Primer probe sets, reagents and detection methods for detecting diarrheal pathogens in children based on digital PCR

Abstract

The present invention provides a primer probe set, reagent and detection method for detecting diarrhea pathogens in children based on digital PCR. The primer probe set includes specific primers and probes for 10 diarrhea pathogens and human internal reference genes; primers and probes for 5 bacteria and primers and probes for human internal reference genes are designed in the same PCR reaction system, and primers and probes for 5 viruses are designed in another PCR reaction system. The present invention designs specific primers and probes in the conservative region of pathogen genes, and has targeted detection and strong specificity; 10 diarrhea pathogens in stool specimens can be quickly detected in two PCR reaction systems, with high sensitivity, greatly reducing costs and shortening detection time, solving the pain point of conventional PCR in detecting multiple pathogens in stool specimens at the same time, overcoming the difficulty of being easily interfered by inhibitors in stool specimen detection, and providing a reliable laboratory basis for diagnosis and treatment of children, epidemic monitoring and infection control.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid amplification technology, and in particular to a primer probe set, reagents and detection method for detecting childhood diarrhea pathogens based on digital PCR. Background Art

[0002] According to the World Health Organization (WHO), diarrheal disease is the third leading cause of death among children under five, killing more than 440,000 children under five and another 50,851 children aged five to nine each year. Diarrhea is a serious threat to children's health. The pathogens that cause diarrhea mainly include bacteria and viruses. Accurate detection of pathogens is helpful for timely diagnosis and treatment of acute diarrhea in children, investigation of diarrheal outbreaks in children, and monitoring and controlling the spread of infection, which is of great significance.

[0003] The current pediatric diarrhea etiology diagnostic technology system mainly includes bacterial isolation and culture combined with biochemical identification technology and viral multiplex real-time fluorescence quantitative PCR detection technology. These methods provide a benchmark basis for microbiological testing for clinical diagnosis. However, in practical applications, traditional detection methods have significant limitations: on the one hand, the bacterial isolation and culture cycle is long (usually 3-5 days), and on the other hand, most viral nucleic acid detection systems can only cover 1-2 common pathogens. When encountering a high incidence of seasonal childhood diarrhea or suspected clustered epidemics, the existing detection schemes are difficult to achieve rapid and high-throughput identification of pathogens, which not only affects the timely blocking of the infection chain, but also delays the public health emergency response process, ultimately leading to delayed clinical diagnosis and treatment decisions and reduced epidemic prevention and control effectiveness.

[0004] The existing clinical etiological detection methods for childhood diarrhea mainly include the following:

[0005] (1) Immunological tests:

[0006] The most widely used immunological detection technology in clinical practice is the gold-label immunochromatography (gold-label method), which is mainly based on the specific binding of antigens and antibodies and the principle of color reaction to detect pathogen antigens such as rotavirus, norovirus, and Clostridium difficile in the feces of children with diarrhea. However, the gold-label method is affected by factors such as the intensity of the color reaction signal and the load of pathogens. Its sensitivity is lower than that of the conventional polymerase chain reaction (PCR), and it is usually a single pathogen test, which cannot provide a comprehensive diagnosis for children with complex infections.

[0007] (2) Cultivation method:

[0008] The culture method is to identify diarrhea-related bacteria and detect drug resistance through bacterial culture. However, the biggest disadvantage of the culture method is the long detection time. It takes several days to obtain the test results, which leads to a delay in the etiological diagnosis of children with acute diarrhea. In addition, the culture method has low sensitivity, and the results are affected by factors such as the use of antimicrobial drugs, pathogen load, and culture medium selectivity, and the manual operation steps are complicated.

[0009] (3) Conventional PCR:

[0010] Reverse transcription PCR (RT-PCR) and real-time fluorescence quantitative PCR (qRT-PCR) are widely used in laboratory diagnosis and research of infectious diarrhea in children, mainly for the identification of single pathogens such as rotavirus and norovirus and differentiation of subtypes. However, these methods can only detect pathogens that are highly suspected clinically, with a single detection target and the possibility of false negatives. In addition, organic solvent residues in the experiment have an inhibitory effect on DNA synthesis, which poses a risk of false negatives.

[0011] (4) High-throughput sequencing:

[0012] High-throughput sequencing is a new technology that sequences thousands to billions of DNA fragments simultaneously. High-throughput sequencing (non-targeted sequencing) has the widest coverage, not only can it detect potential pathogens, but also can detect the drug resistance genes of pathogenic bacteria, and even detect mutations in the genetic genes of pathogens. However, high-throughput sequencing should not be used as the first choice in the detection of pathogens of infectious diarrhea in children, because it is easily affected by interfering genes such as humans, intestinal flora and food, and requires reference to more gene sequence data. At the same time, high-throughput sequencing has a long detection cycle and is expensive. It requires the support of platform construction and bioinformatics teams, which is not conducive to large-scale clinical applications. Therefore, high-throughput sequencing is more used in the detection of pathogens or pathogen gene research where the cause is unclear and cultivation is difficult.

[0013] Therefore, a rapid, high-throughput, economical and practical multiple pathogen detection method is urgently needed in clinical practice. Summary of the invention

[0014] In view of this, the purpose of the present invention is to develop a new method for detecting common diarrhea pathogens and their subtypes in children based on droplet digital PCR, and propose a primer probe set, reagent and detection method for detecting diarrhea pathogens in children based on digital PCR, which can simultaneously detect 5 diarrhea-related viruses and their subtypes including human rotavirus, norovirus GII type, astrovirus, enteric adenovirus, and zaruvirus in stool specimens, as well as 5 common diarrhea-causing bacteria including Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni, and Vibrio parahaemolyticus; design specific primers and probes in the conserved region of pathogen genes for targeted detection with strong specificity; combined with droplet digital PCR technology, 10 diarrhea-causing pathogens in stool specimens can be quickly detected in two PCR reaction systems with high sensitivity; reduce costs and shorten detection time, solve the pain points of conventional PCR in detecting multiple pathogens in stool specimens at the same time, overcome the difficulty of being easily interfered by inhibitors in stool specimen detection, and provide a reliable laboratory basis for diagnosis and treatment of children, epidemic monitoring and infection control.

[0015] The present invention provides a primer probe set for detecting diarrhea pathogens in children based on digital PCR, which is used for simultaneously detecting 10 diarrhea pathogens of common diarrhea in children, wherein the 10 diarrhea pathogens include: 5 bacteria and 5 viruses; wherein the 5 bacteria include: Escherichia coli O157:H7, Salmonella typhimurium, Shigella, Campylobacter jejuni (C.jejuni), Vibrio parahemolyticus; the 5 viruses include: human rotavirus (HRV), norovirus GII type (norovirus GII), astrovirus (astrovirus), enteric adenovirus (EAV) and Sapovirus;

[0016] The primer-probe set includes primers and probes specific to the 10 diarrheal pathogens and a human internal reference gene (β2-microglobulin (B2M) gene); the primers and probes for the five bacteria and the primers and probes for the human internal reference gene are designed in the same PCR reaction system, and the primers and probes for the five viruses are designed in another PCR reaction system;

[0017] The nucleotide sequences of the primers and probes are shown in Table 1:

[0018] Table 1

[0019]

[0020]

[0021] In Table 1, F represents forward primer, R represents reverse primer, Probe represents probe, and the sequences in the table include fluorescent groups and minor groove binders (MGB); D represents degenerate bases, including G / A / T.

[0022] Furthermore, the design regions of the primers and probes for the five bacteria and the primers and probes for the five viruses are in the conserved regions of pathogen genes, as shown in Table 2:

[0023] Table 2

[0024]

[0025] The present invention also provides a reagent for detecting diarrheal pathogens in children based on digital PCR, comprising the primer probe set as described above.

[0026] The present invention also provides a detection method for detecting children's diarrhea pathogens based on digital PCR, using the primer probe set for detecting children's diarrhea pathogens based on digital PCR as described above and the reagent for detecting children's diarrhea pathogens based on digital PCR as described above, comprising the following steps:

[0027] S1. Nucleic acid extraction, including: using Tianlong GeneRotex 96 fully automatic rotary nucleic acid extractor and matching magnetic bead extraction reagents to extract fecal nucleic acids including DNA and RNA nucleic acids of bacteria and viruses;

[0028] S2. Configure the digital PCR reaction system as shown in Table 3:

[0029] Table 3

[0030]

[0031] S3, using an integrated chip-based digital PCR instrument for droplet generation, amplification, reading and analysis to obtain the copy concentrations of the five bacteria, five viruses and human-derived internal reference genes;

[0032] Reaction program: 53°C for 5 min; 95°C for 20 sec; followed by 5 cycles, each cycle including 95°C for 3 sec, 62°C for 30 sec, followed by 35 cycles, each cycle including 95°C for 3 sec, 58°C for 30 sec;

[0033] S4. Construct a multiplex digital PCR method and optimize the reaction system: using the recombinant plasmid mixed with the five bacteria, five viruses and human internal reference genes as a template, perform digital PCR detection according to steps S2-S3;

[0034] The test results of the embodiment of the present invention show that the genes of 5 bacteria, 5 viruses and human internal reference genes are specifically amplified only in their respective primer probes, indicating that the primers and probes of the digital PCR system have good specificity and can be used for subsequent research;

[0035] S5. Performing digital PCR specificity verification on the primers and probes for the five bacteria and five viruses;

[0036] The embodiments of the present invention use a single positive bacterial or viral sample as a control, so that only the positive sample is amplified and no non-specific amplification occurs in other channels. The experiments are performed in this way to verify the specificity of primers and probes. The verification results show that 5 types of bacteria and 5 types of viruses are specifically amplified only in their respective primer probes, indicating that the primers and probes of the digital PCR system have good specificity;

[0037] S6. Perform digital PCR sensitivity verification on the primers and probes of the five bacteria and five viruses.

[0038] Preferably, 5 bacteria and 5 viral plasmid DNA templates are mixed and diluted in 4 gradients of 10, 100, 1000, and 10000 copy / reaction to obtain sample templates, which are amplified in an optimized multiplex digital PCR reaction system.

[0039] The verification results showed that the detection limits LOD of the five viral plasmid DNA templates all reached 10 copy / reaction, and the linear relationship R 2 All of them were greater than 0.999, and the linear relationship was good.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] The primer probe set, reagent and detection method for detecting diarrhea pathogens in children based on digital PCR provided by the present invention can simultaneously detect 5 diarrhea-related viruses and their subtypes including human rotavirus, norovirus GII type, astrovirus, enteric adenovirus, and zaruvirus in stool specimens, as well as 5 common diarrhea-causing bacteria including Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni, and Vibrio parahaemolyticus; specific primers and probes are designed in the conserved region of pathogen genes for targeted detection with strong specificity; combined with droplet digital PCR technology, 10 diarrhea-causing pathogens in stool specimens can be quickly detected in two PCR reaction systems, and the detection limit of the droplet digital PCR method is 10 copies / reaction, with high sensitivity, which greatly reduces costs and shortens detection time, solves the pain point of conventional PCR in detecting multiple pathogens in stool specimens at the same time, overcomes the difficulty of being easily interfered by inhibitors in stool specimen detection, and provides a reliable laboratory basis for diagnosis and treatment of children, epidemic monitoring and infection control, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Various other advantages and benefits will become apparent to those of ordinary skill in the art by reading the following detailed description of the preferred embodiment.The drawings are only for the purpose of illustrating the preferred embodiments and are not to be construed as limiting the invention.

[0043] In the attached picture:

[0044] Figure 1 This is a graph showing the detection results of five types of bacteria detected by digital PCR in an embodiment of the present invention;

[0045] Figure 2 This is a graph showing the detection results of five viruses detected by digital PCR in an embodiment of the present invention;

[0046] Figure 3 Specific amplification diagram of the five bacteria in the examples of the present invention in the primer probes of the respective bacteria;

[0047] Figure 4 Specific amplification diagram of the five viruses in the examples of the present invention in the primer probes of the respective viruses;

[0048] Figure 5 This is a digital PCR sensitivity verification diagram of the primer probes of 5 bacteria in the embodiment of the present invention;

[0049] Figure 6 This is a digital PCR sensitivity verification diagram of the primer probes for 5 viruses in the examples of the present invention. DETAILED DESCRIPTION

[0050] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, the same numbers in different drawings represent the same or similar elements unless otherwise indicated. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present disclosure. Instead, they are merely examples of devices and products consistent with some aspects of the present disclosure as detailed in the appended claims.

[0051] The terms used in this disclosure are for the purpose of describing specific embodiments only and are not intended to limit the disclosure. The singular forms of "a", "said" and "the" used in this disclosure and the appended claims are also intended to include plural forms unless the context clearly indicates otherwise. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0052] It should be understood that although the terms first, second, third, etc. may be used in the present disclosure to describe various information, such information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present disclosure, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0053] The embodiments of the present invention are described in further detail below.

[0054] The embodiment of the present invention provides a primer probe set for detecting diarrhea pathogens in children based on digital PCR, which is used to simultaneously detect 10 diarrhea pathogens of common diarrhea in children, wherein the 10 diarrhea pathogens include: 5 bacteria and 5 viruses; wherein the 5 bacteria include: Escherichia coli O157:H7, Salmonella typhimurium, Shigella, Campylobacter jejuni (C.jejuni), Vibrio Parahemolyticus; the 5 viruses include: human rotavirus (HRV), norovirus GII type (norovirus GII), astrovirus (astrovirus), enteric adenovirus (EAV) and Sapovirus;

[0055] The primer-probe set includes primers and probes specific to the 10 diarrheal pathogens and human-derived internal reference genes; the primers and probes for the five bacteria and the primers and probes for the human-derived internal reference genes are designed in the same PCR reaction system, and the primers and probes for the five viruses are designed in another PCR reaction system;

[0056] The nucleotide sequences of the primers and probes are shown in Table 1:

[0057] Table 1

[0058] In Table 1, F represents forward primer, R represents reverse primer, Probe represents probe, and the sequences in the table include fluorescent groups and minor groove binders (MGB); D represents degenerate bases, including G / A / T.

[0059] Note: The complete sequence of human rotavirus detection probe No. 3 is "ROX-AGTTAAAAGCTAACACTGTCAAA-MGB", where: "ROX" is the fluorescent label at the 5' end, used for signal detection; "MGB" is the minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of the Norovirus GII type detection probe No. 6 is "FAM-TGGGAGGGCGATCGCAATCT-MGB", where: "FAM" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve the probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of the astrovirus detection probe No. 9 is "HEX-CACAGAAGAGCAACTCCATCGC-MGB", where: "HEX" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of enteric adenovirus detection probe No. 12 is "Quasar705-CTGACACGGGCACTCTTCGC-MGB", where: "Quasar705" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of the Zaru virus detection probe No. 15 is "CY5-TGGTTCATAGGTGGTGC-MGB", where: "CY5" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of the Escherichia coli O157:H7 detection probe No. 18 is "CY5-ACTAGGACCGCAGAGGAAAGAGAGGAA-MGB", where: "CY5" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of the Salmonella typhi detection probe No. 21 is "FAM-TCTGCTGAAGTTGAGGATGT-MGB", where: "FAM" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of Shigella detection probe No. 24 is "ROX-TGCGGAGGTCATTTGCTGTCACT-MGB", where: "ROX" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of Campylobacter jejuni detection probe No. 27 is "Quasar705-AGCGATGTTGGAATTCAATGT-MGB", where: "Quasar705" is a fluorescent marker at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of Vibrio parahaemolyticus detection probe 30 is "HEX-CAATGTAAAGGTCTCTGACT-MGB", wherein: "HEX" is a fluorescent label at the 5' end, used for signal detection; "MGB" is a minor groove binder at the 3' end, used to improve probe binding specificity, shorten the probe length and quench the fluorescent signal; The complete sequence of the human reference gene (β2-microglobulin (B2M) gene) detection probe No. 33 is "Cy5 / ROX / ATGTGTCTGGGTTTCATCCATCCGACA-MGB", where: "Cy5" and "ROX" are fluorescent labels at the 5' end for signal detection; "MGB" is a minor groove binder at the 3' end, which is used to improve the probe binding specificity, shorten the probe length and quench the fluorescent signal; Due to the limitation of WIPO Sequence software, the above modification marks cannot be displayed in the computer-readable sequence table, but their functions and effects have been described in detail in the specification.

[0060] The design regions of the primers and probes for the five bacteria and the primers and probes for the five viruses are in the conserved regions of pathogen genes, as shown in Table 2:

[0061] Table 2

[0062]

[0063]

[0064] The embodiment of the present invention also provides a reagent for detecting diarrheal pathogens in children based on digital PCR, including the primer probe set as described above.

[0065] The embodiment of the present invention also provides a detection method for detecting diarrheal pathogens in children based on digital PCR, using the primer probe set for detecting diarrheal pathogens in children based on digital PCR as described above and the reagent for detecting diarrheal pathogens in children based on digital PCR as described above, comprising the following steps:

[0066] S1. Nucleic acid extraction, including: using Tianlong GeneRotex 96 fully automatic rotary nucleic acid extractor and matching magnetic bead extraction reagents to extract fecal nucleic acids including DNA and RNA nucleic acids of bacteria and viruses;

[0067] S2. Configure the digital PCR reaction system as shown in Table 3:

[0068] Table 3

[0069]

[0070] S3, using an integrated chip-based digital PCR instrument for droplet generation, amplification, reading and analysis to obtain the copy concentrations of the five bacteria, five viruses and human-derived internal reference genes;

[0071] Reaction program: 53°C for 5 min; 95°C for 20 sec; followed by 5 cycles, each cycle including 95°C for 3 sec, 62°C for 30 sec, followed by 35 cycles, each cycle including 95°C for 3 sec, 58°C for 30 sec;

[0072] S4. Construct a multiplex digital PCR method and optimize the reaction system: Use the recombinant plasmid mixed with the 5 bacteria, 5 viruses and human internal reference genes as a template and perform digital PCR detection according to steps S2-S3; the detection results are as follows: Figure 1As shown, the genes of 5 bacteria, 5 viruses and human internal reference genes were specifically amplified only in their respective primer probes, indicating that the primers and probes of this digital PCR system have good specificity and can be used for subsequent research;

[0073] Figure 1 The test results are based on 5 bacterial and human internal reference gene recombinant plasmids as templates. Figure 2 These are the test results using 5 viral recombinant plasmids as templates.

[0074] right Figure 1 , Figure 2 The name symbols in are annotated as follows:

[0075] O157: Escherichia coli O157:H7; ST: Salmonella Typhi, Shigella: Shigella, C.jejuni: Campylobacter jejuni, VP: Vibrio parahaemolyticus; HRV: human rotavirus, NOV-GII: Norovirus GII type, Astrovirus: Astrovirus, EAV: Enteric adenovirus; Sapovirus: Sapovirus.

[0076] S5. Performing digital PCR specificity verification on the primers and probes for the five bacteria and five viruses;

[0077] In this embodiment, by using a single positive bacterial or viral sample as a control, only the positive sample is amplified, and other channels have no non-specific amplification, and the experiment is performed in this way to verify the specificity of the primers and probes. Digital PCR detection is performed according to the above steps.

[0078] The results are as follows Figure 3 , Figure 4 As shown, 5 bacteria and 5 viruses were specifically amplified only in their respective primer probes, indicating that the primers and probes of the digital PCR system had good specificity;

[0079] Figure 3 , Figure 4 They are the test results of single positive samples of 5 kinds of bacteria and 5 kinds of viruses.

[0080] right Figure 3 , Figure 4 The name symbols in are annotated as follows:

[0081] O157: Escherichia coli O157:H7; ST: Salmonella typhi, Shigella: Shigella, C.jejuni: Campylobacter jejuni, VP: Vibrio parahaemolyticus; HRV: human rotavirus, NOV-GII: Norovirus GII type, Astrovirus: Astrovirus, EAV: Enteric adenovirus; Sapovirus: Sapovirus;

[0082] S6. Perform digital PCR sensitivity verification on the primers and probes of the five bacteria and five viruses.

[0083] Five bacterial and five viral plasmid DNA templates were mixed and diluted in four gradients of 10, 100, 1000, and 10000 copies / reaction to obtain sample templates, which were amplified in the optimized multiplex digital PCR reaction system. The results are shown in Figure 2. Figure 5 , Figure 6 As shown in the figure, the detection limits LOD of the five viral plasmid DNA templates all reached 10 copy / reaction, and the linear relationship R 2 All of them were greater than 0.999, and the linear relationship was good.

[0084] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments, but it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

[0085] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A primer probe set for detecting diarrheal pathogens in children based on digital PCR, characterized in that: Used to simultaneously detect 10 common diarrheal pathogens in children, the 10 diarrheal pathogens include: 5 bacteria and 5 viruses; wherein the 5 bacteria include: Escherichia coli O157:H7, Salmonella typhi, Shigella, Campylobacter jejuni, Vibrio parahaemolyticus; the 5 viruses include: human rotavirus, norovirus GII type, astrovirus, enteroadenovirus and zaruvirus; The primer-probe set includes primers and probes specific to the 10 diarrheal pathogens and human-derived internal reference genes; the primers and probes for the five bacteria and the primers and probes for the human-derived internal reference genes are designed in the same PCR reaction system, and the primers and probes for the five viruses are designed in another PCR reaction system; The nucleotide sequences of the primers and probes are shown in Table 1: Table 1 ； In Table 1, F represents forward primer, R represents reverse primer, Probe represents probe, and the sequences in the table include fluorescent groups and minor groove binders (MGB); D represents degenerate bases, including G / A / T.

2. A primer probe set for detecting diarrheal pathogens in children based on digital PCR, characterized in that: The design regions of the primers and probes for the five bacteria and the primers and probes for the five viruses are in the conserved regions of pathogen genes, as shown in Table 2: Table 2 。 3. A reagent for detecting diarrheal pathogens in children based on digital PCR, characterized in that: Comprising the primer probe set as described in claim 1 or 2.

4. A method for detecting diarrheal pathogens in children based on digital PCR, using the primer probe set for detecting diarrheal pathogens in children based on digital PCR as claimed in claim 1 or 2 and the reagent for detecting diarrheal pathogens in children based on digital PCR as claimed in claim 3, characterized in that: The following steps are involved: S1. Nucleic acid extraction, including: using Tianlong GeneRotex 96 fully automatic rotary nucleic acid extractor and matching magnetic bead extraction reagents to extract fecal nucleic acids including DNA and RNA nucleic acids of bacteria and viruses; S2. Configure the digital PCR reaction system as shown in Table 3: Table 3 ； S3, using an integrated chip-based digital PCR instrument for droplet generation, amplification, reading and analysis to obtain the copy concentrations of the five bacteria, five viruses and human-derived internal reference genes; Reaction program: 53°C for 5 min; 95°C for 20 sec; followed by 5 cycles, each cycle including 95°C for 3 sec, 62°C for 30 sec, followed by 35 cycles, each cycle including 95°C for 3 sec, 58°C for 30 sec; S4. Construct a multiplex digital PCR method and optimize the reaction system: using the recombinant plasmid mixed with the five bacteria, five viruses and human internal reference genes as a template, perform digital PCR detection according to steps S2-S3; S5. Performing digital PCR specificity verification on the primers and probes for the five bacteria and five viruses; S6. Perform digital PCR sensitivity verification on the primers and probes of the five bacteria and five viruses.

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