (type). Based on the detection of conserved and specific markers, pathogens can be accurately identified and finely typed. As shown in Table 6, three enterovirus markers were detected in all three samples: one conserved marker for the enterovirus genus and two specific markers for human rhinovirus, thus identifying the enterovirus in the samples as human rhinovirus. Therefore, the primer set provided in this embodiment can be used for the typological differentiation of pathogens in wastewater, thereby enabling the monitoring of prevalent pathogen species (types) in wastewater.

[0087] The above description is merely an optional embodiment of this disclosure and is not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A primer set for identifying multiple pathogens in wastewater, characterized in that, The primer set includes primer pairs 1 to 19, each primer pair including 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 19th primer pair and the reverse primer of the 19th primer pair are shown in sequence as SEQ ID NO: 1 to SEQ ID NO: 38 in the sequence listing.

2. A reagent kit for identifying multiple pathogens in wastewater, characterized in that, The kit includes the primer set as described in claim 1.

3. The application of the primer set as described in claim 1 or the kit as described in claim 2, characterized in that, The application includes using the primer set or the kit to identify pathogens in wastewater, the types of which include viruses, bacteria, and fungi.

4. The application according to claim 3, characterized in that, The viruses include: adenovirus type F, coronavirus 229E, coronavirus HKU1, coronavirus NL63, coronavirus OC43, enterovirus 71, coxsackievirus, monkeypox virus, and human rhinovirus.

5. The application according to claim 3, characterized in that, The bacteria include: Haemophilus influenzae, Salmonella enterica, and Pseudomonas aeruginosa.

6. The application according to claim 3, characterized in that, The fungi include Histoplasma capsulatum and Cryptococcus neoformans.

7. The application according to claim 3, characterized in that, The types of pathogens found in the wastewater also include Chlamydia pneumoniae.

8. The application according to claim 3, characterized in that, The applications include using the primer set or the kit for database construction, genetic variation monitoring, and type differentiation of multiple pathogens in wastewater.

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