CN114836581B - Primer combination for detecting pathogens of digestive tract infectious diseases - Google Patents
Primer combination for detecting pathogens of digestive tract infectious diseases Download PDFInfo
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- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
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Abstract
The invention discloses a primer combination for detecting pathogens of digestive tract infectious diseases, which comprises specific primers and TaqMan probes for detecting EB virus, cytomegalovirus, proteus mirabilis, cryptosporidium, salmonella typhi, moraxella catarrhalis, yersinia small intestine and adenovirus; the method of the invention can detect the 3 viruses, 4 bacteria and 1 parasite at the same time; the kit has the advantages of high sensitivity, strong specificity, simple operation, short time and low cost, and has important significance for clinical early diagnosis of patients suffering from digestive tract infection.
Description
Technical Field
The invention belongs to the technical field of pathogen detection, and particularly relates to a primer and a TaqMan probe for simultaneously detecting 8 pathogens of digestive tract infectious diseases.
Background
Digestive tract diseases refer to diseases of the digestive system of human bodies, and are mainly caused by infection due to consumption of water or food polluted by pathogens, wherein the pathogens are discharged out of the body of a patient or a carrier along with excreta, and the pathogens pollute hands, water, food and tableware by living contact to eat into the body. The disease has high incidence rate and wide incidence range, can appear in people of all ages, is easy to repeatedly attack, and has great influence on the physical health of patients. Digestive tract infection is a common disease, most often causing infectious diarrhea, and is mostly caused by bacterial, fungal, viral, parasitic and other infections. Common digestive tract infectious diseases include bacillary dysentery, poliomyelitis (i.e. poliomyelitis), typhoid, paratyphoid, cholera, paramylosis, amebic dysentery, various enterovirus infections (such as Coxsackie virus, epstein-Barr virus, etc.), bacterial food poisoning, various intestinal parasitic diseases (such as ascariasis, taeniasis, enterobiasis, fasciolopsis, etc.), etc. The pathogenic bacteria are various, and the pathogenic bacteria and normal flora are symbiotic, and the pathogenic effects are different. Therefore, the etiology is correctly searched in time, and the significance of formulating a reasonable treatment scheme is great.
There are many methods for detecting pathogens in digestive tract clinically at present, such as traditional direct microscopic examination of specimens, pathogen isolation and culture, immunological examination and the like. At present, the isolated culture method is still used as a gold standard for identifying pathogens, bacterial infection can be cultured and proliferated by using a common culture medium and a selective culture medium, and the detection and identification are convenient. The virus pathogen can be cultured by monkey kidney, human embryo kidney, human amniotic cell, hela and other passage cells, but the method has the advantages of long time consumption, low detection rate, complicated steps, high requirement on conditions, and high requirement on detection personnel with abundant experience, and is difficult to complete in a general laboratory. The immunological detection is to detect the antigens of bacteria, viruses and parasites in the feces and the specific antibodies in the serum by enzyme-linked immunosorbent assay (ELISA), solid-phase radioimmunoassay, latex agglutination assay, chemiluminescence immunoassay and other methods, but the method has a certain window period, needs a certain detection time and has lower detection rate. In recent years, with the development of molecular biology, nucleic acid detection technologies such as loop-mediated isothermal amplification, gene chip technology, digital PCR technology, NGS sequencing technology and real-time fluorescent quantitative PCR technology have been developed. The technology has the advantages of high sensitivity and strong specificity, can detect pathogen nucleic acid, but can easily generate false positive, such as LAPM technology, gene chip, digital PCR technology and the like, and the gene chip has high flux but high price and poor repeatability.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a primer combination for detecting pathogens of digestive tract infectious diseases, which comprises specific primers for detecting EB virus, cytomegalovirus, proteus mirabilis, cryptosporidium, salmonella typhi, moraxella catarrhalis, yersinia small intestine and adenovirus and TaqMan probes, wherein the primer combination is used in multiple qPCR, and a conventional detection reagent of multiple qPCR is also involved in detection.
The specific primers are SEQ ID NO. 1 and SEQ ID NO. 2 for EB virus, SEQ ID NO. 4 and SEQ ID NO. 5 for cytomegalovirus, SEQ ID NO. 7 and SEQ ID NO. 8 for Proteus mirabilis, SEQ ID NO. 10 and SEQ ID NO. 11 for Cryptosporidium, SEQ ID NO. 13 and SEQ ID NO. 14 for Salmonella typhi, SEQ ID NO. 16 and SEQ ID NO. 17 for Moraxella catarrhalis, SEQ ID NO. 19 and SEQ ID NO. 20 for Yersinia small intestine, SEQ ID NO. 22 and SEQ ID NO. 23 for adenovirus;
the TaqMan probes are SEQ ID NO. 3 for EB virus, SEQ ID NO. 6 for cytomegalovirus, SEQ ID NO. 9 for Proteus mirabilis, SEQ ID NO. 12 for Cryptosporidium, SEQ ID NO. 15 for Salmonella typhi, SEQ ID NO. 18 for Moraxella catarrhalis, SEQ ID NO. 21 for Yersinia small intestine and SEQ ID NO. 24 for adenovirus.
Fluorophores that label the TaqMan probes include, but are not limited to FAM, ROX, TAMRA, CY5; labels that quench fluorophores include, but are not limited to, BHQ1, BHQ2, DABCYL.
The method of using the above primer combination is as follows:
1. extracting sample DNA, wherein the sample is feces or anal swab;
2. respectively amplifying target genes by qPCR by taking the DNA extracted in the step 1 as a template;
the amplification system 40. Mu.L was: mix 20 μl, 1 μl each of the upstream primer, 1 μl each of the downstream primer, 5 μl each of the template, and the remainder was filled with water; mixing the PCR amplification system with the DNA extracted in the step 1, adding the mixture to eight rows for PCR reaction under the reaction condition of 30s pre-denaturation at 95 ℃, and then sequentially carrying out 45 cycles at 95 ℃ for 5 s and 58 ℃ for 30s (reading fluorescence);
3. analysis of PCR results
Detecting and interpreting detection results of EB virus, cytomegalovirus, proteus mirabilis, cryptosporidium, salmonella typhi, moraxella catarrhalis, yersinia small intestine and adenovirus according to the amplification curve obtained in the step 2, and judging whether a sample contains pathogen infection to be detected;
the positive result interpretation includes: (1) The Ct value of the internal reference gene is less than or equal to 36, and the negative control group and the template-free control group have no Ct value; if the detection result does not meet the requirement, the mqPCR detection is carried out again, or the nucleic acid is extracted again to carry out the mqPCR detection; (2) The Ct value of the pathogen is less than or equal to 36.0, and if the Ct value is more than 36.0, single qPCR verification is required to be carried out on the pathogen; (3) the amplification curve is standard "S" and free of abnormal fluctuations.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention can rapidly, accurately and sensitively detect a plurality of pathogens in the digestive tract by applying a multiplex fluorescence quantitative PCR technology, has high detection sensitivity and good specificity, and has the advantages of high flux, short time, low cost and the like; the method can qualitatively detect and distinguish the digestive tract, can detect the 3 viruses, the 4 bacteria and the 1 parasite at the same time, has wide coverage, and the detection result can be used for clinical diagnosis and treatment.
Drawings
FIG. 1 shows the results of a single RT-qPCR specificity test of EB virus;
FIG. 2 shows the results of a cytomegalovirus single RT-qPCR specificity assay;
FIG. 3 shows the results of a single qPCR specificity test for Proteus mirabilis;
FIG. 4 shows the results of a single qPCR specificity assay for Cryptosporidium;
FIG. 5 shows the results of a single qPCR specificity test for Salmonella typhi;
FIG. 6 shows the results of a single qPCR specificity test of Moraxella catarrhalis;
FIG. 7 is the results of a single qPCR specificity test of Yersinia small intestine;
FIG. 8 is a single qPCR specificity test result of adenovirus;
FIG. 9 shows the results of a single qPCR specificity assay for GAPDH;
FIG. 10 shows the results of multiplex RT-qPCR specificity assays for EB virus, cytomegalovirus, proteus mirabilis, cryptosporidium and GAPDH;
FIG. 11 is a graph showing the results of multiplex qPCR specificity assays for Salmonella typhi, moraxella catarrhalis, yersinia small intestine, adenovirus, and GAPDH;
FIG. 12 shows the results of a sensitivity test of multiplex fluorescence quantitative PCR of EB virus and cytomegalovirus;
FIG. 13 shows the results of a sensitivity test of multiplex fluorescence quantitative PCR for Proteus mirabilis and Cryptosporidium;
FIG. 14 sensitivity test results of multiplex fluorescence quantitative PCR for Salmonella typhi, moraxella catarrhalis;
FIG. 15 is a sensitivity test result of multiplex fluorescence quantitative PCR for Yersinia small intestine and adenovirus;
FIG. 16 shows the results of a sensitivity experiment for multiplex qPCR of housekeeping gene GAPDH.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described by the following specific embodiments, but the invention is not limited to the scope of the examples; the materials used in the examples below are not limited to the above list, but may be replaced with other similar materials, and the apparatus is not specified, and the person skilled in the art should be aware of the use of conventional materials and apparatus according to conventional conditions, or according to conditions suggested by the manufacturer.
Example 1: design of primer probes
1. Since there are conserved regions in the pathogen genome, the NCBI (national center for Biotechnology information) website
The internal reference sequence of the medium downloaded pathogen is as follows, EB virus NA1 coding gene, cytomegalovirus imate-early protein coding gene, proteus mirabilis ureR gene, cryptosporidium oocyst wall protein coding gene, salmonella typhi fimbrial protein coding gene, moraxella catarrhalis M protein coding gene, yersinia small intestine tadF gene, adenovirus hexon protein coding gene; nucleotide sequence comparison is carried out through Mega5.0 software, a conserved region sequence is selected as a primer probe sequence, analysis is carried out through PrimerSelect analysis software, and the primer probe is initially designed after the following conditions are met.
a. Length of the gene fragment of interest: 50-150 bp;
b. tm value: the Tm value of the probe is 8-10 ℃ higher than that of the primer, wherein the Tm value of the probe is 60 ℃;
c. G+C content: typically not less than 40%;
d. the first base at the 5' end of the probe cannot be G;
c. primer dimer is not generated, and the hairpin structure software evaluation result is OK;
2. and (3) comparing the primer probe nucleotide sequences which are preliminarily designed by using BLAST search functions in NCBI website, and selecting primers and probe sequences with high specificity.
Based on pathogen nucleic acid types, the 5' -end fluorescent group of the 8 digestive tract infectious disease pathogens TaqMan probe is FAM, ROX, TAMRA, CY; 3' end quenching fluorophore selection: selecting 3' -end quenching fluorescent groups to select BHQ1, BHQ2 and DABCYL; finally, the nucleotide sequences of specific primers and probes of 8 digestive tract infectious pathogens and housekeeping genes are obtained, as shown in EQ ID NO: 1-SEQ ID NO: 27, and the following table is provided:
。
4. construction of plasmids
The specific sequences of 8 pathogens (SEQ ID NO: 28-SEQ ID NO: 36) and the sequence of the internal reference gene GAPDH are connected with a pUC57 vector to synthesize a plasmid standard, and plasmid construction is completed by Zhongmeitai and biotechnology Beijing Co., ltd; wherein salmonella typhi and EB virus, yersinia small intestine and proteus mirabilis, adenovirus and GAPDH, cryptosporidium and catarrhal moraxella are synthesized on one plasmid, cytomegalovirus is synthesized on one plasmid alone, concentration measurement is carried out by an ultraviolet spectrophotometer, and the copy number of the plasmid is calculated according to the length and concentration of each plasmid;
copy number results are shown in the following table:
the plasmid is diluted in a gradient way according to a 10-fold dilution method, and six gradients are respectively set to be 10 6 、10 5 、10 4 、10 3 、10 2 On the order of 10 copies/. Mu.L.
Example 2: qPCR amplification and specificity, sensitivity, reproducibility assays
1. Single fluorescent quantitative PCR
Concentration is set to 10 3 Taking 10 mu L of plasmid standard substances of the copies/mu L as templates, respectively, carrying out single fluorescent quantitative PCR detection by using a qPCR kit of a Pro Taq HS premix type probe method of Ai Kerui biological company, and respectively detecting 8 mixed plasmid templates of pathogens by using specific primers and probes of each pathogen, wherein the detection results are shown in figures 1-9; from the results, it can be seen that the amplification curves are all found for 8 pathogens and the reference genes.
2. Multiplex qPCR pathogen grouping and specificity assays
The 8 pathogens were grouped together into 2 groups, see table below:
；
since multiplex fluorescent quantitative PCR is required to detect 4 pathogens and 1 reference gene in one system, the multiplex fluorescent quantitative PCR reaction system is 40. Mu.L, wherein 1. Mu.L of primer probes of the 4 pathogens and 1 reference gene are added, and the template is 4. Mu.L:
the amplification reaction procedure was: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 3s, annealing at 58℃and extension for 30s,40 cycles, fluorescence signals were collected during the annealing extension phase of each cycle.
Concentration is set to 10 3 10 mu L of plasmid standard products with the magnitude of cobies/mu L are respectively taken and mixed as templates, a Ai Kerui biological company Pro Taq HS premixed probe method qPCR kit is used for carrying out multiplex fluorescence quantitative PCR detection, and each group of 4 pathogen primers and probes are respectively used for detecting 8 pathogen mixed plasmid templates, and the results are shown in figures 10-11; from the above results, it can be seen that there is no cross reaction between each group of pathogens, indicating that multiplex fluorescence quantitative PCR has good specificity.
3. Multiplex qPCR sensitivity assay
Gradient 10 by MqPCR method 6 、10 5 、10 4 、10 3 、10 2 The plasmid template of 10 copies/. Mu.L is detected, the lowest plasmid concentration which can be detected by the MqPCR detection method is determined, the result is shown in figures 12-16, and the detection limit of EB virus, cytomegalovirus, proteus mirabilis and Cryptosporidium can be seen from figures 12-13 to reach the order of 10 copies/. Mu.L; FIGS. 14-15 show that Salmonella typhi, moraxella catarrhalis, yersinia small intestine, and adenovirus detection limits reached the order of 10 copies/. Mu.L; the results in FIG. 16 show that the limit of detection of the reference GADPH gene is on the order of 10 copies/. Mu.L.
4. Multiplex qPCR reproducibility assay
To verify the reproducibility of the MqPCR detection method,with 10 3 Experiment is carried out by taking the copies/mu L magnitude plasmid as a template, and the repeated experiments in the group and between the groups are respectively carried out; the specific primer and probe of each group of pathogens are used for detecting the plasmid template, the detection is repeated three times at the same time, the Ct value is observed and recorded, the repeated detection is carried out once every week, the repeated detection is carried out for three weeks continuously, the Ct value is observed and recorded, and the repeated results are shown in the following table:
。
example 3: fecal sample or anal swab sample detection
1. Sample collection
10 stool samples or anal swabs clinically diagnosed as infection with digestive tract pathogens and verified as positive by bacterial culture or first generation sequencing were selected, and 6 stool samples or anal swabs verified as negative by bacterial culture or first generation sequencing.
The patient is in compliance with the prompt of clinician, and fresh feces 3-5g discharged naturally is left or the sampling swab is put into normal saline to be soaked, the collector is put into anus for about 2cm, the collector is rubbed by rotating for several circles, and the collector is taken out and immediately put into a sterile tube for temporary storage at-80 ℃ for long-term storage until detection.
2. Genomic DNA extraction
In this example, the inactivated Virus culture solution and the bacterial culture solution were subjected to DNA extraction using a commercial TGuidE Virus DNA/RNA Kit, and the extraction method was referred to the instructions of the Kit.
3. The 16 clinical samples are subjected to multiple fluorescent quantitative PCR detection by the detection reagent, and the multiple real-time fluorescent quantitative PCR technology is compared with the bacterial culture and first-generation sequencing results, and the results are shown in the following table:
the table shows that the pathogen which can not be detected by bacterial culture or first-generation sequencing can be detected by the detection reagent of the pathogen of the digestive tract infectious disease, and the result of bacterial culture or first-generation sequencing can be supplemented; the invention shows that the multiplex real-time fluorescent quantitative PCR of 8 digestive tract infectious pathogens and internal reference GAPDH genes has good application value.
Sequence listing
<110> university of Kunming engineering
<120> primer set for detecting pathogens of digestive tract infectious diseases
<160> 36
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> Artificial sequence (Artifical)
<400> 1
aggatgcgat taaggacctt gtt 23
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 2
ggaaaccagg gaggcaaatc t 21
<210> 3
<211> 24
<212> DNA
<213> Artificial sequence (Artifical)
<400> 3
tgacaaagcc cgctcctacc tgca 24
<210> 4
<211> 24
<212> DNA
<213> Artificial sequence (Artifical)
<400> 4
tcatccacac taggagagca gact 24
<210> 5
<211> 17
<212> DNA
<213> Artificial sequence (Artifical)
<400> 5
gccaagcggc ctctgat 17
<210> 6
<211> 28
<212> DNA
<213> Artificial sequence (Artifical)
<400> 6
actgggcaaa gaccttcatg cagatctc 28
<210> 7
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<400> 7
ccatcagatt atgtcattca a 21
<210> 8
<211> 23
<212> DNA
<213> Artificial sequence (Artifical)
<400> 8
gaggaaaatg caatttatct tta 23
<210> 9
<211> 25
<212> DNA
<213> Artificial sequence (Artifical)
<400> 9
cacaccctac ccaacattca tttca 25
<210> 10
<211> 23
<212> DNA
<213> Artificial sequence (Artifical)
<400> 10
caaggcctcc aatgtataca aac 23
<210> 11
<211> 23
<212> DNA
<213> Artificial sequence (Artifical)
<400> 11
cgattgcttg gactgaaata cag 23
<210> 12
<211> 28
<212> DNA
<213> Artificial sequence (Artifical)
<400> 12
cctgtctgcc ctccaggtac agtattac 28
<210> 13
<211> 22
<212> DNA
<213> Artificial sequence (Artifical)
<400> 13
cgaagtcaga gtcgacatag gc 22
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence (Artifical)
<400> 14
aagacctcaa cgccgatcac 20
<210> 15
<211> 25
<212> DNA
<213> Artificial sequence (Artifical)
<400> 15
catttgttct ggagcaggct gacgg 25
<210> 16
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 16
gtgagtgccg cttttacaac c 21
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence (Artifical)
<400> 17
tgtatcgcct gccaagacaa 20
<210> 18
<211> 27
<212> DNA
<213> Artificial sequence (Artifical)
<400> 18
tgcttttgca gctgttagcc agcctaa 27
<210> 19
<211> 19
<212> DNA
<213> Artificial sequence (Artifical)
<400> 19
gacgatacct tggtatagc 19
<210> 20
<211> 19
<212> DNA
<213> Artificial sequence (Artifical)
<400> 20
gacgatacct tggtatagc 19
<210> 21
<211> 30
<212> DNA
<213> Artificial sequence (Artifical)
<400> 21
ccggaaccga cacatcagtg ctaaatagat 30
<210> 22
<211> 23
<212> DNA
<213> Artificial sequence (Artifical)
<400> 22
gccscagtgg tcttacatgc aca 23
<210> 23
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 23
caccgtgggg tttctaaact t 21
<210> 24
<211> 22
<212> DNA
<213> Artificial sequence (Artifical)
<400> 24
tacctgagcc ccgggctggt gc 22
<210> 25
<211> 19
<212> DNA
<213> Artificial sequence (Artifical)
<400> 25
gaaggtgaag gtcggagtc 19
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence (Artifical)
<400> 26
gaagatggtg atgggatttc 20
<210> 27
<211> 21
<212> DNA
<213> Artificial sequence (Artifical)
<400> 27
acggatttgg tcgtattggg c 21
<210> 28
<211> 106
<212> DNA
<213> Artificial sequence (Artifical)
<400> 28
aggatgcgat taaggacctt gttatgacaa agcccgctcc tacctgcaat atcagggtga 60
ctgtgtgcag ctttgacgat ggagtagatt tgcctccctg gtttcc 106
<210> 29
<211> 137
<212> DNA
<213> Artificial sequence (Artifical)
<400> 29
tcatccacac taggagagca gactctcaaa ggatcggccc ccagaatgta ctgggcaaag 60
accttcatgc agatctcctc aatgcggcgc ttcattacac taataacctc aggcttggtt 120
atcagaggcc gcttggc 137
<210> 30
<211> 88
<212> DNA
<213> Artificial sequence (Artifical)
<400> 30
ccatcagatt atgtcattca aaatcaccga gaaaataatg aaatgaatgt tgggtagggt 60
gtgaataaag ataaattgca ttttcctc 88
<210> 31
<211> 92
<212> DNA
<213> Artificial sequence (Artifical)
<400> 31
caaggcctcc aatgtataca aactagttct gctccaactc aacctgtctg ccctccaggt 60
acagtattac aagataatgc ctgtatttca gt 92
<210> 32
<211> 130
<212> DNA
<213> Artificial sequence (Artifical)
<400> 32
cgaagtcaga gtcgacatag gcatagattt tcaggccata cattaatttg ccaaggttgc 60
tataaacatt tgttctggag caggctgacg gaaattccgt gaactcgctg gtgatcggcg 120
ttgaggtctt 130
<210> 33
<211> 72
<212> DNA
<213> Artificial sequence (Artifical)
<400> 33
gtgagtgccg cttttacaac cactgctttt gcagctgtta gccagcctaa ggttgtcttg 60
gcaggcgata ca 72
<210> 34
<211> 69
<212> DNA
<213> Artificial sequence (Artifical)
<400> 34
gacgatacct tggtatagca atctatttag cactgatgtg tcggttccgg caataaagtc 60
atcagctat 69
<210> 35
<211> 130
<212> DNA
<213> Artificial sequence (Artifical)
<400> 35
gccgcagtgg tcttacatgc acatctcggg ccaggacgcc tcggagtacc tgagccccgg 60
gctggtgcag tttgcccgcg ccaccgagac gtacttcagc ctgaataaca agtttagaaa 120
ccccacggtg 130
<210> 36
<211> 226
<212> DNA
<213> Artificial sequence (Artifical)
<400> 36
gaaggtgaag gtcggagtca acagatttgg tcgtattggg cgcctggtta ccagggctgc 60
ttttaactct ggtaaagtgg atattgtcgc catcaatgat cccttcattg acctcaacta 120
catggtctac atgttccggt atgattccac ccatggcaaa ttccatggca ctgtcaaggc 180
tgagaacgag aagcttgtca acaatggaaa tcccatcacc atcttc 226
Claims (3)
1. A primer set for detecting a pathogen of an infectious disease of the digestive tract, characterized in that: specific primers and TaqMan probes for detecting EB virus, cytomegalovirus, proteus mirabilis, cryptosporidium, salmonella typhi, moraxella catarrhalis, yersinia small intestine and adenovirus;
the specific primers are SEQ ID NO. 1 and SEQ ID NO. 2 for EB virus, SEQ ID NO. 4 and SEQ ID NO. 5 for cytomegalovirus, SEQ ID NO. 7 and SEQ ID NO. 8 for Proteus mirabilis, SEQ ID NO. 10 and SEQ ID NO. 11 for Cryptosporidium, SEQ ID NO. 13 and SEQ ID NO. 14 for Salmonella typhi, SEQ ID NO. 16 and SEQ ID NO. 17 for Moraxella catarrhalis, SEQ ID NO. 19 and SEQ ID NO. 20 for Yersinia small intestine, SEQ ID NO. 22 and SEQ ID NO. 23 for adenovirus;
the TaqMan probes are SEQ ID NO. 3 for EB virus, SEQ ID NO. 6 for cytomegalovirus, SEQ ID NO. 9 for Proteus mirabilis, SEQ ID NO. 12 for Cryptosporidium, SEQ ID NO. 15 for Salmonella typhi, SEQ ID NO. 18 for Moraxella catarrhalis, SEQ ID NO. 21 for Yersinia small intestine and SEQ ID NO. 24 for adenovirus.
2. The primer combination for detecting pathogens of digestive tract infectious diseases according to claim 1, wherein: the fluorescent group labeled by the TaqMan probe is selected from FAM, ROX, TAMRA, CY; the label for quenching the fluorophore is selected from BHQ1, BHQ2, DABCYL.
3. The primer combination for detecting pathogens of digestive tract infectious diseases according to claim 2, wherein: specific primers and probes of EB virus, cytomegalovirus, proteus mirabilis and Cryptosporidium are used simultaneously; specific primers and probes for Salmonella typhi, moraxella catarrhalis, yersinia small intestine, adenovirus are used simultaneously.
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