MNP marker site for identifying multiple pathogens in intestinal tract, primer set and application thereof

Abstract

The application provides a MNP marker site for identifying multiple pathogens in the intestinal tract, a primer set and application. The MNP marker site comprises MNP-1 to MNP-15, the primer set comprises the first primer pair to the 15th primer pair, and the nucleotide sequence is shown in SEQ ID NO:1 to SEQ ID NO:30. The primer set is amplified by multiplex PCR and combined with high-throughput sequencing, so that multiple intestinal pathogens such as adenovirus F type, group A rotavirus, sapovirus, Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhi, Klebsiella pneumoniae, Shigella flexneri, Shigella sonnei, Cryptosporidium tyzzi and Entamoeba histolytica can be detected simultaneously and synchronously in one time, and the primer set has the characteristics of high detection throughput, good sensitivity and the ability of identifying pathogen types.

Description

Technical Field

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

[0002] In the fields of public health and clinical diagnosis and treatment, early detection of enteric pathogens is of great significance for disease prevention and control. However, the types of pathogens in the gut are complex, encompassing various types such as viruses and bacteria, which poses a significant challenge to accurate diagnosis.

[0003] Currently, commonly used detection technologies mainly include PCR-based nucleic acid detection and metagenomic sequencing. PCR detection typically targets a single pathogen at a single site, resulting in low throughput and difficulty in capturing sequence variations or polymorphisms. Metagenomic sequencing, on the other hand, is prone to false negatives due to low viral nucleic acid content and easy genome degradation, making it particularly unsuitable for identifying viral pathogens. These limitations severely restrict the development of simultaneous detection technologies for multiple intestinal pathogens.

[0004] Therefore, there is an urgent need to develop a novel detection method that can efficiently, comprehensively, and simultaneously detect multiple pathogens in the gut. Multinucleotide polymorphism (MNP) labeling technology, with its advantages of multi-site parallel analysis, strong resistance to degradation, and ability to distinguish variants, provides a feasible solution to the above problems. Summary of the Invention

[0005] To address the problems of existing technologies, the present invention aims to provide an MNP marker site, primer set, and application for identifying multiple pathogens in the gut.

[0006] The specific technical solution is as follows:

[0007] On one hand, the present invention provides a combination of MNP marker sites for identifying multiple pathogens in the gut, the combination of MNP marker sites including MNP-1 to MNP-15, the positions of MNP-1 to MNP-15 on the reference sequence are shown in Table 1.

[0008] On the other hand, the present invention provides a primer set for identifying multiple pathogens in the gut, the primer set comprising primer pairs 1 to 15, each primer pair comprising a forward primer and a reverse primer, the nucleotide sequences of primer pairs 1 to 15 being shown in SEQ ID NO: 1 to SEQ ID NO: 30.

[0009] In another aspect, the present invention provides a kit for identifying multiple pathogens in the gut, the kit comprising the aforementioned primer set.

[0010] Furthermore, this invention provides applications of the aforementioned MNP marker sites, primer sets, or kits for identifying multiple pathogens in the gut. These applications, through multiplex PCR amplification combined with high-throughput sequencing, can simultaneously identify multiple pathogens in the gut in a single operation, including viruses, bacteria, and parasites.

[0011] Specifically, the viruses include: adenovirus type F, zaruvirus, and group A rotavirus.

[0012] Specifically, the bacteria include: Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhi, Klebsiella pneumoniae, Shigella flexneri, and Shigella sonnei.

[0013] Specifically, the parasites include Cryptosporidium taizeri and Entamoeba histolytica.

[0014] Specifically, the applications also include: using the MNP marker site combination, the primer set, or the kit for the construction of an intestinal pathogen MNP fingerprint database, genetic variation monitoring, and fine typing.

[0015] The beneficial effects of this invention are:

[0016] The MNP marker sites provided by this invention are species-specific markers obtained by screening the genome of target pathogens. These markers exhibit multiple nucleotide polymorphisms within a species, demonstrating high polymorphism and strong species-discriminating ability. Screening 1-3 MNP marker sites for each pathogen can achieve specific differentiation of multiple pathogens in the gut.

[0017] The primer set provided by this invention has good amplification compatibility and can perform ultramultiplex PCR amplification in a single reaction system, simultaneously amplifying multiple MNP marker sites. Combined with a high-throughput sequencing platform for sequence analysis, it enables simultaneous identification and type differentiation of multiple pathogens in the gut in a single reaction.

[0018] Compared with traditional fluorescent PCR methods (which typically detect only one marker site for a single pathogen in a single reaction and rely on fluorescence signal interpretation), the primer set and kit provided by this invention detect 1 to 3 marker sites for each pathogen, which not only improves detection throughput and accuracy, but also allows direct acquisition of sequence information of amplified products, thereby effectively identifying sequence variations of pathogens and distinguishing between types. It is suitable for efficient identification, genetic variation monitoring, database construction and fine typing of multiple pathogens in the intestine. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this disclosure clearer, the embodiments of this disclosure will be described in further detail below.

[0020] Example 1

[0021] This embodiment provides a combination of MNP marker sites for identifying multiple pathogens in the gut, comprising 15 MNP marker sites, named MNP-1 to MNP-15 sequentially. The position information of MNP-1 to MNP-15 on the reference sequence is shown in Table 1.

[0022] This embodiment also provides a primer set for detecting the MNP marker site combination, comprising primer pairs 1 to 15, totaling 15 primer pairs. Each primer pair consists of one forward primer and one reverse primer. The nucleotide sequences of primer pairs 1 to 15 are shown in SEQ ID NO: 1 to SEQ ID NO: 30 in the sequence listing, respectively. The sequences of each primer pair and their corresponding MNP marker sites are detailed in Table 1.

[0023] Table 1 shows the primer sequence and the location of the corresponding MNP marker sites.

[0024]

[0025] Example 2

[0026] This embodiment provides a kit for identifying multiple pathogens in the gut, which contains the primer set described in the embodiment.

[0027] Example 3

[0028] This embodiment provides an application of the above-described primer set or kit in identifying multiple pathogens in the gut. These multiple pathogens include viruses, bacteria, and parasites.

[0029] Specifically, the viruses include: adenovirus type F, zaruvirus, and group A rotavirus.

[0030] Specifically, the bacteria include: Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhi, Klebsiella pneumoniae, Shigella flexneri, and Shigella sonnei.

[0031] Specifically, the parasites include Cryptosporidium taizeri and Entamoeba histolytica.

[0032] In this embodiment, the primer set was used to detect positive samples of 11 target pathogens. The target pathogens include: adenovirus type F, group A rotavirus, zarjor virus, Vibrio cholerae, Vibrio parahaemolyticus, Salmonella typhi, Klebsiella pneumoniae, Shigella flexneri, Shigella sonnei, Cryptosporidium taizeri, and Entamoeba histolytica. The positive samples of the target pathogens were nucleic acids from pathogen isolates and cultures that had been identified as positive by digital PCR and whose copy numbers had been quantified.

[0033] The nucleic acids of the aforementioned target pathogens were mixed in equal amounts to form a simulated multipathogen pooled sample (hereinafter referred to as the simulated pooled sample). The simulated pooled sample was diluted to three copy gradients so that the copy number of each pathogen was 1 copy / reaction, 10 copies / reaction, and 100 copies / reaction, respectively, and was named 1 copy / reaction sample, 10 copy / reaction sample, and 100 copy / reaction sample.

[0034] The kit described in Example 2 was used to detect three simulated mixed samples. The detection method for MNP markers is as follows:

[0035] First, the total nucleic acids in the simulated mixed sample were reverse transcribed to convert the RNA component into cDNA. Reverse transcription was performed using a commercial reverse transcription kit, and the procedure was followed according to the kit's instructions. In this example, the 20 μL reverse transcription system consisted of: 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, and storage at 10℃. This step yielded a mixed template containing both cDNA and DNA.

[0036] Then, using the obtained mixed template as a template, a first multiplex PCR amplification was performed using the primer set described in Example 1 to obtain the first multiplex amplification product. The amplification system for the first multiplex PCR amplification included: 4 μL mixed template, 4 μL primer set (each forward and reverse primer had a concentration of 4 μM), 4 μL E1 Mix, and 6 μL Nuclease-free ddH2O. The amplification program for the first multiplex PCR amplification is shown in Table 2.

[0037] Table 2 shows the amplification procedure for the first multiplex PCR amplification.

[0038]

[0039] The obtained first multiplex amplification product was subjected to second multiplex PCR amplification using the primer set described in Example 1 to obtain the second multiplex amplification product. The amplification system for the second multiplex PCR amplification included: 4 μL of the first multiplex amplification product, 4 μL of the primer set (each forward and reverse primer had a concentration of 4 μM), 10 μL of E1 Mix, and 16 μL of Nuclease-free ddH2O. The amplification program for the second multiplex PCR amplification is shown in Table 3.

[0040] Table 3 shows the amplification procedure for the second multiplex PCR amplification.

[0041]

[0042] After purification of the second multiplex amplification product, sequencing libraries were constructed using standard methods. High-throughput sequencing was performed on the obtained libraries to acquire sequencing data. Simultaneously, a blank control was set up by replacing the template with an equal volume of sterile water, recorded as 0 copies / reaction. Three replicate libraries were constructed for each sample daily for three consecutive days, resulting in a total of nine sequencing data sets per sample. Specific detection results are shown in Table 4.

[0043] Table 4. Result evaluation for each simulated mixed sample

[0044]

[0045] Table 4 summarizes the number of MNP marker sites aligned to the detected sequences, 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. The normalized sequence number refers to the number of sequences aligned to pathogen MNP marker sites per 100,000 sequences. Table 4 shows instances where the number of detected markers exceeds the number of pathogens covered. This is because this embodiment designed multiple MNP marker sites for some pathogens to ensure true positive results. When multiple markers are detected for a single pathogen, the number of detected markers will exceed the number of pathogens covered.

[0046] Sensitivity assessment

[0047] As shown in Table 4, when the copy number reaches 10 copies / reaction, all 11 pathogens were detected in 9 repeated tests; however, when the copy number is as low as 1 copy / reaction, detection cannot be guaranteed. Therefore, the primer set described in this embodiment has a detection sensitivity of 10 copies / reaction for the target pathogen.

[0048] Reproducibility and accuracy assessment

[0049] The reproducibility and accuracy of the detection method in this embodiment were evaluated by comparing whether the genotypes of common detection sites were consistent in any two repeated detections under the same copy gradient. Specifically, pairwise comparisons were performed on the nine groups of sequencing data from the 100 copy / reaction samples in Table 4, and the results are shown in Table 5.

[0050] Table 5 shows the reproducibility and accuracy assessment of primer set detection of pathogen genotypes in the gut.

[0051]

[0052] As shown in Table 5, the number of MNP loci with different major genotypes between any two replicate tests of 100 copies / reaction samples was 0. In this embodiment, the reproducibility of the major genotype between two replicate experiments was used as the criterion for accuracy. The reproducibility rate *r* was defined as the ratio of the number of reproducible major genotype loci to the number of shared loci, and the accuracy *a* = 1 - (1 - *r*) / 2 = 0.5 + 0.5 *r*. In this embodiment, the logarithm of the major genotype difference of MNP markers between different libraries and different library preparation batches was 0, the reproducibility rate *r* = 100%, and the corresponding accuracy *a* = 100%.

[0053] Specificity assessment

[0054] As shown in Table 4, in the three simulated mixed samples, when the pathogen copy number was ≥10 copies / reaction, the sequencing sequences, after alignment analysis, specifically matched the MNP marker sequences of the 11 target pathogens, and no cross-matches with non-target pathogens were found. This indicates that the primer set and detection method provided in this embodiment have high specificity for the detection of target pathogens in complex template backgrounds.

[0055] Detection thresholds for multiple pathogens in the gut using primer sets

[0056] Table 4 shows that in samples with a 1 copy / reaction ratio, most sequences aligned to enteric pathogens were detected. However, a small number of enteric pathogen sequences were also detected in some blank controls. This may be due to environmental aerosol contamination or sequencing index mismatch. Because the MNP marker detection method is extremely sensitive, false positives are easily caused by trace contamination during the detection process; therefore, strict quality control and judgment criteria must be established.

[0057] This embodiment performs quality control analysis on the test results of simulated samples with different copy numbers over three consecutive days. The quality control plan is as follows:

[0058] 1) The sequencing data volume should be no less than 30 Mb. The calculation is based on the following: a maximum of 15 MNP marker sites are detected per sample, and the length of a single sequencing fragment is 300 bp. When the data volume reaches 30 Mb, it can be guaranteed that the number of sequencing fragments for each MNP marker site covers a depth of more than 6600 times, meeting the needs for accurate analysis of the base sequence at each site.

[0059] 2) Calculate the signal-to-noise ratio (SNR) of each intestinal pathogen based on the sequence number S of each intestinal pathogen in the test sample and the sequence number N of the pathogen in the blank control.

[0060] Based on the sequencing data in Table 4, when the pathogen copy number is ≥10 copies / reaction, the SNR value of each pathogen is ≥10. Therefore, taking into account both accuracy and sensitivity, the primer set and detection method provided in this embodiment are used to determine the positivity of enteric pathogens as follows: when the signal-to-noise ratio (SNR) of a certain enteric pathogen in the sample is ≥10, the nucleic acid of the pathogen is detected in the sample.

[0061] Application of primer sets in the detection of multiple pathogens in the gut

[0062] Using the primer set provided by this invention, pathogen detection was performed on 20 intestinal samples (anal swabs). The sample numbers are shown in Table 6. Three negative controls using sterile water as a template were also included. Total nucleic acid was extracted from each sample using a commercially available pathogen total nucleic acid extraction kit.

[0063] As a methodological comparison, the extracted total nucleic acid of pathogens in the samples to be tested was first detected using the traditional qPCR method, with the detection targets being group A rotavirus and adenovirus type F. The results showed that among the 20 samples, 9 were positive for group A rotavirus, 3 were positive for adenovirus type F, and the remaining 8 were negative for both pathogens.

[0064] Subsequently, the primer set provided by this invention was used to detect the above 20 intestinal samples. Specifically, the extracted total nucleic acid of the pathogen was reverse transcribed using a commercial reverse transcription kit to obtain a mixed template containing cDNA and DNA. Following the method described in Example 3, first and second multiplex PCR amplifications were performed using the mixed template. The resulting multiplex amplification products were purified and used to construct a sequencing library, which was then subjected to high-throughput sequencing to obtain sequencing data. Simultaneously, three negative controls underwent the same procedures. The results showed that the target pathogen sequence was not detected in any of the negative controls. The detection results of the 20 samples are shown in Table 6.

[0065] Table 6 shows the normalized sequence counts (unit: sequences) detected in 20 samples using the primer set.

[0066]

[0067] Statistical analysis of the sequencing data showed that, among the 20 intestinal samples tested, 9 samples positive for group A rotavirus and 3 samples positive for adenovirus type F pathogen, identified by qPCR, were accurately detected using the primer set provided in this embodiment, with a positive concordance rate of 100%. The remaining 8 samples that tested negative by qPCR also did not detect these two pathogens using this method, with a negative concordance rate of 100%. These results indicate that the primer set provided in this embodiment is completely consistent with the qPCR method in detecting group A rotavirus and adenovirus type F.

[0068] In addition, the primer set provided in this embodiment also detected multiple other intestinal pathogens in 20 samples. The pathogen detection results of the 20 real intestinal samples are shown in Table 7.

[0069] Table 7 shows the statistics of pathogens detected in 20 real intestinal samples.

[0070]

[0071] As shown in Tables 6 and 7, in the 20 samples, in addition to group A rotavirus and adenovirus type F, the primer set provided in Example 1 also detected Klebsiella pneumoniae, Shigella flexneri, Cryptosporidium tizenii, Shigella sonnei, Zaju virus, Vibrio parahaemolyticus, and Entamoeba histolytica. This demonstrates the advantage of this primer set in terms of comprehensive detection.

[0072] Example 4

[0073] This embodiment provides the application of the MNP marker site, primer set, or kit in monitoring genetic variations of pathogen strains in the gut.

[0074] Using the primer set, 20 intestinal samples were tested, and the major genotypes of the MNP marker sites of the pathogens in each sample were obtained. Table 6 shows that adenovirus type F was detected in 3 samples (S18, S19, and S20). Comparative analysis of the major genotypes of the MNP marker sites of adenovirus type F in these 3 samples revealed that the adenovirus type F in samples S18 and S20 had the same genotype, while the genotype of adenovirus type F in sample S19 differed from both. Therefore, the primer set can effectively distinguish genotype differences of the same pathogen in different samples and can be used for monitoring genetic variation of pathogens.

[0075] Example 5

[0076] This embodiment provides the application of the MNP marker sites, primer sets, or kits in constructing a database of pathogens in the gut.

[0077] As shown in Table 7, after testing the samples using this primer set, the major genotypes of each MNP marker locus for each pathogen in each sample were obtained. The major genotypes of the same pathogen in different samples were compared, and the major genotypes with discrepancies were entered into the database, thus forming the MNP fingerprint database for that pathogen. In subsequent tests, if the major genotype of the MNP marker locus of a new sample or strain differs from the existing records in the database, its fingerprint pattern can also be added to the database. Therefore, the MNP fingerprint database can be continuously updated and enriched.

[0078] Because this database is built based on the actual gene sequences of pathogens and is compatible with all high-throughput sequencing data, it has the characteristics of being co-built, shareable, and updated in real time.

[0079] Example 6

[0080] This embodiment provides the application of the MNP marker sites, primer sets, or kits in the fine typing of intestinal pathogens.

[0081] Viral pathogens, a type of enteric pathogen, can be further classified into species, subspecies, and subtypes. For example, rotaviruses are known to have 10 types (A, J), with type A being the predominant type infecting humans. The primer set provided in this embodiment features three specific MNP marker sites designed for group A rotaviruses. By detecting these specific markers, accurate identification and fine typing of the pathogen can be achieved. As shown in Table 6, the three specific markers for group A rotaviruses were detected in all nine samples, thus accurately identifying the rotaviruses in these samples as type A. Therefore, the primer set provided in this embodiment can effectively distinguish between different types of enteric pathogens and can be used for monitoring the prevalence of enteric pathogen types.

[0082] 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 the gut, characterized in that: The primer set includes primer pairs 1 to 15, each primer pair including a forward primer and a reverse primer; the nucleotide sequences of primer pairs 1 to 15 are shown in SEQ ID NO: 1 to SEQ ID NO:

30.

2. A kit for identifying multiple pathogens in the gut, characterized in that: It includes the primer set as described in claim 1.

3. The use of a primer set as described in claim 1 or a kit as described in claim 2 in the preparation of a product for identifying multiple pathogens in the gut, characterized in that: The pathogens include viruses, bacteria, and parasites.

4. The application according to claim 3, characterized in that: The viruses include: adenovirus type F, zaruvirus, and group A rotavirus.

5. The application according to claim 3, characterized in that: The bacteria include: Vibrio parahaemolyticus, Vibrio cholerae, Salmonella typhi, Klebsiella pneumoniae, Shigella flexneri, and Shigella sonnei.

6. The application according to claim 3, characterized in that: The parasites include Cryptosporidium taizeri and Entamoeba histolytica.

7. The application according to claim 3, characterized in that: The product is used to construct an MNP fingerprint database of intestinal pathogens, monitor genetic variations of intestinal pathogens, and perform fine typing of intestinal pathogens.

8. A method for identifying multiple pathogens for non-disease diagnostic purposes, characterized in that: Includes the following steps: (1) Extract total nucleic acid from the sample to be tested; (2) The total nucleic acid was reverse transcribed to obtain a mixed template containing cDNA and DNA; (3) Using the mixed template as a template, the primer set described in claim 1 is used to perform a first multiplex PCR amplification to obtain a first multiplex amplification product; (4) Using the first multiplex amplification product as a template, a second multiplex PCR amplification is performed using the primer set described in claim 1 to obtain the second multiplex amplification product; (5) Construct a sequencing library using the second multiplex amplification product and perform high-throughput sequencing; (6) Analyze the sequencing data to identify the types of pathogens in the sample to be tested.

9. The method according to claim 8, characterized in that: The amplification program for the first multiplex PCR amplification in step (3) is as follows: heat at 105℃; 25℃ for 10 min; 95℃ for 3 min; 95℃ for 20 s, 60℃ for 4 min, 20 cycles; 72℃ for 4 min; store at 10℃.

10. The method according to claim 8, characterized in that: The amplification program for the second multiplex PCR amplification in step (4) is as follows: open the hot lid at 105℃; 95℃ for 3 min; 95℃ for 15 s, 58℃ for 15 s, 70℃ for 30 s, 10 cycles; 72℃ for 5 min; store at 10℃.

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