Primer set for detecting intestinal infection-associated pathogens and its applications

Abstract

This invention discloses a primer set for detecting intestinal infection-related pathogens and its applications. The primer set includes amplification primers for 70 intestinal infection-related pathogens. Using the primer set of this invention at specific working concentrations, combined with high-throughput sequencing technology, low concentrations of pathogens and mixed infections can be effectively detected. The detection is highly sensitive and specific, and exhibits strong resistance to endogenous and exogenous interference. This invention can identify and type 70 pathogens in a single test sample, significantly shortening the testing cycle, improving testing efficiency, and reducing the pain and cost of multiple sampling tests for patients. Furthermore, a single run can test hundreds of samples, providing high throughput and effectively meeting the needs of large-scale sample testing in medical institutions, disease control centers, and other scenarios, thus providing technical support for large-scale screening and prevention of intestinal infections.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically to the field of high-throughput sequencing and gene detection technology. More specifically, this invention relates to a primer set for simultaneously detecting 70 intestinal infection-related pathogens and its application. Background Technology

[0002] Diarrhea is a major global health problem and the second leading cause of death in children under five. Statistics show that approximately 1.7 billion cases of childhood diarrhea occur globally each year, with about 525,000 children under five dying from it. Gastrointestinal infections caused by bacteria, viruses, or parasites are the main causative agents of diarrhea, and its spread is often linked to contaminated food, drinking water, and poor sanitation. In my country, cholera is classified as a Class A infectious disease, typhoid and paratyphoid fever as Class B infectious diseases, and infectious diarrhea other than cholera, bacterial and amoebic dysentery, typhoid and paratyphoid fever as Class C infectious diseases. This demonstrates the significant public health importance of controlling intestinal infection-related diseases.

[0003] The most serious harm of infectious diarrhea is the dehydration and electrolyte imbalance it causes, which can be life-threatening if not addressed promptly. Therefore, rapid and accurate detection of gastrointestinal pathogens not only provides strong support for clinical diagnosis and helps patients receive targeted treatment, but also provides a basis for developing infection control and epidemiological prevention and control measures, thereby reducing disease transmission and lowering the risk of infection. Because intestinal infections caused by different pathogens have highly similar clinical manifestations, clinical presentation alone is insufficient to accurately distinguish the cause of infection; laboratory diagnostic methods are essential to identify the causative agent.

[0004] Currently, clinical laboratory diagnosis of gastrointestinal infections mainly relies on traditional techniques, including microscopic examination, pathogen culture, and immunoassay. Microscopic examination is simple and inexpensive, but its sensitivity is poor, making it difficult to detect low concentrations of pathogens. Pathogen culture cycles are lengthy (usually several days), failing to provide rapid clinical results and potentially delaying treatment. While immunoassay is rapid and convenient, its sensitivity and specificity are low, leading to false positives or false negatives.

[0005] Molecular biology detection methods offer significant advantages over traditional diagnostic methods, including speed, specificity, and sensitivity. However, conventional single / multiplex fluorescent PCR is limited by the number of fluorescence detection channels in the instrument, allowing a single tube to identify only one or a few enteric pathogens. This fails to meet the clinical needs for detecting complex intestinal infections (such as mixed infections with multiple pathogens or rare pathogen infections). When dealing with complex intestinal infection samples, directly increasing the number of primer pairs to increase the number of targets may result in the generation of numerous primer dimers. Furthermore, cross-complementary interference between primers can lead to amplification imbalances, low data validity, and missed detection of low-abundance pathogens, making it impossible to simultaneously amplify dozens of pathogens. Summary of the Invention

[0006] Based on this, the purpose of this invention is to provide a primer set that can be used to accurately and specifically detect 70 intestinal infection-related pathogens simultaneously.

[0007] The technical solutions for achieving the above-mentioned objectives include the following.

[0008] In a first aspect, the present invention provides a primer set comprising: primers for detecting BK polyomavirus with sequences as shown in SEQ ID NO:1~SEQ ID NO:2; primers for detecting cytomegalovirus with sequences as shown in SEQ ID NO:3~SEQ ID NO:4; primers for detecting human parvovirus B19 with sequences as shown in SEQ ID NO:5~SEQ ID NO:6; primers for detecting EB virus with sequences as shown in SEQ ID NO:7~SEQ ID NO:8; primers for detecting human adenovirus with sequences as shown in SEQ ID NO:9~SEQ ID NO:10; primers for detecting human adenovirus type 40 with sequences as shown in SEQ ID NO:11~SEQ ID NO:12; primers for detecting human adenovirus type 41 with sequences as shown in SEQ ID NO:13~SEQ ID NO:14; primers for detecting human adenovirus group F with sequences as shown in SEQ ID NO:15~SEQ ID NO:16; primers for detecting human herpesvirus type 6 with sequences as shown in SEQ ID NO:17~SEQ ID NO:18; and primers for detecting human herpesvirus type 6 with sequences as shown in SEQ ID NO:19~SEQ ID NO:10. Primers for detecting herpes simplex virus type 1 (SEQ ID NO:20); primers for detecting echovirus (SEQ ID NO:21-SEQ ID NO:22); primers for detecting enterovirus (SEQ ID NO:23-SEQ ID NO:24); primers for detecting enterovirus (SEQ ID NO:25-SEQ ID NO:26); primers for detecting enterovirus A71 (SEQ ID NO:27-SEQ ID NO:28); primers for detecting coxsackievirus (SEQ ID NO:29-SEQ ID NO:30); primers for detecting human astrovirus (SEQ ID NO:31-SEQ ID NO:32); primers for detecting norovirus group GI (SEQ ID NO:31-SEQ ID NO:32); primers for detecting norovirus group GII (SEQ ID NO:33-SEQ ID NO:34); primers for detecting rotavirus type A (SEQ ID NO:35-SEQ ID NO:36); primers for detecting rotavirus type A (SEQ ID NO:37-SEQ ID NO:28); primers for detecting human astrovirus (SEQ ID NO:29-SEQ ID NO:30); primers for detecting norovirus group GI (SEQ ID NO:31-SEQ ID NO:32); primers for detecting norovirus group GII (SEQ ID NO:33-SEQ ID NO:34); primers for detecting rotavirus type A (SEQ ID NO:35-SEQ ID NO:36); primers for detecting human astrovirus (SEQ ID NO:37-SEQ ID NO:28); primers for detecting human astrovirus (SEQ ID NO:29-SEQ ID NO:30 ... Primers for detecting rotavirus type B as shown in NO:38; primers for detecting rotavirus type C as shown in SEQ ID NO:39~SEQ ID NO:40; primers for detecting Sapporo virus genotype GI as shown in SEQ ID NO:41~SEQ ID NO:42; primers for detecting Sapporo virus genotype GII as shown in SEQ ID NO:43~SEQ ID NO:44;Primers for detecting Bacillus cereus with sequences as shown in SEQ ID NO:45~SEQ ID NO:46; primers for detecting the tcdA gene of Clostridium difficile enterotoxin with sequences as shown in SEQ ID NO:47~SEQ ID NO:48; primers for detecting the tcdB gene of Clostridium difficile cytotoxin with sequences as shown in SEQ ID NO:49~SEQ ID NO:50; primers for detecting Clostridium perfringens with sequences as shown in SEQ ID NO:51~SEQ ID NO:52; primers for detecting Clostridium neonate with sequences as shown in SEQ ID NO:53~SEQ ID NO:54; primers for detecting Listeria monocytogenes with sequences as shown in SEQ ID NO:55~SEQ ID NO:56; primers for detecting Staphylococcus aureus with sequences as shown in SEQ ID NO:57~SEQ ID NO:58; primers for detecting Staphylococcus aureus enterotoxin A with sequences as shown in SEQ ID NO:59~SEQ ID NO:60; primers for detecting Clostridium difficile enterotoxin A with sequences as shown in SEQ ID NO:61~SEQ ID NO:58. Primers for detecting Staphylococcus aureus enterotoxin B as shown in NO:62; primers for detecting Staphylococcus aureus enterotoxin C as shown in SEQ ID NO:63~SEQ ID NO:64; primers for detecting Staphylococcus aureus enterotoxin D as shown in SEQ ID NO:65~SEQ ID NO:66; primers for detecting Staphylococcus aureus enterotoxin E as shown in SEQ ID NO:67~SEQ ID NO:68; primers for detecting Aeromonas hydrophila as shown in SEQ ID NO:69~SEQ ID NO:70; primers for detecting Campylobacter coli as shown in SEQ ID NO:71~SEQ ID NO:72; primers for detecting Campylobacter jejuni as shown in SEQ ID NO:73~SEQ ID NO:74; primers for detecting Cronobacter sakazakii as shown in SEQ ID NO:75~SEQ ID NO:76; primers for detecting Shiga toxin-producing Escherichia coli as shown in SEQ ID NO:77~SEQ ID NO:78; primers for detecting Shiga toxin-producing Escherichia coli as shown in SEQ ID NO:62; primers for detecting Campylobacter coli B as shown in SEQ ID NO:63~SEQ ID NO:64; primers for detecting Campylobacter jejuni as shown in SEQ ID NO:75~SEQ ID NO:76; primers for detecting Shiga toxin-producing Escherichia coli as shown in SEQ ID NO:77~SEQ ID NO:78; primers for detecting Campylobacter coli B as shown in SEQ ID NO:62; primers for detecting Campylobacter coli B as shown in SEQ ID NO:64; primers for detecting Campylobacter jejuni as shown in SEQ ID NO:75~SEQ ID NO:76; primers for detecting Cronobacter sakazakii as shown in SEQ ID NO:75~SEQ Primers for detecting enterotoxigenic Escherichia coli as shown in NO:79~SEQ ID NO:80; primers for detecting enterohemorrhagic Escherichia coli as shown in SEQ ID NO:81~SEQ ID NO:82; primers for detecting enteroaggregative Escherichia coli as shown in SEQ ID NO:83~SEQ ID NO:84; primers for detecting enteroinvasive Escherichia coli as shown in SEQ ID NO:85~SEQ ID NO:86.Primers for detecting enteropathogenic Escherichia coli with sequences as shown in SEQ ID NO:87~SEQ ID NO:88; primers for detecting diffusely adhesive Escherichia coli with sequences as shown in SEQ ID NO:89~SEQ ID NO:90; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:91~SEQ ID NO:92; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:93~SEQ ID NO:94; primers for detecting Salmonella spp. with sequences as shown in SEQ ID NO:95~SEQ ID NO:96; primers for detecting Salmonella typhi with sequences as shown in SEQ ID NO:97~SEQ ID NO:98; primers for detecting Salmonella paratyphi A with sequences as shown in SEQ ID NO:99~SEQ ID NO:100; primers for detecting Salmonella paratyphi B with sequences as shown in SEQ ID NO:101~SEQ ID NO:102; primers for detecting Salmonella paratyphi B with sequences as shown in SEQ ID NO:103~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:103~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:87~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:103~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:104; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:105~SEQ ID NO:96; primers for detecting Salmonella spp. with sequences as shown in SEQ ID NO:105~SEQ ID NO:96; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:105~SEQ ID NO:98; primers for detecting Shigella Primers for detecting Salmonella paratyphi C as shown in NO:104; primers for detecting non-typhoid Salmonella as shown in SEQ ID NO:105~SEQ ID NO:106; primers for detecting Salmonella enteritidis as shown in SEQ ID NO:107~SEQ ID NO:108; primers for detecting Salmonella typhimurium as shown in SEQ ID NO:109~SEQ ID NO:110; primers for detecting Vibrio vulnificus as shown in SEQ ID NO:111~SEQ ID NO:112; primers for detecting Vibrio parahaemolyticus as shown in SEQ ID NO:113~SEQ ID NO:114; primers for detecting Vibrio mimicus as shown in SEQ ID NO:115~SEQ ID NO:116; primers for detecting Vibrio cholerae as shown in SEQ ID NO:117~SEQ ID NO:118; primers for detecting Vibrio cholerae as shown in SEQ ID NO:119~SEQ ID NO:110. Primers for detecting Yersinia enterocolitica (NO:120); primers for detecting Aegyptiformis duodenalis (SEQ ID NO:121~SEQ ID NO:122); primers for detecting Clonorchis sinensis (SEQ ID NO:123~SEQ ID NO:124); primers for detecting Clonorchis sinensis (SEQ ID NO:125~SEQ ID NO:126); primers for detecting Cryptosporidium (SEQ ID NO:127~SEQ ID NO:128); primers for detecting Entamoeba histolytica (SEQ ID NO:129~SEQ ID NO:130).Primers for detecting Entamoeba histolytica with sequences shown in SEQ ID NO:131~SEQ ID NO:132; primers for detecting Intestinal worms with sequences shown in SEQ ID NO:133~SEQ ID NO:134; primers for detecting Fasciolopsis buski with sequences shown in SEQ ID NO:135~SEQ ID NO:136; primers for detecting Balantidium coli with sequences shown in SEQ ID NO:137~SEQ ID NO:138; and primers for detecting Strongyloides stercoralis with sequences shown in SEQ ID NO:139~SEQ ID NO:140.

[0009] A second aspect of the present invention provides the application of the above-described primer set in the preparation of products for detecting intestinal infection-related pathogens.

[0010] A third aspect of the present invention provides a kit for detecting intestinal infection-related pathogens, comprising the aforementioned primer set.

[0011] The inventors of this invention designed specific amplification primers targeting 70 pathogenic microorganisms associated with intestinal infections (covering five major categories of microorganisms: DNA viruses, RNA viruses, Gram-positive pathogens, Gram-negative pathogens, and intestinal parasites). After extensive screening and optimization of primer sequences and concentrations, 140 primers (70 pairs) at specific working concentrations were obtained. These 140 primers can perform multiplex PCR reactions in a single tube, and the amplicon corresponding to each target can be amplified efficiently and specifically. Combined with high-throughput sequencing technology, the identification and typing of 70 pathogens in the sample (feces or anal swab) can be completed in a single test, greatly shortening the testing cycle (the entire testing process takes only about 12 hours), improving testing efficiency, and reducing the pain and cost of multiple sampling tests for patients. Moreover, hundreds of samples can be tested in a single run, with high throughput, which can well meet the needs of large-scale sample testing in medical institutions, disease control centers, and other scenarios, providing technical support for large-scale screening and prevention of intestinal infectious diseases.

[0012] In addition, the primer set with specific working concentrations of the present invention can effectively detect low concentrations of pathogens and mixed infection cases. Compared with traditional culture techniques, it has higher detection sensitivity (the lowest detection limit for viruses can reach 250 copies / mL, and the detection limit for bacteria can be as low as 1000 CFU / mL) and specificity, and has strong resistance to endogenous and exogenous interference. Attached Figure Description

[0013] Figure 1 This is a peak diagram showing the fragment length distribution of the detection library in Embodiment 2 of the present invention. Detailed Implementation

[0014] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0015] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0016] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in Green and Sambrook et al., *Molecular Cloning: A Laboratory Manual* (2013), or as recommended by the manufacturer. All commonly used chemical reagents used in the examples are commercially available products.

[0017] In this invention, specific amplification primers were designed based on 70 pathogen targets related to intestinal infections (comprehensively covering the vast majority of intestinal infection-related pathogens currently used in clinical practice). By optimizing primer sequences and concentrations, the optimal amplification conditions for all primers were made consistent, ensuring efficient amplification of the amplicons corresponding to each target in a single amplification system. Simultaneously, mutual interference between primers was minimized, reducing non-specific amplification. Ultimately, a total of 142 primers (71 pairs) were used for multiplex PCR reactions in one tube. Combining multiplex PCR amplification and enrichment technology with targeted next-generation sequencing (tNGS) technology, the amplified pathogen nucleic acids were sequenced and analyzed using bioinformatics, further improving the accuracy and specificity of detection. The primer set of this invention enables efficient and specific detection of 70 pathogen nucleic acids in samples (feces or anal swabs). The entire detection process (from nucleic acid extraction to the issuance of test results) takes only about 12 hours. It has the advantages of wide detection range, high efficiency, low cost and high throughput. It is particularly suitable for people with intestinal infection and diarrhea, especially for the investigation of the cause of diarrhea in children. It can effectively avoid the cumbersome problem of multiple sampling and testing for multiple pathogens in traditional tests, and provide strong support for rapid clinical diagnosis and precise treatment.

[0018] In some embodiments of the present invention, a primer set is disclosed, comprising: primers for detecting BK polyomavirus with sequences as shown in SEQ ID NO:1~SEQ ID NO:2; primers for detecting cytomegalovirus with sequences as shown in SEQ ID NO:3~SEQ ID NO:4; primers for detecting human parvovirus B19 with sequences as shown in SEQ ID NO:5~SEQ ID NO:6; primers for detecting EB virus with sequences as shown in SEQ ID NO:7~SEQ ID NO:8; primers for detecting human adenovirus with sequences as shown in SEQ ID NO:9~SEQ ID NO:10; primers for detecting human adenovirus type 40 with sequences as shown in SEQ ID NO:11~SEQ ID NO:12; primers for detecting human adenovirus type 41 with sequences as shown in SEQ ID NO:13~SEQ ID NO:14; primers for detecting human adenovirus group F with sequences as shown in SEQ ID NO:15~SEQ ID NO:16; primers for detecting human herpesvirus type 6 with sequences as shown in SEQ ID NO:17~SEQ ID NO:18; and primers for detecting human herpesvirus type 6 with sequences as shown in SEQ ID NO:19~SEQ ID NO:10. Primers for detecting herpes simplex virus type 1 (SEQ ID NO:20); primers for detecting echovirus (SEQ ID NO:21-SEQ ID NO:22); primers for detecting enterovirus (SEQ ID NO:23-SEQ ID NO:24); primers for detecting enterovirus (SEQ ID NO:25-SEQ ID NO:26); primers for detecting enterovirus A71 (SEQ ID NO:27-SEQ ID NO:28); primers for detecting coxsackievirus (SEQ ID NO:29-SEQ ID NO:30); primers for detecting human astrovirus (SEQ ID NO:31-SEQ ID NO:32); primers for detecting norovirus group GI (SEQ ID NO:31-SEQ ID NO:32); primers for detecting norovirus group GII (SEQ ID NO:33-SEQ ID NO:34); primers for detecting rotavirus type A (SEQ ID NO:35-SEQ ID NO:36); primers for detecting rotavirus type A (SEQ ID NO:37-SEQ ID NO:28); primers for detecting human astrovirus (SEQ ID NO:29-SEQ ID NO:30); primers for detecting norovirus group GI (SEQ ID NO:31-SEQ ID NO:32); primers for detecting norovirus group GII (SEQ ID NO:33-SEQ ID NO:34); primers for detecting rotavirus type A (SEQ ID NO:35-SEQ ID NO:36); primers for detecting human astrovirus (SEQ ID NO:37-SEQ ID NO:28); primers for detecting human astrovirus (SEQ ID NO:29-SEQ ID NO:30 ... Primers for detecting rotavirus type B as shown in NO:38; primers for detecting rotavirus type C as shown in SEQ ID NO:39~SEQ ID NO:40; primers for detecting Sapporo virus genotype GI as shown in SEQ ID NO:41~SEQ ID NO:42; primers for detecting Sapporo virus genotype GII as shown in SEQ ID NO:43~SEQ ID NO:44;Primers for detecting Bacillus cereus with sequences as shown in SEQ ID NO:45~SEQ ID NO:46; primers for detecting the tcdA gene of Clostridium difficile enterotoxin with sequences as shown in SEQ ID NO:47~SEQ ID NO:48; primers for detecting the tcdB gene of Clostridium difficile cytotoxin with sequences as shown in SEQ ID NO:49~SEQ ID NO:50; primers for detecting Clostridium perfringens with sequences as shown in SEQ ID NO:51~SEQ ID NO:52; primers for detecting Clostridium neonate with sequences as shown in SEQ ID NO:53~SEQ ID NO:54; primers for detecting Listeria monocytogenes with sequences as shown in SEQ ID NO:55~SEQ ID NO:56; primers for detecting Staphylococcus aureus with sequences as shown in SEQ ID NO:57~SEQ ID NO:58; primers for detecting Staphylococcus aureus enterotoxin A with sequences as shown in SEQ ID NO:59~SEQ ID NO:60; primers for detecting Clostridium difficile enterotoxin A with sequences as shown in SEQ ID NO:61~SEQ ID NO:58. Primers for detecting Staphylococcus aureus enterotoxin B as shown in NO:62; primers for detecting Staphylococcus aureus enterotoxin C as shown in SEQ ID NO:63~SEQ ID NO:64; primers for detecting Staphylococcus aureus enterotoxin D as shown in SEQ ID NO:65~SEQ ID NO:66; primers for detecting Staphylococcus aureus enterotoxin E as shown in SEQ ID NO:67~SEQ ID NO:68; primers for detecting Aeromonas hydrophila as shown in SEQ ID NO:69~SEQ ID NO:70; primers for detecting Campylobacter coli as shown in SEQ ID NO:71~SEQ ID NO:72; primers for detecting Campylobacter jejuni as shown in SEQ ID NO:73~SEQ ID NO:74; primers for detecting Cronobacter sakazakii as shown in SEQ ID NO:75~SEQ ID NO:76; primers for detecting Shiga toxin-producing Escherichia coli as shown in SEQ ID NO:77~SEQ ID NO:78; primers for detecting Shiga toxin-producing Escherichia coli as shown in SEQ ID NO:62; primers for detecting Campylobacter coli B as shown in SEQ ID NO:63~SEQ ID NO:64; primers for detecting Campylobacter jejuni as shown in SEQ ID NO:75~SEQ ID NO:76; primers for detecting Cronobacter sakazakii as shown in SEQ ID NO:77~SEQ ID NO:78; primers for detecting Shiga toxin-producing Escherichia coli as shown in SEQ ID NO:62; primers for detecting Campylobacter coli B as shown in SEQ ID NO:64; primers for detecting Campylobacter jejuni as shown in SEQ ID NO:75~SEQ ID NO:76; primers for detecting Shiga toxin-producing Escherichia coli as shown in SEQ ID NO: Primers for detecting enterotoxigenic Escherichia coli as shown in NO:79~SEQ ID NO:80; primers for detecting enterohemorrhagic Escherichia coli as shown in SEQ ID NO:81~SEQ ID NO:82; primers for detecting enteroaggregative Escherichia coli as shown in SEQ ID NO:83~SEQ ID NO:84; primers for detecting enteroinvasive Escherichia coli as shown in SEQ ID NO:85~SEQ ID NO:86.Primers for detecting enteropathogenic Escherichia coli with sequences as shown in SEQ ID NO:87~SEQ ID NO:88; primers for detecting diffusely adhesive Escherichia coli with sequences as shown in SEQ ID NO:89~SEQ ID NO:90; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:91~SEQ ID NO:92; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:93~SEQ ID NO:94; primers for detecting Salmonella spp. with sequences as shown in SEQ ID NO:95~SEQ ID NO:96; primers for detecting Salmonella typhi with sequences as shown in SEQ ID NO:97~SEQ ID NO:98; primers for detecting Salmonella paratyphi A with sequences as shown in SEQ ID NO:99~SEQ ID NO:100; primers for detecting Salmonella paratyphi B with sequences as shown in SEQ ID NO:101~SEQ ID NO:102; primers for detecting Salmonella paratyphi B with sequences as shown in SEQ ID NO:103~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:103~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:87~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:103~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:104; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:105~SEQ ID NO:96; primers for detecting Salmonella spp. with sequences as shown in SEQ ID NO:104; primers for detecting Shigella spp. with sequences as shown in SEQ ID NO:105~SEQ ID NO:98; primers for detecting Shigella spp. with sequences as shown Primers for detecting Salmonella paratyphi C as shown in NO:104; primers for detecting non-typhoid Salmonella as shown in SEQ ID NO:105~SEQ ID NO:106; primers for detecting Salmonella enteritidis as shown in SEQ ID NO:107~SEQ ID NO:108; primers for detecting Salmonella typhimurium as shown in SEQ ID NO:109~SEQ ID NO:110; primers for detecting Vibrio vulnificus as shown in SEQ ID NO:111~SEQ ID NO:112; primers for detecting Vibrio parahaemolyticus as shown in SEQ ID NO:113~SEQ ID NO:114; primers for detecting Vibrio mimicus as shown in SEQ ID NO:115~SEQ ID NO:116; primers for detecting Vibrio cholerae as shown in SEQ ID NO:117~SEQ ID NO:118; primers for detecting Vibrio cholerae as shown in SEQ ID NO:119~SEQ ID NO:110. Primers for detecting Yersinia enterocolitica (NO:120); primers for detecting Aegyptiformis duodenalis (SEQ ID NO:121~SEQ ID NO:122); primers for detecting Clonorchis sinensis (SEQ ID NO:123~SEQ ID NO:124); primers for detecting Clonorchis sinensis (SEQ ID NO:125~SEQ ID NO:126); primers for detecting Cryptosporidium (SEQ ID NO:127~SEQ ID NO:128); primers for detecting Entamoeba histolytica (SEQ ID NO:129~SEQ ID NO:130).Primers for detecting Entamoeba histolytica with sequences shown in SEQ ID NO:131~SEQ ID NO:132; primers for detecting Intestinal worms with sequences shown in SEQ ID NO:133~SEQ ID NO:134; primers for detecting Fasciolopsis buski with sequences shown in SEQ ID NO:135~SEQ ID NO:136; primers for detecting Balantidium coli with sequences shown in SEQ ID NO:137~SEQ ID NO:138; and primers for detecting Strongyloides stercoralis with sequences shown in SEQ ID NO:139~SEQ ID NO:140.

[0019] In one embodiment, the primer set further includes primers for detecting internal standards with sequences as shown in SEQ ID NO:141~SEQ ID NO:142.

[0020] In one embodiment, the 5' ends of the primers in the primer set are connected to a common sequence as shown in SEQ ID NO:143.

[0021] In other embodiments of the present invention, the application of the above-described primer set in the preparation of products for detecting intestinal infection-related pathogens is disclosed.

[0022] In one embodiment, the product is a reagent kit.

[0023] In other embodiments of the present invention, a kit for detecting intestinal infection-related pathogens is disclosed, comprising the aforementioned primer set.

[0024] In one embodiment, the kit further includes PCR reaction buffer, purification reagents, and sequencing adapters.

[0025] In one embodiment, the PCR reaction buffer is an integrated amplification premix system, including Taq DNA polymerase, dNTPs, magnesium ions, potassium chloride, and other essential components for the PCR reaction.

[0026] In one embodiment, the working concentrations of primers for detecting BK polyomavirus were all 0.19 μM to 0.23 μM; the working concentrations of primers for detecting cytomegalovirus, Staphylococcus aureus, and Salmonella paratyphi C were all 0.89 μM to 0.93 μM; the working concentrations of primers for detecting human parvovirus B19 and Ahroptera duodenalis were all 0.46 μM to 0.50 μM; the working concentrations of primers for detecting Epstein-Barr virus (EBV), human adenovirus group F, human astrovirus, Staphylococcus aureus enterotoxin E, Shigella-like bacteria, Enterobacter helminthes, and Strongyloides stercoralis were all 0.59 μM to 0.63 μM; the working concentrations of primers for detecting human adenovirus and rotavirus type A were all 0.86 μM to 0.90 μM; and the working concentrations of primers for detecting human adenovirus type 40 were all 1.16 μM to 1.20 μM. The working concentrations of primers for detecting human adenovirus type 41, herpes simplex virus type 1, Clostridium difficile enterotoxin tcdA gene, and Campylobacter coli were all 0.56 μM–0.60 μM; the working concentrations of primers for detecting human herpesvirus type 6, echovirus, enteroinvasive Escherichia coli, and Salmonella typhi were all 0.68 μM–0.72 μM; the working concentrations of primers for detecting enteroviruses were all 0.37 μM–0.41 μM; the working concentrations of primers for detecting enterovirus A71, Clostridium neonate, Listeria monocytogenes, and Cronobacter sakazakii were all 0.74 μM–0.78 μM; and the working concentrations of primers for detecting Coxsackievirus, rotavirus type C, and diffusely adherent Escherichia coli were all 0.71 μM–0.75 μM. The working concentrations of primers for detecting norovirus GI, norovirus GII, Salmonella spp., Vibrio vulnificus, and Fasciolopsis buski were all 0.43 μM–0.47 μM; the working concentrations of primers for detecting rotavirus type B and enterotoxigenic Escherichia coli were all 1.07 μM–1.11 μM; the working concentrations of primers for detecting Sapporo virus genotype GI and Clostridium difficile cytotoxin tcdB gene were all 0.92 μM–0.96 μM; the working concentrations of primers for detecting Sapporo virus genotype GII, Salmonella typhimurium, Vibrio cholerae, and Yersinia enterocolitica were all 0.80 μM–0.84 μM; the working concentrations of primers for detecting Bacillus cereus were all 0.31 μM–0.35 μM; the working concentrations of primers for detecting Clostridium perfringens were all 0.34 μM–0.38 μM; and the working concentrations of primers for detecting Staphylococcus aureus enterotoxin A were all 0.50 μM. The working concentrations of primers for detecting Staphylococcus aureus enterotoxin B, enterocolitically aggregated Escherichia coli, enteropathogenic Escherichia coli, Vibrio parahaemolyticus, and Cryptosporidium were all 0.65 μM to 0.69 μM; the working concentrations of primers for detecting Staphylococcus aureus enterotoxin C were all 0.22 μM to 0.54 μM.The working concentrations of primers for detecting Staphylococcus aureus enterotoxin D were 0.83 μM–0.87 μM; the working concentrations of primers for detecting Aeromonas hydrophila, Shigella spp., and Clonorchis sinensis were 0.53 μM–0.57 μM; the working concentrations of primers for detecting Campylobacter jejuni and Salmonella paratyphi A were 1.04 μM–1.08 μM; the working concentrations of primers for detecting Shiga toxin-producing Escherichia coli and Vibrio mimicus were 1.01 μM–1.05 μM; the working concentrations of primers for detecting enterohemorrhagic Escherichia coli were 0.40 μM–0.44 μM; the working concentrations of primers for detecting Salmonella paratyphi B were 0.28 μM–0.32 μM; and the working concentrations of primers for detecting non-typhoid Salmonella, Salmonella enteritidis, and Balantidium coli were 0.98 μM–1.02 μM. μM; the working concentrations of primers for detecting *Bacillus humanis*, *Entamoeba coli*, and *Entamoeba histolytica* are all 0.77 μM–0.81 μM.

[0027] In one embodiment, the working concentrations of primers for detecting BK polyomavirus are all 0.20 μM to 0.22 μM; the working concentrations of primers for detecting cytomegalovirus, Staphylococcus aureus, and Salmonella paratyphi C are all 0.90 μM to 0.92 μM; the working concentrations of primers for detecting human parvovirus B19 and Ahroptera duodenalis are all 0.47 μM to 0.49 μM; the working concentrations of primers for detecting Epstein-Barr virus (EBV), human adenovirus group F, human astrovirus, Staphylococcus aureus enterotoxin E, Shigella-like bacteria, Enterobacter helminthes, and Strongyloides stercoralis are all 0.60 μM to 0.62 μM; the working concentrations of primers for detecting human adenovirus and rotavirus type A are all 0.87 μM to 0.89 μM; and the working concentrations of primers for detecting human adenovirus type 40 are all 1.17 μM to 1.19 μM. The working concentrations of primers for detecting human adenovirus type 41, herpes simplex virus type 1, Clostridium difficile enterotoxin tcdA gene, and Campylobacter coli were all 0.57 μM–0.59 μM; the working concentrations of primers for detecting human herpesvirus type 6, echovirus, enteroinvasive Escherichia coli, and Salmonella typhi were all 0.69 μM–0.71 μM; the working concentrations of primers for detecting enteroviruses were all 0.38 μM–0.40 μM; the working concentrations of primers for detecting enterovirus A71, Clostridium neonate, Listeria monocytogenes, and Cronobacter sakazakii were all 0.75 μM–0.77 μM; and the working concentrations of primers for detecting Coxsackievirus, rotavirus type C, and diffusely adherent Escherichia coli were all 0.72 μM–0.74 μM. The working concentrations of primers for detecting norovirus GI, norovirus GII, Salmonella spp., Vibrio vulnificus, and Fasciolopsis buski were all 0.44 μM–0.46 μM; the working concentrations of primers for detecting rotavirus type B and enterotoxigenic Escherichia coli were all 1.08 μM–1.10 μM; the working concentrations of primers for detecting Sapporo virus genotype GI and Clostridium difficile cytotoxin tcdB gene were all 0.93 μM–0.95 μM; the working concentrations of primers for detecting Sapporo virus genotype GII, Salmonella typhimurium, Vibrio cholerae, and Yersinia enterocolitica were all 0.81 μM–0.83 μM; the working concentrations of primers for detecting Bacillus cereus were all 0.32 μM–0.34 μM; the working concentrations of primers for detecting Clostridium perfringens were all 0.35 μM–0.37 μM; and the working concentrations of primers for detecting Staphylococcus aureus enterotoxin A were all 0.51 μM. The working concentrations of primers for detecting Staphylococcus aureus enterotoxin B, enterocolitically aggregated Escherichia coli, enteropathogenic Escherichia coli, Vibrio parahaemolyticus, and Cryptosporidium were all 0.66 μM to 0.68 μM; the working concentrations of primers for detecting Staphylococcus aureus enterotoxin C were all 0.23 μM to 0.53 μM.The working concentrations of primers for detecting Staphylococcus aureus enterotoxin D were all 0.84 μM–0.86 μM; the working concentrations of primers for detecting Aeromonas hydrophila, Shigella spp., and Clonorchis sinensis were all 0.54 μM–0.56 μM; the working concentrations of primers for detecting Campylobacter jejuni and Salmonella paratyphi A were all 1.05 μM–1.07 μM; the working concentrations of primers for detecting Shiga toxin-producing Escherichia coli and Vibrio mimicus were all 1.02 μM–1.04 μM; the working concentrations of primers for detecting enterohemorrhagic Escherichia coli were all 0.41 μM–0.43 μM; the working concentrations of primers for detecting Salmonella paratyphi B were all 0.29 μM–0.31 μM; and the working concentrations of primers for detecting non-typhoid Salmonella, Salmonella enteritidis, and Balantidium coli were all 0.99 μM–1.01 μM. μM; the working concentrations of primers for detecting *Bacillus humanis*, *Entamoeba coli*, and *Entamoeba histolytica* are all 0.78 μM–0.80 μM.

[0028] In one implementation, the working concentration of the primers for detecting the internal standard is 0.77 μM to 0.81 μM.

[0029] In one implementation, the working concentration of the primers for detecting the internal standard is 0.78 μM to 0.80 μM.

[0030] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Example 1 Primer set for detecting intestinal infection-related pathogens

[0032] Includes the following steps:

[0033] 1. A total of 70 pathogen targets were identified as being associated with intestinal infections. (See Table 1.)

[0034] Table 1

[0035]

[0036]

[0037]

[0038]

[0039]

[0040] 2. Specific amplification primers were designed for the 70 pathogen targets in Table 1 (the specific amplification primer sequences are shown in Table 2, and the concentration of each amplification primer in the detection system is shown in Table 3, where the pooling coefficient is the volume ratio coefficient when the primers are mixed). The optimal amplification conditions of these amplification primers are similar, and each amplicon can be successfully amplified under single amplification conditions. Moreover, the interaction between primer combinations is small, and all primers can obtain good detection performance in a single system.

[0041] Each amplification primer has a universal sequence (5'-GACTGCCGCTGGTTGGATG-3', SEQ ID NO:143) attached to its 5' end for nucleic acid sequencing library construction. This universal sequence is complementary to the 3' end of the sequencing adapter primers on the sequencing platform. Its function is to provide binding sites for the sequencing adapter primers in subsequent library amplification steps.

[0042] Table 2

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Table 3

[0052]

[0053]

[0054]

[0055] Example 2: Detection of intestinal infection-related pathogens in clinical samples using the primer set from Example 1.

[0056] Includes the following steps:

[0057] 1. Sample collection

[0058] In the gastroenterology department of the hospital, a retrospective search was conducted for the remaining samples of patients who had undergone molecular testing within the past month. A total of 100 stool samples and 80 anal swab samples were collected, for a total of 180 samples.

[0059] 2. Sample pretreatment

[0060] For fecal samples, take a sample the size of a soybean into a 1.5 mL centrifuge tube, add 1 mL of physiological saline, shake to mix, and take 500 μL of the lower solid-liquid mixture for extraction.

[0061] For anal swabs, place the sampling tube containing the swab on a vortex mixer and vortex for 30 seconds. Take 500 μL of the lower solid-liquid mixture for extraction.

[0062] 3. Nucleic acid extraction

[0063] Extract using nucleic acid extraction or purification reagents (Guangzhou Jinqirui Biotechnology Co., Ltd., registration number Yue Sui Xie Bei 20220884) following the instructions.

[0064] 4. Prepare primer mixture

[0065] Mix all primers in Table 2 (ambush primers with a common sequence at the 5' end) according to the concentrations in Table 3 to prepare a 450 μL primer mixture. The primer mixture is prepared as follows: dilute each primer to 100 μM, then pipette 0.7 μL of 1F and 1R, then 3.0 μL of 2F and 2R, and so on, until finally pipette 2.6 μL of 71F and 71R. Mix thoroughly and store at 4°C.

[0066] 5. Multiplex PCR amplification

[0067] Prepare multiplex PCR amplification reagent system 1 (1.5 μL primer mixture + 12.5 μL PCR reaction buffer + 11 μL nucleic acid to be tested), and perform multiplex PCR amplification on the extracted nucleic acid to be tested (amplification program is shown in Table 4) to obtain the target region fragment of the pathogenic microorganism.

[0068] Table 4

[0069]

[0070] After purifying the PCR amplification product, prepare library amplification reagent system 2 (1 μL adapter primer + 12.5 μL PCR reaction buffer + 11.5 μL purified amplification product) for a second round of library amplification (amplification program is shown in Table 5), and finally obtain a library with the structure "sequencing adapter-common sequence-target region-common sequence-sequencing adapter".

[0071] Table 5

[0072]

[0073] 6. Purification

[0074] The libraries for each sample were purified (using magnetic bead purification reagent) and then mixed to obtain a library for detecting intestinal infection-related pathogens. The fragment length peaks of the library are shown in the figure. Figure 1 As shown, the results indicate that the main peak of the library is concentrated in the range of 200-400 bp, which is the size of the product after adding the length of the original primer product to the length of the common sequence and sequencing adapter. There is no large fragment contamination or obvious small fragment primer dimers, which meets the requirements for sequencing.

[0075] 7. Sequencing

[0076] The KM MiniSeqDx-CN sequencer manufactured by Guangzhou Jinqirui Biotechnology Co., Ltd. was used, along with a universal sequencing reaction kit (Guangzhou Jinqirui Biotechnology Co., Ltd., catalog number: KS107-CXR, specification MR100), and the library was sequenced according to the instructions.

[0077] 8. Analysis of sequencing data

[0078] (1) Bioinformatics Analysis: For the FastQ files obtained from sequencing, the FastP software was first used to count the amount of raw data, while removing adapter sequences and filtering low-quality data from the raw sequencing results. Then, a script was written to screen and retain valid reads containing primer sequences in the sequencing products. Next, the MEM alignment algorithm of the BWA alignment software was used to align the valid read product sequences to the self-built database of this invention, and the number of reads covered by each pathogen was counted based on the BED file. The self-built database mentioned above consists of genomes matched to the primer product sequences, sourced from the NCBI Nucleotide database. Each pathogen target contains at least one matching genome, and multiple genomes are added for the same target to ensure that all possible primer product sequences can be perfectly aligned to the database. The BED file format is a widely used text file format in bioinformatics, specifically designed to describe genomic features and regions, generated based on the genomic region location information aligned to the self-built database according to primer theory.

[0079] (2) Sample quality control: Quality control was performed on the analyzed data. The criteria for acceptance were as follows: the minimum number of sequencing reads per sample was ≥50,000; otherwise, the raw data was deemed unacceptable. The sequencing quality value Q30 was ≥75%; otherwise, the sequencing quality was deemed unacceptable. The number of homogenized reads for the internal control was ≥50; otherwise, the internal control was deemed unacceptable. For samples that met the above quality control criteria, pathogen identification was then performed.

[0080] (3) Pathogen identification: For each pathogen within the detection range, the number of original reads allocated is used to calculate the normalized read count. The pathogen's positivity or negativity is determined based on the normalized read count. A normalized read count ≥ 10 indicates a positive result; otherwise, it is considered negative. The formula for normalizing the read count is:

[0081]

[0082] in, The total number of raw reads sequenced for a given sample. This refers to the number of reads in the sample that have been confirmed to fall within a specific pathogen target region through comparison. In this embodiment, the normalization coefficient is used. = 100000, for The number of reads is normalized. Normalization eliminates the differences between samples caused by variations in the original data volume.

[0083] 9. Test Results

[0084] An analysis of 180 samples was conducted, and all samples passed quality control. The statistical results are shown in Table 6.

[0085] Table 6

[0086]

[0087] As shown in Table 6, the primer set of Example 1 and the detection method of this example can successfully detect fecal and anal swab samples with a pathogen positive detection rate of up to 99% (178 / 180).

[0088] The specific results of the detection of related pathogens are shown in Table 7.

[0089] Table 7

[0090]

[0091]

[0092]

[0093] As shown in Table 7, the primer set of this invention enables the simultaneous detection of multiple intestinal infection-related pathogens under the same amplification system and procedure. Compared with prior results based on previous clinical testing (metagenomic sequencing mNGS methodology), the primer set and detection method of this invention not only detected all the pathogens recorded in the prior results, but also detected low concentrations of mixed pathogens in multiple samples. BLAST alignment of these pathogen sequences with NCBI confirmed that they indeed aligned with the target species, not other species. Therefore, the bioinformatics workflow determined that all results were specific.

[0094] The results of this embodiment show that the primer set of the present invention has higher detection sensitivity, and can achieve comprehensive and accurate detection of a variety of intestinal pathogens in a single reaction system and procedure.

[0095] Example 3: Detection performance of the primer set from Example 1

[0096] 1. Sensitivity and accuracy

[0097] (1) Preparation of artificial simulated samples

[0098] Six bacterial cultures (all purchased from Henan Beina Biotechnology, including Shiga toxin-producing Escherichia coli (STEC), Salmonella enteritidis, Clostridium difficile, Vibrio vulnificus, Vibrio parahaemolyticus, and Yersinia enterocolitica), four viral cultures (all purchased from Guangzhou Bondsheng Biotechnology, including rotavirus type A, norovirus group GI, enterovirus type A71, and human adenovirus type 41), and two parasites (from Guangzhou Jijian Biotechnology, including Clonorchis sinensis and Acletostoma duodenale) were mixed with a confirmed negative mixed fecal sample (this means that various impurities in the fecal sample were retained to approximate the real sample as closely as possible, rather than directly using culture samples for testing) to prepare artificial simulated samples.

[0099] (2) Extraction of nucleic acid and serial dilution

[0100] Nucleic acid was extracted from the artificially simulated sample according to the method in Example 2, and serial dilutions were performed. The detection concentration gradients for bacteria were set at 1000 CFU / mL, 2000 CFU / mL, and 4000 CFU / mL; the detection concentration gradients for viruses and parasites were set at 250 copies / mL, 500 copies / mL, and 1000 copies / mL.

[0101] For each concentration gradient of each pathogen, 20 replicate tests were performed, resulting in a total of 720 library data (12 pathogens × 3 concentrations × 20 replicates).

[0102] (3) Result determination

[0103] Following the analytical method of Example 2, the normalized read count for each pathogen was counted. A normalized read count ≥ 10 was considered a positive detection; otherwise, it was considered a negative detection. The detection rate at each concentration was calculated.

[0104] The results are shown in Tables 8 and 9.

[0105] Table 8

[0106]

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] Table 9

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] As shown in Tables 8 and 9, the primer set of this invention achieved 100% accuracy in detecting 12 artificially simulated positive pathogens at the highest concentration in artificially simulated samples. Through serial dilutions and 20 replicate experiments, the detection performance was confirmed: for Clostridium difficile, Vibrio vulnificus, Vibrio parahaemolyticus, and Yersinia enterocolitica, the detection rate reached 100% (20 / 20) at a concentration of 1000 CFU / mL; for rotavirus type A and human adenovirus type 41, the detection rate still reached 100% (20 / 20) at a low concentration of 250 copies / mL; and for Clonorchis sinensis, the detection rate reached 100% (20 / 20) at a concentration of 500 copies / mL.

[0119] Therefore, using the primer set and detection method of the present invention, not only can the target pathogen be accurately detected, but the detection limit for some pathogens can be as low as 250 copies / mL, demonstrating excellent detection performance.

[0120] 2. Cross-reactivity specificity

[0121] Ten closely related non-target pathogens (non-diarrheal Escherichia coli, Staphylococcus epidermidis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus faecalis, Candida albicans, Candida glabrata, Saccharomyces cerevisiae, and adenovirus type 5) commonly found in fecal samples and with high homology to the detection targets of this invention were selected and cultured (Guangzhou Jijian Biotechnology) and prepared into a concentration of 10. 4 Simulated positive samples with CFU / mL or copies / mL were tested using the method in Example 2, with each sample group tested three times.

[0122] The results are shown in Table 10.

[0123] Table 10

[0124]

[0125] As shown in Table 10, the primer set of this invention is effective against a variety of common fecal proximal non-target pathogens.

[0126] No cross-amplification, no false positive results, primers with extremely high specificity, effectively avoiding homologous sequence interference, and preserving...

[0127] To improve the accuracy of pathogen detection.

[0128] 3. Endogenous (hemoglobin) anti-interference properties

[0129] Four clinically confirmed mixed fecal samples and four mixed anal swab samples were selected. Each sample was divided into two groups: a control group (sample mixed with physiological saline at a volume ratio of 3:1) and a hemoglobin interference group (sample mixed with hemoglobin solution at a volume ratio of 3:1). All samples were processed and tested under the same conditions, and each group was tested three times. The differences between the detection results of the two groups and the sequencing homogenized reads data were compared.

[0130] The results are shown in Table 11.

[0131] Table 11

[0132]

[0133] As shown in Table 11, common endogenous hemoglobin impurities in fecal and anal swab samples do not interfere with the amplification and sequencing results of the detection system of this invention. The sample detection rate and data stability are normal, and the system has good endogenous anti-interference ability.

[0134] 4. Exogenous (drug) anti-interference performance

[0135] Four clinically confirmed mixed fecal samples and four mixed anal swab samples were selected. Each sample was divided into five aliquots, with a blank control group and four drug interference groups. The blank control group received no interfering substances, while the other groups received norfloxacin, ampicillin, clindamycin, and ribavirin, respectively, at final concentrations of 3 mg / L, 6 mg / L, 15 mg / L, and 2 mg / L. All samples were processed uniformly before testing, with each group tested three times.

[0136] The results are shown in Table 12.

[0137] Table 12

[0138]

[0139] As shown in Table 12, commonly used intestinal anti-infective drugs in clinical practice do not inhibit or interfere with the multiplex amplification and sequencing detection system of this invention, and will not cause missed pathogen detection, false negatives or abnormal data. The system has excellent resistance to external interference.

[0140] Based on the above results of this embodiment, the primer set and detection system for detecting intestinal infection-related pathogens of the present invention have high accuracy, high sensitivity, and strong specificity, with no cross-reaction with closely related pathogens. They can effectively resist interference from endogenous impurities in hemoglobin in fecal and anal swab samples as well as exogenous interference from commonly used clinical drugs. The detection performance is stable, the anti-interference ability is outstanding, and it is highly adaptable to the detection scenarios of complex samples in clinical practice.

[0141] Example 4: Comparison of the impact of different detection systems on detection results

[0142] The inventors of this invention designed multiple pairs of candidate primers for each target pathogen and conducted extensive screening and optimization experiments on single primer sequences, mixed primer sequences, and primer concentrations. Ultimately, they obtained a detection system consisting of the primer sets in Table 2 of Example 1 and the primer concentrations in Table 3. Under this detection system, the optimal amplification conditions for all primers are similar, there is minimal interference between primers, no significant non-specific amplification, and all amplicons can be successfully amplified under the same amplification conditions.

[0143] The following uses a detection system consisting of a primer set different from that in Example 1 (see Table 13; the primer sequences and concentrations of the remaining unlisted pathogens are the same as in Example 1 and are marked as the control group) as an example to compare the effects of using different detection systems on 10 different types of clinical samples (the detection method is the same as in Example 2). The specific results are shown in Table 14.

[0144] Table 13

[0145]

[0146]

[0147]

[0148] Table 14

[0149]

[0150]

[0151] As shown in Table 14, the overall dimer proportion in the control group was as high as 48.4%, the average effective data rate was only 23%, the highest average proportion of a single dimer combination was 15.1%, the number of normalized reads for the internal standard was low (27-61), and there were cases of missed pathogen detection. In contrast, the overall dimer proportion in the system of Example 1 decreased to 18%, the average effective data rate increased to 64%, the highest average proportion of a single dimer combination was 6%, the number of normalized reads for the internal standard increased significantly to 260-900, and there were no missed pathogens compared to the prior results.

[0152] The results of this embodiment demonstrate that the detection system composed of primer sets of a specific concentration according to the present invention has significantly better detection performance.

[0153] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0154] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A primer set, characterized by, include: Primers for detecting BK polyomavirus with sequences shown in SEQ ID NO:1~SEQ ID NO:2; Primers for detecting cytomegalovirus with sequences as shown in SEQ ID NO:3~SEQ ID NO:4; Primers for detecting human parvovirus B19, with sequences shown in SEQ ID NO:5~SEQ ID NO:6; Primers for detecting EB virus, with sequences shown in SEQ ID NO:7~SEQ ID NO:8; Primers for detecting human adenovirus with sequences as shown in SEQ ID NO:9~SEQ ID NO:10; Primers for detecting human adenovirus type 40, with sequences shown in SEQ ID NO:11~SEQ ID NO:12; Primers for detecting human adenovirus type 41, with sequences shown in SEQ ID NO:13~SEQ ID NO:14; Primers for detecting human adenovirus group F, with sequences shown in SEQ ID NO:15~SEQ ID NO:16; Primers for detecting human herpesvirus 6, with sequences shown in SEQ ID NO:17~SEQ ID NO:18; Primers for detecting herpes simplex virus type 1, with sequences shown in SEQ ID NO:19~SEQ ID NO:20; Primers for detecting echovirus with sequences as shown in SEQ ID NO:21~SEQ ID NO:22; Primers for detecting enteroviruses, with sequences shown in SEQ ID NO:23~SEQ ID NO:24; Primers for detecting enterovirus A71, with sequences shown in SEQ ID NO:25~SEQ ID NO:26; Primers for detecting Coxsackievirus with sequences as shown in SEQ ID NO:27~SEQ ID NO:28; Primers for detecting human astrovirus with sequences as shown in SEQ ID NO:29~SEQ ID NO:30; Primers for detecting norovirus GI group, with sequences as shown in SEQ ID NO:31~SEQ ID NO:32; Primers for detecting norovirus group GII, with sequences shown in SEQ ID NO:33~SEQ ID NO:34; Primers for detecting rotavirus type A, with sequences shown in SEQ ID NO:35~SEQ ID NO:36; Primers for detecting rotavirus type B, with sequences shown in SEQ ID NO:37~SEQ ID NO:38; Primers for detecting rotavirus type C, with sequences shown in SEQ ID NO:39~SEQ ID NO:40; Primers for detecting Sapporo virus genotype GI, with sequences shown in SEQ ID NO:41~SEQ ID NO:42; Primers for detecting Sapporo virus genotype GII, with sequences shown in SEQ ID NO:43~SEQ ID NO:44; Primers for detecting Bacillus cereus, with sequences as shown in SEQ ID NO:45~SEQ ID NO:46; Primers for detecting the Clostridium difficile enterotoxin tcdA gene, with sequences shown in SEQ ID NO:47~SEQ ID NO:48; Primers for detecting the Clostridium difficile cytotoxin tcdB gene, with sequences shown in SEQ ID NO:49~SEQ ID NO:50; Primers for detecting Clostridium perfringens, with sequences as shown in SEQ ID NO:51~SEQ ID NO:52; Primers for detecting Clostridium neonate, with sequences shown in SEQ ID NO:53~SEQ ID NO:54; Primers for detecting Listeria monocytogenes, with sequences as shown in SEQ ID NO:55~SEQ ID NO:56; Primers for detecting Staphylococcus aureus with sequences as shown in SEQ ID NO:57~SEQ ID NO:58; Primers for detecting Staphylococcus aureus enterotoxin A, with sequences as shown in SEQ ID NO:59~SEQ ID NO:60; Primers for detecting Staphylococcus aureus enterotoxin B, with sequences as shown in SEQ ID NO:61~SEQ ID NO:62; Primers for detecting Staphylococcus aureus enterotoxin C, with sequences as shown in SEQ ID NO:63~SEQ ID NO:64; Primers for detecting Staphylococcus aureus enterotoxin D, with sequences as shown in SEQ ID NO:65~SEQ ID NO:66; Primers for detecting Staphylococcus aureus enterotoxin E, with sequences as shown in SEQ ID NO:67~SEQ ID NO:68; Primers for detecting Aeromonas hydrophila with sequences as shown in SEQ ID NO:69~SEQ ID NO:70; Primers for detecting Campylobacter coli with sequences as shown in SEQ ID NO:71~SEQ ID NO:72; Primers for detecting Campylobacter jejuni with sequences as shown in SEQ ID NO:73~SEQ ID NO:74; Primers for detecting Cronobacter sakazakii with sequences as shown in SEQ ID NO:75~SEQ ID NO:76; Primers for detecting Shiga toxin-producing Escherichia coli, with sequences as shown in SEQ ID NO:77~SEQ ID NO:78; Primers for detecting enterotoxigenic Escherichia coli with sequences as shown in SEQ ID NO:79~SEQ ID NO:80; Primers for detecting enterohemorrhagic Escherichia coli with sequences as shown in SEQ ID NO:81~SEQ ID NO:82; Primers for detecting enteroaggregative Escherichia coli with sequences as shown in SEQ ID NO:83~SEQ ID NO:84; Primers for detecting enteroinvasive Escherichia coli with sequences as shown in SEQ ID NO:85~SEQ ID NO:86; Primers for detecting enteropathogenic Escherichia coli with sequences as shown in SEQ ID NO:87~SEQ ID NO:88; Primers for detecting diffusely adhesive Escherichia coli with sequences as shown in SEQ ID NO:89~SEQ ID NO:90; Primers for detecting Shigella species, with sequences as shown in SEQ ID NO:91~SEQ ID NO:92; Primers for detecting *Shigella* species, with sequences shown in SEQ ID NO:93~SEQ ID NO:94; Primers for detecting Salmonella, with sequences as shown in SEQ ID NO:95~SEQ ID NO:96; Primers for detecting Salmonella typhi, with sequences as shown in SEQ ID NO:97~SEQ ID NO:98; Primers for detecting Salmonella paratyphi A, with sequences as shown in SEQ ID NO:99~SEQ ID NO:100; Primers for detecting Salmonella paratyphi B, with sequences shown in SEQ ID NO:101~SEQ ID NO:102; Primers for detecting Salmonella paratyphi C, with sequences shown in SEQ ID NO:103~SEQ ID NO:104; Primers for detecting non-typhoidal Salmonella, with sequences as shown in SEQ ID NO:105~SEQ ID NO:106; Primers for detecting Salmonella enteritidis, with sequences as shown in SEQ ID NO:107~SEQ ID NO:108; Primers for detecting Salmonella Typhimurium with sequences as shown in SEQ ID NO:109~SEQ ID NO:110; Primers for detecting Vibrio vulnificus, with sequences as shown in SEQ ID NO:111~SEQ ID NO:112; Primers for detecting Vibrio parahaemolyticus, with sequences as shown in SEQ ID NO:113~SEQ ID NO:114; Primers for detecting Vibrio mimicus, with sequences shown in SEQ ID NO:115~SEQ ID NO:116; Primers for detecting Vibrio cholerae, with sequences as shown in SEQ ID NO:117~SEQ ID NO:118; Primers for detecting Yersinia enterocolitica, with sequences as shown in SEQ ID NO:119~SEQ ID NO:120; Primers for detecting Aegyptiformis duodenalis, with sequences as shown in SEQ ID NO:121~SEQ ID NO:122; Primers for detecting human budding protozoa, with sequences as shown in SEQ ID NO:123~SEQ ID NO:124; Primers for detecting Clonorchis sinensis, with sequences as shown in SEQ ID NO:125~SEQ ID NO:126; Primers for detecting Cryptosporidium, with sequences as shown in SEQ ID NO:127~SEQ ID NO:128; Primers for detecting colonic amoebae, with sequences as shown in SEQ ID NO:129~SEQ ID NO:130; Primers for detecting Entamoeba histolytica with sequences as shown in SEQ ID NO:131~SEQ ID NO:132; Primers for detecting Intestinal nematodes, with sequences as shown in SEQ ID NO:133~SEQ ID NO:134; Primers for detecting Fasciola hepatica with sequences as shown in SEQ ID NO:135~SEQ ID NO:136; Primers for detecting baculocilia, with sequences as shown in SEQ ID NO:137~SEQ ID NO:138; Primers for detecting Strongyloides stercoralis, with sequences shown in SEQ ID NO:139~SEQ ID NO:

140.

2. The primer set according to claim 1, characterized in that, It also includes primers for detecting internal standards with sequences as shown in SEQ ID NO:141~SEQ ID NO:

142.

3. The primer set according to claim 1 or 2, characterized in that, The primers in the primer set have a common sequence at their 5' ends, as shown in SEQ ID NO:

143.

4. The use of the primer set according to any one of claims 1 to 3 in the preparation of products for detecting intestinal infection-related pathogens.

5. Use according to claim 4, characterized in that, The product in question is a reagent kit.

6. A kit for detecting enterically infecting pathogenic microorganisms, characterized by, It includes the primer set as described in any one of claims 1 to 3.

7. The kit of claim 6, wherein The kit also includes PCR reaction buffer, purification reagents, and sequencing adapters.

8. The reagent kit according to claim 6, characterized in that, The working concentrations of the primers used to detect BK polyomavirus were all 0.19 μM to 0.23 μM; The working concentrations of the primers for detecting cytomegalovirus, Staphylococcus aureus, and Salmonella paratyphi C were all 0.89 μM to 0.93 μM. The working concentrations of primers for detecting human parvovirus B19 and hookworm were both 0.46 μM to 0.50 μM. The working concentrations of primers for detecting EB virus, human adenovirus group F, human astrovirus, Staphylococcus aureus enterotoxin E, Shigella spp., Enterobacter helminthes, and Strongyloides stercoralis were all 0.59 μM to 0.63 μM. The working concentrations of the primers for detecting human adenovirus and rotavirus type A are both 0.86 μM to 0.90 μM; The working concentrations of the primers for detecting human adenovirus type 40 were all 1.16 μM to 1.20 μM; The working concentrations of the primers for detecting human adenovirus type 41, herpes simplex virus type 1, Clostridium difficile enterotoxin tcdA gene, and Campylobacter colon were all 0.56 μM to 0.60 μM. The working concentrations of primers for detecting human herpesvirus 6, echovirus, enteroinvasive Escherichia coli, and Salmonella typhi were all 0.68 μM to 0.72 μM. The working concentrations of the primers for detecting enteroviruses were all 0.37 μM to 0.41 μM; The working concentrations of primers for detecting enterovirus A71, Clostridium neonate, Listeria monocytogenes, and Cronobacter sakazakii were all 0.74 μM to 0.78 μM. The working concentrations of primers for detecting Coxsackievirus, Rotavirus C, and Diffuse Adhesive Escherichia coli were all 0.71 μM to 0.75 μM. The working concentrations of primers for detecting norovirus GI group, norovirus GII group, Salmonella spp., Vibrio vulnificus and Fasciola hepatica were all 0.43 μM to 0.47 μM. The working concentrations of the primers for detecting rotavirus type B and enterotoxigenic Escherichia coli were both 1.07 μM to 1.11 μM. The working concentrations of the primers used to detect Sapporo virus genotype GI and Clostridium difficile cytotoxin tcdB gene were 0.92 μM to 0.96 μM. The working concentrations of primers for detecting Sapporo virus genotype GII, Salmonella typhimurium, Vibrio cholerae, and Yersinia enterocolitica were all 0.80 μM to 0.84 μM. The working concentrations of the primers for detecting Bacillus cereus were all 0.31 μM to 0.35 μM; The working concentrations of the primers for detecting Clostridium perfringens were all 0.34 μM to 0.38 μM; The working concentrations of the primers for detecting Staphylococcus aureus enterotoxin A were all 0.50 μM to 0.54 μM; The working concentrations of primers for detecting Staphylococcus aureus enterotoxin B, enteroaggregative Escherichia coli, enteropathogenic Escherichia coli, Vibrio parahaemolyticus, and Cryptosporidium were all 0.65 μM to 0.69 μM. The working concentrations of the primers for detecting Staphylococcus aureus enterotoxin C were all 0.22 μM to 0.26 μM; The working concentrations of the primers for detecting Staphylococcus aureus enterotoxin D were all 0.83 μM to 0.87 μM; The working concentrations of the primers for detecting Aeromonas hydrophila, Shigella spp., and Clonorchis sinensis were all 0.53 μM to 0.57 μM. The working concentrations of the primers for detecting Campylobacter jejuni and Salmonella paratyphi A were both 1.04 μM to 1.08 μM. The working concentrations of the primers for detecting Shiga toxin-producing Escherichia coli and Vibrio mimicus were both 1.01 μM to 1.05 μM. The working concentrations of the primers for detecting enterohemorrhagic Escherichia coli were all 0.40 μM to 0.44 μM; The working concentrations of the primers for detecting Salmonella paratyphi B were all 0.28 μM to 0.32 μM; The working concentrations of primers for detecting non-typhoidal Salmonella, enterobacterial Salmonella, and balanitis ciliates were all 0.98 μM to 1.02 μM. The working concentrations of primers for detecting *Bacillus humanis*, *Entamoeba histolytica*, and *Entamoeba histolytica* were all 0.77 μM to 0.81 μM.

9. The reagent kit according to claim 8, characterized in that, The working concentrations of the primers used to detect BK polyomavirus were all 0.20 μM to 0.22 μM; The working concentrations of primers for detecting cytomegalovirus, Staphylococcus aureus, and Salmonella paratyphi C were all 0.90 μM to 0.92 μM. The working concentrations of the primers for detecting human parvovirus B19 and hookworm were both 0.47 μM to 0.49 μM. The working concentrations of primers for detecting EB virus, human adenovirus group F, human astrovirus, Staphylococcus aureus enterotoxin E, Shigella spp., Enterobacter helminthes, and Strongyloides stercoralis were all 0.60 μM to 0.62 μM. The working concentrations of the primers for detecting human adenovirus and rotavirus type A are both 0.87 μM to 0.89 μM. The working concentrations of the primers for detecting human adenovirus type 40 were all 1.17 μM to 1.19 μM; The working concentrations of the primers for detecting human adenovirus type 41, herpes simplex virus type 1, Clostridium difficile enterotoxin tcdA gene, and Campylobacter colon were all 0.57 μM to 0.59 μM. The working concentrations of primers for detecting human herpesvirus 6, echovirus, enteroinvasive Escherichia coli, and Salmonella typhi were all 0.69 μM to 0.71 μM. The working concentrations of the primers for detecting enteroviruses were all 0.38 μM to 0.40 μM; The working concentrations of primers for detecting enterovirus A71, Clostridium neonate, Listeria monocytogenes, and Cronobacter sakazakii were all 0.75 μM to 0.77 μM. The working concentrations of primers for detecting Coxsackievirus, Rotavirus C, and Diffuse Adhesive Escherichia coli were all 0.72 μM to 0.74 μM. The working concentrations of primers for detecting norovirus GI group, norovirus GII group, Salmonella spp., Vibrio vulnificus and Fasciolopsis buski were all 0.44 μM to 0.46 μM. The working concentrations of the primers for detecting rotavirus type B and enterotoxigenic Escherichia coli were both 1.08 μM to 1.10 μM. The working concentrations of the primers used to detect Sapporo virus genotype GI and Clostridium difficile cytotoxin tcdB gene were 0.93 μM to 0.95 μM. The working concentrations of primers for detecting Sapporo virus genotype GII, Salmonella typhimurium, Vibrio cholerae, and Yersinia enterocolitica were all 0.81 μM to 0.83 μM. The working concentrations of the primers for detecting Bacillus cereus were all 0.32 μM to 0.34 μM; The working concentrations of the primers for detecting Clostridium perfringens were all 0.35 μM to 0.37 μM; The working concentrations of the primers for detecting Staphylococcus aureus enterotoxin A were all 0.51 μM to 0.53 μM; The working concentrations of primers for detecting Staphylococcus aureus enterotoxin B, enterocolitically aggregated Escherichia coli, enteropathogenic Escherichia coli, Vibrio parahaemolyticus, and Cryptosporidium were all 0.66 μM to 0.68 μM. The working concentrations of the primers for detecting Staphylococcus aureus enterotoxin C were all 0.23 μM to 0.25 μM; The working concentrations of the primers for detecting Staphylococcus aureus enterotoxin D were all 0.84 μM to 0.86 μM; The working concentrations of the primers for detecting Aeromonas hydrophila, Shigella spp., and Clonorchis sinensis were all 0.54 μM to 0.56 μM. The working concentrations of the primers for detecting Campylobacter jejuni and Salmonella paratyphi A were both 1.05 μM to 1.07 μM. The working concentrations of the primers for detecting Shiga toxin-producing Escherichia coli and Vibrio mimicus were both 1.02 μM to 1.04 μM. The working concentrations of the primers for detecting enterohemorrhagic Escherichia coli were all 0.41 μM to 0.43 μM; The working concentrations of the primers for detecting Salmonella paratyphi B were all 0.29 μM to 0.31 μM; The working concentrations of primers for detecting non-typhoidal Salmonella, enterobacterial Salmonella, and balanitis ciliates were all 0.99 μM to 1.01 μM. The working concentrations of primers for detecting *Bacillus humanis*, *Entamoeba histolytica*, and *Entamoeba histolytica* were all 0.78 μM to 0.80 μM.

10. The kit of claim 6, wherein The working concentrations of the primers used to detect the internal standard were all 0.78 μM to 0.80 μM.

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