CN103074449B - Kit for synchronously detecting thirteen diarrhea viruses and detection method of kit - Google Patents

Abstract

The invention discloses a kit for synchronously detecting thirteen diarrhea viruses and a detection method of the kit. The kit comprises DEPC (diethylpyrocarbonate) water, a 5*RT (reverse transcription) buffer, a reverse transcription primer, a reverse transcriptase, an X solution, a 10*PCR (polymerase chain reaction) buffer, a PCR primer, a 25mM magnesium chloride solution, a DNA (deoxyribonucleic acid) polymerase and a positive control, and is characterized in that the reverse transcription primer comprises RT amplification primers of the eleven diarrhea RNA viruses and a human RNA (ribonucleic acid) internal reference, and has a gene sequence shown as SEQ ID NO. 1-12 (sequence identifier number 1-12), and the PCR primer comprises forward and reverse PCR amplification primers of the rest two diarrhea viruses, a human DNA internal reference and a reaction internal reference, and PCR amplification primers of the eleven diarrhea RNA viruses and the human RNA internal reference, and has a gene sequence shown as SEQ ID NO. 13-32. The kit and the detection method have the advantages of high specificity, sensitivity, flux and reliability, low cost, and no false negative results.

Description

Test kit and the detection method thereof of 13 kinds of diarrhea viruses of a kind of synchronous detection

Technical field

The present invention relates to detection kit and the detection method thereof of diarrhea virus, especially relate to a kind of test kit and detection method thereof of the 13 kinds of diarrhea viruses of synchronous detection based on GeXP multiple gene expression genetic analysis systems.

Background technology

Diarrhoea is that a kind of sickness rate alimentary infection high and that be popular in is all over the world sick, its sickness rate is only second to upper respiratory tract infection, viral diarrhea is common disease and the frequently-occurring disease of China, cause of disease more complicated, especially larger to infant crowd's harm, therefore need badly and set up diarrhea virus diagnostic method rapidly and efficiently.

At present, the traditional detection method of diarrhea virus mainly comprises following several:

(1) stool for routine inspection: stool for routine test procedure is to estimate most probable pathogenic agent according to stool proterties, stool examination, age of onset and epidemic season.During light microscopic microscopy, mucus just, pus and blood stool or bloody stool, can have that volume is red, white corpuscle, be more common in the diarrhoea due to the bacteriums such as salmonella, enteroinvasive E．Coli, enterohemorrhagic Escherichia coli, Campylobacter spp, Yersinia and some virus etc.; Rare just, watery stool, can have a small amount of or without red, white corpuscle, be more common in the diarrhoea due to enterotoxigenic E.Coli, rotavirus, Cryptosporidium, Aeromonas etc., there is cost low, the advantage low to laboratory hardware requirement, but there is following shortcoming: 1) sensitivity is low, often occur false negative; 2) accuracy is lower, easily causes mistake to examine or mistaken diagnosis.

(2) etiological examination: directly do image and examination of living tissue, or pathogen isolation cultivation, at microscope, electric Microscopic observation, make diagnosis afterwards.Advantage: cost is low; Low to laboratory hardware requirement.Shortcoming: 1) length consuming time: generally need 3-5 days or longer; 2) diagnosis efficiency is low: can only single bacterium pure culture; Pathogenic agent to some phenotypic characteristic variations, is difficult to distinguish by morphology experience; In addition, due to antibiotic abuse, bacterium grows and is suppressed in testing process, causes false-negative result; 3) workload is huge: due to every kind of bacterium of each sampling observation sample must be detected, workload is very huge, and particularly part requires comparatively harsh bacterium to culture environment, has more strengthened the workload and the difficulty that detect.

(3) Serological testing: most widely used is Enzyme-multiplied immune technique (ELISA): generally first use primary antibodie and antigen generation specific binding, then by two of general enzyme labelling, resist and primary antibodie generation specific bindings, then by enzyme colour developing, afterwards observations.Advantage: 1) sample flux is higher: utilize 96 hole enzyme plates, one flat plate can complete the detection of a plurality of samples; 2) more responsive, to utilize enzyme to join characteristic, can amplify original antigen signals; 3) quick: in time, than traditional substratum microorganism culturing test procedure, will to shorten a lot; But also there is following shortcoming: 1) be prone to false positive: due to washing and antigen coated operation, or because antigenic surface determinant is similar, cross reaction occurs, therefore be prone to false positive; 2) take time and effort: making antibody is the work taking time and effort very much; 3) sensitivity is uncertain: the quality of antibody is depended in the sensitivity of experiment, and the quality of each antibody differs, and difficult quality is controlled; 4) cannot detect the virus of multi-Vari: variability is virus faster, as some RNA viruses, has multiple hypotype, and often variation, variation causes antibody to lose efficacy, cannot detectable antigens.

(4) molecular biology---PCR detection method: at present often application have real-time fluorescence quantitative PCR, immuno-PCR, a reverse transcription PCR etc., be all that the specific target gene for pathogenic agent detects.Wherein, fluorescent quantitative PCR detection method is the most ripe.The advantage of fluorescent quantitative PCR technique: susceptibility is high, and can accurate quantification, in to the detection of Listeria monocytogenes, Salmonellas, Clostridium botulinum, intestinal bacteria etc., all obtained good result.2004, China issued the national standard of implementing fluorescence quantitative PCR detection bird flu H7 hypotype.2009, U.S. FDA was ratified the test kit by fluorescence quantitative PCR detection porcine influenza H1N1 hypotype.But also there is following shortcoming: 1) flux is low: once can only detect a cause of disease composition, as needs detect multiple pathogenic bacteria, just need multiple pc R instrument to work simultaneously, efficiency is low, and the cycle is long, and the sample of large batch of multiple pathogen contamination is had no way of doing it especially; 2) cost is relatively high: because once detecting a pathogenic agent, when a sample needs to detect multiple pathogenic bacteria simultaneously, must detect one by one, cost increases relatively.

(5) molecular biology---gene chips: gene chip is to pass through micro-processing technology, by the DNA fragmentation of the particular sequence of ten hundreds of and even 1,000,000 (gene probe), arrange and be fixed on the upholders such as silicon chip, slide regularly, the two-dimentional DNA probe array forming, utilize the biological sample of this class chip and mark to hybridize, can carry out fast qualitative and quantitative analysis to the gene expression profile bioinformation of sample.Advantage: 1) high-flux parallel detects: when a sample needs to detect multiple pathogenic bacteria simultaneously, once experiment can draw whole results; 2) easy and simple to handle quick: whole detection only needs 4-8 hour substantially can go out result; But also there is following shortcoming: 1) technical costs is expensive, complicated: each sample needs a chip, and cost great Yu $1000/ sample, is unfavorable for large-scale promotion; 2) the synthetic and fixing more complicated of probe, particularly makes highdensity probe array, is main rate-limiting step; 3) can not accurate quantification, poor repeatability; 4) sensitivity is lower: chip method needs nucleic acid amount larger, generally must first do multiplex PCR amplification, because primer is more, easily self produces dimer, hairpin structure, or because Tm value is different, and the object fragment efficiency that causes increasing is different, and then the sensitivity of impact detection; 5), because the kind of chip is more, be difficult to formulate a unified quality control standard.

GenomeLab ^tMcapillary electrophoresis separation technology and the highly sensitive laser Induced Fluorescence Technology research and development of GeXP multiple gene expression genetic analysis systems based on Beckman company maturation form, the kapillary display and design in a branch of 8 roads takes full advantage of the alignment characteristics of 96 orifice plates, has reduced cost and the complicacy of using larger display to bring.Adopt multiple PCR method, by Beckman Coulter dye marker, in same EP pipe, analyze the expression of a plurality of genes simultaneously, can fast and effeciently detect the expression situation of gene, the defect that has overcome the traditional detection method existence of above-mentioned diarrhea virus, has the following advantages:

1, high-throughput: native system adopts two (96 hole) plates, automatic sample and sample tracer technique, realize a single reaction detection 30-40 site, can do simultaneously 192 reactions (as 192 patient's samples, 30 kinds of diarrhea viruses of each sample detection, 30 sites), within one day, go out result; For co-infected patients, present method can disposablely provide accurate report, avoids undetected.

2, accuracy is strong: GeXP adopts capillary electrophoresis to carry out separation detection to PCR product, can non-specific amplification product, primer dimer and specific amplification products is separated, at utmost reduce false positive;

3, susceptibility is high, and result is reproducible: GeXP system has overcome the deviation that the unequal amplification of normal PCR amplification method causes, and has improved a set of goal gene is carried out to quantitative speed and susceptibility, adopts laser induced fluorescence(LIF)-PMT, has hypersensitivity;

4, method is easy, uses economical: GeXP provides from a complete set of experimental programs such as reagent, multiple PCR primer design, result and quantitative expression spectrum analysis; The testing cost Shao Yu $50 of each sample, is beneficial to large-scale promotion;

5, accurate quantification, handiness are strong: can accurate quantification pathogen gene copy number, can adjust according to demand at any time the target gene of detection.

6, easily be automated: with regard to sample preparation, Biomek series automated fluid processing instrument can mate completely with GeXP analyser and Ampligrid amplification instrument, and integrated bar code reader has guaranteed that sample is followed the trail of and report the test accurately.

At present, both at home and abroad also not about the test kit of 13 kinds of diarrhea viruses of synchronous detection based on GeXP multiple gene expression genetic analysis systems and the correlative study of detection method report thereof.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of high specificity, highly sensitive, flux is high, reliability is strong, cost is low, without test kit and the detection method thereof of the 13 kinds of diarrhea viruses of synchronous detection based on GeXP multiple gene expression genetic analysis systems of false negative result.

The present invention solves the problems of the technologies described above adopted technical scheme: the test kit of 13 kinds of diarrhea viruses of a kind of synchronous detection, comprise DEPC water, 5 * RT damping fluid, reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, PCR primer, 25mM magnesium chloride solution, archaeal dna polymerase and positive reference substance, described reverse transcription primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references in following table 1, described PCR primer comprises remaining two kinds of diarrhoea DNA virus in table 1, people DNA internal reference, reaction forward and reverse pcr amplification primer of internal reference and the pcr amplification primer of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references, gene order is as shown in table 1 below.

Described X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Oppositely amplimer sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.

Described positive reference substance is the cloning vector of pMD18-T of gene conserved sequence that is connected with the primer of design above-mentioned 13 kinds of diarrhea viruses, RNA internal reference, DNA internal reference and reaction internal reference.

The method of detection diarrhea virus of utilizing the test kit of 13 kinds of diarrhea viruses of synchronous detection, specifically comprises the following steps:

(1) collecting sample extract nucleic acid

The separation and Culture thing that gathers ight soil, vomitus or the blood of diarrhea patient extracts nucleic acid from separation and Culture thing;

(2) take patient's nucleic acid carries out RT reaction as template

Get the nucleic acid samples RNA5 μ L of 5-20ng/ul, DEPC water 8 μ L, 5 * RT damping fluid, 4 μ L, RT primer solution 2 μ L, join in 96 hole sample panel and carry out reverse transcription, reaction conditions after RT enzyme 1 μ L mixes: 48 ° of C1 minute; 42 ° of C60 minute; 95 ° of C5 minute; 4 ° of C are until collect RT product, in wherein reverse transcription primer solution, each RT primer concentration is 500nM, described RT primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references, and gene order is as shown in SEQ ID NO.1～NO.12 in sequence table;

(3) take reverse transcription product carries out PCR reaction as template

Get RT product 8.6 μ L, 10 * PCR damping fluid, 2 μ L, the magnesium chloride 4 μ L of 25mM, PCR primer solution 2 μ L, archaeal dna polymerase 1.4 μ L, join the enterprising performing PCR reaction of 96 hole sample panel, reaction conditions: 95 ° of C2 minute after X solution 2 μ L mix; In 94 ° of C30 seconds, in 60 ° of C30 seconds, 70 ° of C1 minute, circulate 35 times; 70 ° of C1 minute; 4 ° of C are until collect PCR product; Wherein said X solution is for comprising triphosphate deoxy-nucleotide and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Oppositely amplimer sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark, in PCR primer solution, each PCR primer concentration is 200nM, PCR primer comprises remaining two kinds of diarrhoea DNA virus, people DNA internal reference, reaction forward and reverse pcr amplification primer of internal reference and pcr amplification primers of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references, and gene order is as shown in SEQ ID NO.13～NO.32 in sequence table;

(4) GeXP genetic analyzer electrocapillary phoresis sample separation

Get PCR product 0.1-1 μ L, the sample-loading buffer 38.75 μ L that GeXP genetic analyzer is supporting, DNA standard substance 0.5 μ L, after one, mineral oil mixes, join on 96 hole parting liquid plates and carry out electrocapillary phoresis sample separation, the collection of illustrative plates that the software of GeXP genetic analyzer is obtained and standard diagram contrast, the kind of judgement diarrhea virus.

Compared with prior art, the invention has the advantages that: the present invention discloses test kit and the detection method thereof of 13 kinds of diarrhea viruses of a kind of synchronous detection first, because this test kit has been introduced for people's enteric coronavirus virus, EAd 40, 41 types, enterovirus and hypotype thereof (coxsackie virus A 16-type), Astrovirus, rotavirus A, B, C group, norovirus, human parechovirus, human bocavirus 2 types, bed ripples virus, little binodal RNA viruses etc., designed specificity amplification primer, can for 13 kinds of diarrhea viruses, detect simultaneously, within one day, can complete the detection of 192 patient's samples, both production cost and testing cost had been saved, improved again detection efficiency and shortened the time, the contrast internal reference of people DNA and people RNA integrity, guarantees the judgement to sample quality in checkout procedure, avoids false negative, normal reaction contrast internal reference (RXN control): monitoring PCR reaction efficiency, avoid false negative, make detection there is better sensitivity and specificity, thereby avoided the not high problem of other detection method specificitys.

In sum, the present invention is based on test kit and the detection method thereof of 13 kinds of diarrhea viruses of synchronous detection of GeXP multiple gene expression genetic analysis systems, can for 13 kinds of diarrhea viruses, detect simultaneously, detection sensitivity is high, specificity is good, reduce the false positive rate of conventional pcr amplification, can also effectively solve the easy pollution problem of conventional PCR; There is Noncompetitive internal comparison system, reliability is strong, without false negative result, utilize that GeXP genetic analysis systems is sensitive, accurate quantitative analysis, quick, high-throughout technical superiority, Jiang Wei Disease Control and Prevention Center, hospital and other medical institutions provide a kind of sensitive, accurate, quick and multiple gene test scheme cheaply.

Accompanying drawing explanation

Fig. 1 is the electrocapillary phoresis sample separation result standard collection of illustrative plates of GeXP genetic analyzer.

Embodiment

Below in conjunction with accompanying drawing, embodiment is described in further detail the present invention.

Specific embodiment one

The test kit of 13 kinds of diarrhea viruses of a kind of synchronous detection of the present invention, comprise DEPC water, 5 * RT damping fluid, reverse transcription primer (RT primer mix), RT enzyme (full name ThermoScript II), solution X, 10 * PCR damping fluid, PCR primer, 25mM magnesium chloride solution, archaeal dna polymerase, positive reference substance (the DNA fragmentation of particular sequence, for the whole reaction system of Quality Control), 5 * RT damping fluid, 10 * PCR damping fluid, 25mM magnesium chloride solution, archaeal dna polymerase Ding Yu sigma company.Above-mentioned reverse transcription primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references in following table 1, above-mentioned PCR primer comprises remaining two kinds of diarrhoea DNA virus, people DNA internal reference, reaction forward and reverse pcr amplification primer of internal reference and pcr amplification primers of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references in table, and gene order is as shown in table 1 below.(also can canonical sequence table shown in)

Table 1 diarrhea virus Multiple detection target site and primer nucleic acid sequence table

Above-mentioned X solution comprises triphosphate deoxy-nucleotide (dNTPs) and universal primer, universal primer forward amplimer sequence is AGGTGACACTATAGAATA, oppositely amplimer sequence is GTACGACTCACTATAGGGA, universal primer forward amplimer band fluorescent mark.

Above-mentioned positive reference substance is the cloning vector of pMD18-T of gene conserved sequence that is connected with the primer of design above-mentioned 13 kinds of diarrhea viruses, RNA internal reference, DNA internal reference and reaction internal reference.

Said gene target site design: the interior conservative gene design primer of special between seed selection, kind

(1) people's enteric coronavirus virus site: everyone enteric coronavirus virus-positive sample can be detected

(2) all enteroviruses site: all enterovirus positive can be detected

(3) EAd 40,41 type sites: all EAd 40,41 type positive can be detected

(4) Astrovirus site: all Astrovirus positive can be detected

(5) rotavirus C group site: all rotavirus C group positive can be detected

(6) norovirus site: all norovirus positive can be detected

(7) human parechovirus site: all human parechovirus's positive can be detected

(8) human bocavirus 2 type sites: everyone bocavirus 2 type positive can be detected

(9) bed ripples virus site: all bed ripples virus-positive samples can be detected

(10) little binodal RNA viruses site: all little binodal RNA viruses positive can be detected

(11) rotavirus B group site: all rotavirus B group positive can be detected

(12) coxsackie virus A 16-type site: all coxsackie virus A 16-type positive can be detected

(13) rotavirus A group site: all rotavirus A group positive can be detected

(14) people DNA internal reference: select mankind RNaseP gene, can detect the integrity of human DNA in sample;

(15) people RNA internal reference: select mankind B2M(beta-2-microglobulin) gene, can detect the integrity of human rna in sample;

(16) reaction internal reference: select pcDNA3.1, whether monitoring is successfully completed whole reactive system and pcr amplification.

Specific embodiment two

The detection method of 13 kinds of diarrhea viruses of synchronous detection that the present invention is a kind of, the virus detecting comprises people's enteric coronavirus virus, EAd 40, 41 types, enterovirus and hypotype thereof (coxsackie virus A 16-type), Astrovirus, rotavirus A, B, C group, norovirus, human parechovirus, human bocavirus 2 types, bed ripples virus, little binodal RNA viruses etc. (in Table 1), gather patient's sample (stool sample, vomitus, blood etc.), extract nucleic acid, the patient's nucleic acid of take carries out reverse transcription and PCR reaction as template, final by electrocapillary phoresis method sample separation, concrete steps are as follows:

1, the test kit of producing the 13 kinds of diarrhea viruses of synchronous detection based on GeXP multiple gene expression genetic analysis systems, the component that test kit comprises is with above-described embodiment 1;

2, collecting sample extract nucleic acid

The separation and Culture thing that gathers ight soil, vomitus or the blood of diarrhea patient extracts nucleic acid from separation and Culture thing;

3, take patient's nucleic acid carries out reverse transcription (RT) reaction as template

1) in following ratio, in 96 hole sample panel, add reagent and sample (RT plate is in Table 2):

Table 2RT reaction reagent and sample mix ratio

RT reaction reagent	Amount/hole
		DEPC(Dnase/Rnase Free) water	8μL
5 * RT damping fluid	4μL
		RT primer (each RT primer concentration is 500nM)	2μL
RT enzyme	1μL
		Sample RNA (5-20ng/ul)	5μL
Total	20μL

Note: add positive reference substance in RT reaction, positive reference substance each target pathogenic agent clone gained of serving as reasons, comprises target fragment plasmid, and consumption is 1 μ L/ reaction.

2) by following temperature, hatch (in Table 3) after mixing:

Table 3RT reaction conditions

Step temperature	Time
		148°C	1 minute
242°C	60 minutes
		395°C	5 minutes
44°C	Continue: until collect RT product

4, take reverse transcription product carries out PCR reaction as template

1) in following ratio, in 96 hole sample panel, add reagent and sample (PCR plate is in Table 4):

Table 4PCR reaction reagent and sample mix ratio

PCR reaction reagent	Amount/hole
		10 * PCR damping fluid	2μL
25mM?MgCl2	4μL
		PCR primer	2μL
Archaeal dna polymerase	1.4μL
		X solution	2μL
RT product	8.6μL
		Total	20μL

Note: X solution comprises triphosphate deoxy-nucleotide (dNTPs) and universal primer, universal primer forward amplimer sequence is AGGTGACACTATAGAATA, oppositely amplimer sequence is GTACGACTCACTATAGGGA, universal primer forward amplimer band fluorescent mark.

2) by following temperature, carry out thermal cycle reaction (in Table 5) after mixing:

Table 5PCR reaction conditions

Step	Temperature	Time
			1	95°C	2 minutes
2	94°C	30 seconds
			3	60°C	30 seconds
4	70°C	1 minute
			5	N/A	Repeat 2-4 step 34 time (totally 35 times)
6	70°C	1 minute
			7	4°C	Continue: until collect PCR product

5, GeXP genetic analyzer electrocapillary phoresis sample separation

1) prepare GeXP sample (in Table 5):

Table 5GeXP sample mix ratio

GeXP sample	Amount/hole
		Sample-loading buffer (Beckman Gexp system support)	38.75μL
DNA size criteria 400	0.5μL

PCR product	0.1-1μL
		Mineral oil	1

2) electrocapillary phoresis sample separation

GeXP sample is added in the hole of proper number on 96 hole capillary electrophoresis separation plates and carry out capillary electrophoresis separation; Capillary electrophoresis separation is that a class be take the Novel liquid-phase isolation technique that kapillary is motivating force as split tunnel, the high-voltage dc of take, and specific procedure is 90 ℃ of sex change 120 seconds, sample introduction voltage 2kv, 30 seconds, separation voltage 6kv, 35 minutes.

6, interpretation of result (seeing GeNO.meLab GeXP genetic analyzer specification sheets)

According to the parameter of giving tacit consent on the own software of GeXP genetic analyzer, result is carried out to clip size analysis, its X-coordinate represents clip size, and ordinate zou is that signal is strong and weak.The collection of illustrative plates that the software of GeXP genetic analyzer is obtained and standard diagram contrast, the kind of judgement diarrhea virus.As shown in Figure 1, its result can accurately detect 13 kinds of target diarrhea viruses to standard diagram, and each target fragment size interval is moderate, and signal is unlikely to supersaturation, and between each target, signal is relatively fair, and there is no broad peak, the phenomenon such as bimodal.

Specific embodiment three

Detection kit sensitivity, specificity analyses

Sensitivity analysis: positive control, by after certain copy number doubling dilution, is detected through pcr amplification and capillary electrophoresis until can't detect signal, and this copy number is lowest detection line, namely the sensitivity of test kit.Sensitivity reaches 40 copies/uL.

Specificity analyses: it is the unimodal of target fragment size that substance pcr amplification detects through capillary electrophoresis.

Above-mentioned explanation is not limitation of the present invention, and the present invention is also not limited to above-mentioned giving an example.Those skilled in the art are in essential scope of the present invention, and the variation of making, remodeling, interpolation or replacement, also should belong to protection scope of the present invention.

Claims (2)

1. the test kit of 13 kinds of diarrhea viruses of a synchronous detection, comprise DEPC water, 5 * RT damping fluid, reverse transcription primer, ThermoScript II, X solution, 10 * PCR damping fluid, PCR primer, 25 mM magnesium chloride solutions, archaeal dna polymerase and positive reference substance, it is characterized in that described reverse transcription primer comprises the RT amplimer of 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references in following table, described PCR primer comprises remaining two kinds of diarrhoea DNA virus in table, people DNA internal reference, reaction forward and reverse pcr amplification primer of internal reference and the pcr amplification primer of above-mentioned 11 kinds of suffer from diarrhoea RNA viruses and people RNA internal references, gene order is as shown in the table,

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Wherein RT reactive system and reaction conditions are: nucleic acid samples RNA 5 uL of 5-20ng/ul, DEPC water 8 uL, 5 * RT damping fluid, 4 uL, RT primer solution 2 uL, after RT enzyme 1 uL mixes, join in 96 hole sample panel and carry out reverse transcription, reaction conditions: 48oC 1 minute; 42oC 60 minutes; 95oC 5 minutes; 4oC is until collect RT product, and in reverse transcription primer solution, each RT primer concentration is 500nM;

PCR reactive system and reaction conditions are: RT product 8.6 uL, 10 * PCR damping fluid, 2 uL, magnesium chloride 4 uL of 25mM, PCR primer solution 2 uL, archaeal dna polymerase 1.4 uL, join the enterprising performing PCR reaction of 96 hole sample panel, reaction conditions: 95oC 2 minutes after X solution 2 uL mix; In 30 seconds of 94oC, in 30 seconds of 60oC, 70oC 1 minute, circulates 35 times; 70oC 1 minute; 4oC is until collect PCR product, and in PCR primer solution, each PCR primer concentration is 200nM; Described X solution is for comprising triphosphate deoxy-nucleotide (dNTPs) and universal primer, and described universal primer forward amplimer sequence is AGGTGACACTATAGAATA; Oppositely amplimer sequence is GTACGACTCACTATAGGGA, described universal primer forward amplimer band fluorescent mark.

2. the test kit of 13 kinds of diarrhea viruses of a kind of synchronous detection according to claim 1, is characterized in that: described positive reference substance is the cloning vector of pMD18-T of gene conserved sequence that is connected with the primer of the above-mentioned 13 kinds of diarrhea viruses of design, RNA internal reference, DNA internal reference and reaction internal reference.

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