CN107058632A - A kind of multiplex PCR detects three kinds of diarrhea virus kits and its detection method - Google Patents

Abstract

The invention belongs to nucleic acid detection technique field, and in particular to the real-time fluorescence multiple PCR fast detection kit of A group rotavirus, astrovirus and intestinal adenovirus.A kind of multiplex PCR disclosed by the invention detects three kinds of diarrhea virus kits, positive control and negative control are provided with kit, a variety of primer sets and the mixture of probe sequence for detecting one or more pathogen in A group rotavirus, astrovirus and intestinal adenovirus are additionally provided with.Compared with prior art, detection kit involved in the present invention, avoid the problem of producing crosslinking mutually between different primers/probe, every group of primer/probe will not produce cross homology to other targets and non-targeted nucleotide sequence, primary first-order equation can be achieved and detects three kinds of common causatives for causing diarrhoea simultaneously, short the time required to detection, accuracy is high, and strong detection instrument is provided for testing laboratory.

Description

A kind of multiplex PCR detects three kinds of diarrhea virus kits and its detection method

Technical field

Detect that three kinds of diarrhea viruses are used the present invention relates to nucleic acid detection technique field, more particularly to a kind of multiplex PCR Kit and its detection method.

Background technology

Diarrhoea is the common disease of China, frequently-occurring disease, and the cause of disease of infection is mainly virus etc., and the infected is low from Juan Dai ﹑ Fa Li ﹑ Different symptoms can be presented in the Re Deng ﹐ even vitals Shou Sun ﹐ such as full body Gan Ran ﹐ Nao ﹑ Ji Sui ﹑ Xin ﹑ livers, and prognosis compare Cha ﹐ simultaneously can Leave sequelae or cause death.Therefore the fast and accurately diagnosis to diarrhoeal diseases substance is very necessary.While diarrhoeal diseases substance Species it is various, bring huge challenge to diagnosis.People's astrovirus (Human Astrovirus, HAstV) be cause infant, One of important pathogeny of the elderly and immunodeficiency crowd diarrhoea, is widely present, can both cause distributing in the world Property diarrhoea, fulminant can also be caused to suffer from diarrhoea.Adenovirus hominis has 51 serotypes, adheres to 6 subgroups of A~F separately.F groups are divided into again The type of adenovirus 40 (Ad40) and 41 types (Ad41), referred to as intestinal adenovirus (Enteric Adenovirus, EAdV).EAdV is to draw The major reason of global infant's acute severe diarrhoea is played, is considered as with distributing diarrhoea in kindergarten, school and hospital and breaking out Diarrhoea is relevant.Rotavirus (Rotavirus, RV) is the most common cause of disease of autumn and winter infantile diarrhea, occupies baby diarrhea One (accounting for 40%).7 different groups of A~G can be divided into according to virus coat antigen, A groups in terms of epidemiology and vaccine most To be important.RV global ranges are widely present.

At present, the diarrhoea pathogenic body detecting method clinically commonly used is test strips method (enzyme linked immunosorbent assay, ELISA), Although this method time is short, sensitiveness is relatively low, and by the dynamic (dynamical) influence of antibody.Fluorescence nucleic acid round pcr has sensitivity Property high, the advantages of detecting quick, it is contemplated that will be widely used in clinical diagnosis.However, traditional fluorescent real time PCR is every Secondary to expand a kind of nucleic acid of pathogen, accurately to find out disease-producing pathogens need to repeatedly attempt, and take a substantial amount of time and essence Power, and can not effectively be distinguished for infection caused by multiple pathogens.New multiple real time fluorescence PCR can effectively make up Substance PCR these defects, are a kind of diagnostic methods for having very much an application prospect.

Chinese patent《Multiplex PCR detects seven kinds of diarrhea virus primer sets and kit and its detection method》(document number CN106191316；Publication date on December 7th, 2016) a kind of kit and its detection method are disclosed, predominantly detect Nova virus G1, Nova virus G2, adenovirus, astrovirus and rotation virus, according to patent document, 5 kinds of enteropathogen detections point For two reactions, reaction one includes Nova virus G1, Nova virus G2 and internal reference；Reaction two includes adenovirus, astrovirus And rotavirus.This patent carries out two three joint inspections surveys actually.But this patent does not add internal standard, it is impossible to which sample nucleic acid is entered Row control, in addition, Cy5, FAM and YAK fluorescein are marked adenovirus, astrovirus and rotavirus by this patent respectively, and Cy5 The fluorescent value of fluorescein in itself is relatively low, can cause fluorescent value too low in low concentration and be considered negative by instrument, so as to cause gland The false negative result of virus.

Successful implementation multiplex PCR is wanted, it is necessary to solve several technical problems：(1) due to the optimal amplification of each amplicon Condition is all different, it is necessary to coordinate each condition individually reacted to ensure that each amplicon can be carried out under single amplification condition Successful amplification.(2) possibility of crosslinking is produced between multigroup primer/probe combinations, different primers/probe mutually due to needing to use Property it is very big, therefore design when need consider eliminate this possibility.(3) every group of primer/probe can not be to other targets and non- Target nucleic acid sequence has cross homology, to prevent the result of false positive, influences the diagnosis to disease.(4) one is needed Fixed method excludes the problem of coming from terms of nucleic acid extraction, and this has required internal reference as reference.

The content of the invention

Present invention aim to address there is provided one kind diarrhoea the problem of producing crosslinking between different primers/probe mutually Viral diagnosis primer and kit and its detection method.

The present invention is achieved by the following technical solutions：A kind of multiplex PCR detects three kinds of diarrhea virus kits, examination Positive control and negative control are provided with agent box, it is characterised in that：Be additionally provided with the kit for detect A group rotavirus, The a variety of primer sets and the mixture of probe sequence of one or more pathogen in astrovirus and intestinal adenovirus.

It is preferred that, the primer sequence detected for A group rotavirus is respectively SEQ ID NO：1 and SEQ ID NO：2, Taqman probe sequences are SEQ ID NO：3, fluorescence labeling is ROX；

The primer sequence detected for astrovirus is respectively SEQ ID NO：4 and SEQ ID NO：5, Taqman probe sequences It is classified as SEQ ID NO：6, fluorescence labeling is FAM；

The primer sequence detected for intestinal adenovirus is respectively SEQ ID NO：7 and SEQ ID NO：8, Taqman probes Sequence is SEQ ID NO：9, fluorescence labeling is HEX；

The primer sequence detected for people's internal standard gene is respectively SEQ ID NO：10 and SEQ ID NO：11, Taqman visit Pin sequence is SEQ ID NO：12, fluorescence labeling is Cy5.

It is preferred that, the kit includes PCR amplifing reagents and contrast agents, and the PCR amplifing reagents react including PCR Buffer solution, HAstV/EAdV/RV primed probes mixed liquor and enzyme system, the contrast agents include negative control and HAstV/ EAdV/RV positive controls, the negative control is cell culture fluid, and the positive control is the base containing HAstV, EAdV and RV mesh Because of the plasmid bacterial of fragment.

Preferably, the PCR reaction buffers include 5 × RT-PCR buffer and dNTPs, the HAstV/EAdV/ RV primed probes mixed liquor is HAstV/EAdV/RV, interior label primer and probe, and the enzyme system includes archaeal dna polymerase, reverse transcription Enzyme, RNase inhibitor.

The detection method of three kinds of diarrhea viruses, it is characterised in that：This method comprises the following steps：

1) PCR reaction solutions are prepared by following composition (n is reaction tube number)

PCR reaction buffers 1 are 16 μ l × n, and HAstV/EAdV/RV primed probes mixed liquor is 2 μ l × n, and enzyme system 2 is 2 μ l×n；(note：Ensure that PCR reaction buffers, HAstV/EAdV/RV primed probes mixed liquor fully dissolve and mixed before use, Enzyme system before that need to centrifuge to ensure that all enzymes concentrate on bottom)

2) reaction system is dispensed into PCR reaction tubes by 20 μ l/ pipes, the reaction tube that will be equipped with PCR reaction solutions moves to sample Present treatment area；

3) sample-adding (sample process area carry out), taken respectively with the suction nozzle with filter core processed good sample, negative control, Each 5 μ l of positive control supernatant are added separately in the PCR reaction tubes equipped with PCR reaction solutions, cover lid, expansion is moved to after the centrifugation several seconds Increase detection zone；

4) fluorescent quantitative PCR (being carried out in augmentation detection area), PCR reaction tubes are put into fluorescent PCR amplification instrument Row augmentation detection.

It is preferred that, step 1) the final concentration of primer be 0.2uM, the final concentration of probe is 0.1uM.

Preferably, step 4) reaction condition include following a-g program：

a：50 DEG C keep 25min (reverse transcription)；

b：95 DEG C keep 5min (thermal starting)；

c：95 DEG C maintain 10s (denaturation)；

d：55 DEG C maintain 15s (annealing)；

e：72 DEG C maintain 30s (extension), carry out 5 circulations (pre- amplification)；

f：95 DEG C maintain 10s (denaturation)；

g：60 DEG C maintain 45s (annealing/extension gathers fluorescence signal), carry out 40 circulations.

Detected the invention provides a kind of multiplex PCR and positive control is provided with three kinds of diarrhea virus kits, kit And negative control, it is additionally provided with for detecting one or more pathogen in A group rotavirus, astrovirus and intestinal adenovirus The mixture of a variety of primer sets and probe sequence.There is advantages below compared with prior art：This kit, i.e. rotavirus (A) Group/astrovirus/intestinal adenovirus kit for detecting nucleic acid (fluorescent PCR method), at the same detect astrovirus, intestinal adenovirus, Rotavirus A groups and people internal standard gene actin, are 4 link detection reagent kits.This kit adds internal standard, and sample can be controlled System, in the case where astrovirus, intestinal adenovirus and rotavirus A groups are all for feminine gender, if internal standard is also feminine gender, illustrates sample This degraded or nucleic acid content it is less or exist suppress, it is necessary to again extract nucleic acid or carry out purification process.This kit is from normal FAM, HEX and ROX fluorescein mark astrovirus, intestinal adenovirus and rotavirus A groups respectively, ensure that pathogen There is obvious amplification curve in low concentration；Fluorescent value less high Cy5 itself is marked into internal standard, because actin concentration is general Higher, Cy5 has normal amplification curve, generally speaking, detection kit involved in the present invention, it is to avoid different primers/spy The problem of producing crosslinking mutually between pin, every group of primer/probe will not produce intersection to other targets and non-targeted nucleotide sequence Homology, can be achieved primary first-order equation and detects three kinds of common causatives for causing diarrhoea, short, accuracy the time required to detection simultaneously Height, strong detection instrument is provided for testing laboratory.

Embodiment

Embodiments of the invention are elaborated below, the present embodiment is carried out lower premised on technical solution of the present invention Implement, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to following implementations Example.

Set in rotavirus (A) group/astrovirus/intestinal adenovirus kit for detecting nucleic acid (fluorescent PCR method), kit There are positive control and negative control, be additionally provided with the kit for detecting A group rotavirus, astrovirus and intestinal adenovirus A variety of primers of middle one or more pathogen and the mixture of probe.Wherein, the primer sequence detected for A group rotavirus Respectively SEQ ID NO：1 and SEQ ID NO：2, Taqman probe sequences are SEQ ID NO：3, fluorescence labeling is ROX；For The primer sequence of astrovirus detection is respectively SEQ ID NO：4 and SEQ ID NO：5, Taqman probe sequences are SEQ ID NO：6, fluorescence labeling is FAM；The primer sequence detected for intestinal adenovirus is respectively SEQ ID NO：7 and SEQ ID NO： 8, Taqman probe sequences are SEQ ID NO：9, fluorescence labeling is HEX；The primer sequence difference detected for people's internal standard gene For SEQ ID NO：10 and SEQ ID NO：11, Taqman probe sequences are SEQ ID NO：12, fluorescence labeling is Cy5；

Embodiment 1：Detect A group rotavirus：Using A group rotavirus positive controls as sample, corresponding primer/probe Embodiment 2 is shown in combination and the acquisition of positive control, using QIAamp MinElute Virus Spin Kit (QIAGEN) from sample Nucleic acid is extracted in this, each primer/probe combinations (2 μ L), enzyme system (2 μ L) are then separately added into reaction buffer 1 (16 μ L) And the nucleic acid (5 μ L) that sample or negative control are extracted, primer ultimate density is 0.2 μM, and probe ultimate density is 0.1 μM.With Afterwards by each reaction tube according to following procedure at suitable real-time fluorescence PCR instrument (such as ABI 7500, Applied Biosystems) Enterprising performing PCR reaction：50 DEG C keep 25min (reverse transcription), and 95 DEG C keep 5min (thermal starting)；95 DEG C maintain 10s (denaturation), 55 DEG C 15s (annealing) is maintained, 72 DEG C maintain 30s (extension), 5 circulations (expand in advance)；95 DEG C maintain 10s (denaturation), 60 DEG C of maintenances 45s (annealing/extension gathers fluorescence signal), 40 circulations.As a result it can detect A group rotavirus.The inspection of remaining each pathogen Survey same as Example 1.

Embodiment 2：One-time detection rotavirus (A) group, astrovirus and enteron aisle adenopathy substance.Design primer/probe groups Close：According to the nucleotide sequence of each pathogen, carry out with the sequence alignment of inter-species to find conservative region, and use Primer The primer and Taqman probes that the softwares of Express 3.0 carry out multiplex PCR to these conservative regions are designed, and resulting is respectively drawn Thing/probe combinations carry out BLAST on NCBI websites and are analysed to ensure that with other microorganisms that may be present in sample do not occur Cross reaction.Finally by its performance of experimental verification, the design of internal reference uses same method.

1) each group primer/probe designed by is as follows：Containing in rotavirus (A) group, astrovirus, intestinal adenovirus and people Mark gene actin

2) each positive control is prepared：The nucleic acid of various pathogen is separated first, and prepares PCR reaction systems, then will Various pathogen nucleic acids, which are separately added into reaction system, enters performing PCR reaction, and reaction product is reclaimed and uses electroresis appraisal, then The plasmid for being connected with each target pathogen sequence is obtained using suitable TA Cloning Kits (such as pGEM-T carriers, Promega) Carrier, is used as the positive control of PCR reactions.

3) reaction main mixture is prepared：Using reaction buffer 1, Buffer and dNTPs containing premix are contained using enzyme system There are a Taq archaeal dna polymerases of premix, reverse transcriptase, the component such as RNase inhibitor.

4) pcr amplification reaction is carried out：Use appropriate RNA/DNA extractive techniques (such as QIAamp MinElute Virus Spin Kit, QIAGEN) nucleic acid is extracted from sample, be then separately added into reaction main mixture (16 μ L) enzyme system (2 μ L), Each primer/probe combinations (2 μ L) and the nucleic acid (5 μ L) extracted from sample or negative control, it is right to the positive in the same scale Row married operation is shone into, ultimate density positive control is 5 μM, and primer concentration is 0.2 μM, and concentration and probe concentration is 0.1 μM.Then will be each Reaction tube is carried out according to following procedure on suitable real-time fluorescence PCR instrument (such as ABI7500, Applied Biosystems) PCR reacts：50 DEG C keep 25min (reverse transcription), and 95 DEG C keep 5min (thermal starting)；95 DEG C maintain 10s (denaturation), 55 DEG C of maintenances 15s (annealing), 72 DEG C maintain 30s (extension), 5 circulations (pre- amplification)；95 DEG C maintain 10s (denaturation), and 60 DEG C maintain 45s (to move back Fire/extension, gathers fluorescence signal), 40 circulations.

Result judgement：The signal value of all negative controls should all be less than threshold value.All positive control signals should all have index Rise and Ct values are necessarily less than 33.Otherwise explanation amplification is unsuccessful.As the corresponding fluorescence signal of certain pathogen is produced on index Rise, be then the positive, be otherwise feminine gender.

Kit is mainly constituted

This kit is according to RT-PCR and Taqman probe for real-time fluorescence PCR technologies, for people's astrovirus, enteron aisle adenopathy Specific primer and probe, people's astrovirus, enteron aisle adenopathy are designed in poison and rotavirus (A groups) genome conserved region for target region Poison and universal probe difference flag F AM, HEX and ROX fluorescein of rotavirus (A groups), internal standard probe mark Cy5 fluoresceins, The compositions such as reverse transcriptase, thermal starting Taq DNA polymerase are equipped with, viral RNA first through reverse transcription formation cDNA, expands subsequently into PCR Increase, so as to carry out qualitative detection to people's astrovirus, intestinal adenovirus and rotavirus (A groups) nucleic acid in sample.

Detection method is as follows：

1st, PCR reaction solutions are prepared by following composition (n is reaction tube number).

PCR reaction buffers 1 are 16 μ l × n, and HAstV/EAdV/RV primed probes mixed liquor is 2 μ l × n, and enzyme system 2 is 2 μ l×n.(note：Ensure that PCR reaction buffers, HAstV/EAdV/RV primed probes mixed liquor fully dissolve and mixed before use, Enzyme system before that need to centrifuge to ensure that all enzymes concentrate on bottom).

2nd, reaction system is dispensed into PCR reaction tubes by 20 μ l/ pipes, the reaction tube that will be equipped with PCR reaction solutions moves to sample Present treatment area.

3rd, sample-adding (sample process area carry out), taken respectively with the suction nozzle with filter core processed good sample, negative control, Each 5 μ l of positive control supernatant are added separately in the PCR reaction tubes equipped with PCR reaction solutions.Lid is covered, expansion is moved to after the centrifugation several seconds Increase detection zone.

4th, fluorescent quantitative PCR (being carried out in augmentation detection area), PCR reaction tubes are put into fluorescent PCR amplification instrument Row augmentation detection.Program：50 DEG C keep 25min (reverse transcription), and 95 DEG C keep 5min (thermal starting)；95 DEG C maintain 10s (denaturation), 55 DEG C maintain 15s (annealing), and 72 DEG C maintain 30s (extension), 5 circulations (pre- amplification)；95 DEG C maintain 10s (denaturation), 60 DEG C of dimensions Hold 45s (annealing/extension gathers fluorescence signal), 40 circulations.Experiment can directly read result after terminating from instrument.

Quality control standard

1st, negative control：As a result it is feminine gender, internal standard result is positive and Ct values<30.

2nd, positive control：As a result it is the positive, positive control Ct value≤28.

3rd, two need to simultaneously meet in once testing more than, and otherwise, this experiment is invalid, and experiment should be re-started.

Interpretation of result

1st, after experiment terminates, analyzed, more than regulation noise margin to baseline noise, made according to the software of pertinent instruments Negative control Ct values occur without any numerical value.

2nd, threshold value setting principle is just above the highest of normal negative control curve (random noise line) with threshold line Point is defined.FAM, HEX/VIC, ROX and Cy5 baseline choose 6~15 race ways.

3rd, register instrument automatically analyzes the sample Ct values calculated.

The reference value of this kit of reference value is that Ct values are equal to 33.

The explanation of assay

1. experiment is both needed to together detect the negative control in kit, positive control and sample to be checked every time, detection knot Fruit should meet its performance indications, and otherwise this experiment is invalid.

2. experimental result determination methods

2.1. negative findings judges：If sample is in FAM passages, HEX/VIC passages and ROX passages amplification curve not into S types, Ct values are UNDET or ＞ 35.00, and internal standard Cy5 passage amplification curves are S-type and result Ct values<30, then result is the moon Property；If internal standard Cy5 passages amplification curve is not S-type or result Ct value >=30, sample should be rechecked.

2.2. positive findings judges：If sample is FAM passage amplification curves are S-type and Ct value≤33, result is HAstV is positive, if sample is HEX/VIC passage amplification curves are S-type and Ct value≤33, result is the EAdV positives, if Sample is ROX passage amplification curves are S-type and Ct value≤33, then result is RV positive.

2.3. gray area is tested：If sample FAM passages, HEX/VIC passages or ROX passage amplification curves it is S-type and 35 ＞ Ct values ＞ 33, then result, which is located at, tests gray area, and sample should be rechecked.

2.4. " reinspection " operation is as follows：It is recommended that carrying out concentration to sample, the preservation liquid after sample is vortexed fully is collected After 4 DEG C of centrifugation 5min of 1ml, 12000rpm, supernatant is carefully removed, the μ l of layer about 200 that keep on file are as sample, and subsequent treatment is with general Sample to be tested processing method, if rechecking, result amplification curve is S-type, and result of determination is the positive, if amplification curve is not S-type, Then result of determination is feminine gender.Feminine gender represents not detect, and concentration of specimens is less than the test limit of kit.

Properties of product：This kit, which can be detected and reached, distinguishes people's astrovirus, intestinal adenovirus and rotavirus (A groups), Other viruses identical or similar infection symptoms with infection site, such as norovirus, letter such as virus, enterovirns type 71 and Ke Sa The no cross reactions such as strange virus A 16-type.The endogenous material such as excrement that may be present and department of antiviral drugs Austria in sample to be tested The exogenous materials such as his Wei, zanamivir are noiseless to the testing result of this kit, wherein, test limit is not higher than 1.0 × 103PFU/ml。

For example, detection 10 parts of enterprise's positive reference product, 10 parts of enterprise's negative reference product, coincidence rate is 100%；1 part of enterprise Sensitivity reference material repeats detection 20 times, at least 17 times positives of testing result；2 parts of enterprise's precision reference materials repeat detection 10 It is secondary, the as a result Ct values coefficient of variation (CV)≤5%.

In summary, detect and be provided with three kinds of diarrhea virus kits, kit the invention provides a kind of multiplex PCR Positive control and negative control, are additionally provided with one or more in A group rotavirus, astrovirus and intestinal adenovirus for detecting The a variety of primer sets and the mixture of probe sequence of pathogen.There is advantages below compared with prior art：This kit, i.e. colyliform Viral (A) group/astrovirus/intestinal adenovirus kit for detecting nucleic acid (fluorescent PCR method), while detecting astrovirus, enteron aisle Adenovirus, rotavirus A groups and people internal standard gene actin, are 4 link detection reagent kits.This kit adds internal standard, can be to sample It is controlled, in the case where astrovirus, intestinal adenovirus and rotavirus A groups are all for feminine gender, if internal standard is also feminine gender, Illustrate sample breakdown or nucleic acid content is less or presence suppresses, it is necessary to extract nucleic acid again or carry out purification process.This kit Astrovirus, intestinal adenovirus and rotavirus A groups are marked respectively from conventional FAM, HEX and ROX fluorescein, ensure that Pathogen has obvious amplification curve in low concentration；Fluorescent value less high Cy5 itself is marked into internal standard, it is dense because of actin Degree is general higher, Cy5 has normal amplification curve, generally speaking, detection kit involved in the present invention, it is to avoid difference is drawn The problem of producing crosslinking mutually between thing/probe, every group of primer/probe will not be produced to other targets and non-targeted nucleotide sequence Cross homology, can be achieved primary first-order equation and detects three kinds of common causatives for causing diarrhoea simultaneously, short the time required to detection, accurately Property it is high, strong detection instrument is provided for testing laboratory.

The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention Any modifications, equivalent substitutions and improvements made within refreshing and principle etc., should be included in the scope of the protection.

Claims (7)

1. a kind of multiplex PCR, which is detected, is provided with positive control and negative control in three kinds of diarrhea virus kits, kit, it is special Levy and be：It is additionally provided with the kit for detecting one or more diseases in A group rotavirus, astrovirus and intestinal adenovirus The a variety of primer sets and the mixture of probe sequence of substance.

2. a kind of multiplex PCR according to claim 1 detects three kinds of diarrhea virus kits, it is characterised in that：For A The primer sequence of group rotavirus detection is respectively SEQ ID NO：1 and SEQ ID NO：2, Taqman probe sequences are SEQ ID NO：3, fluorescence labeling is ROX；

The primer sequence detected for astrovirus is respectively SEQ ID NO：4 and SEQ ID NO：5, Taqman probe sequences are SEQ ID NO：6, fluorescence labeling is FAM；

The primer sequence detected for intestinal adenovirus is respectively SEQ ID NO：7 and SEQ ID NO：8, Taqman probe sequences For SEQ ID NO：9, fluorescence labeling is HEX；

The primer sequence detected for people's internal standard gene is respectively SEQ ID NO：10 and SEQID NO：11, Taqman probe sequences It is classified as SEQ ID NO：12, fluorescence labeling is Cy5.

3. a kind of multiplex PCR according to claim 1 detects three kinds of diarrhea virus kits, it is characterised in that：It is described Kit includes PCR amplifing reagents and contrast agents, and the PCR amplifing reagents include PCR reaction buffers, HAstV/EAdV/ RV primed probes mixed liquor and enzyme system, the contrast agents include negative control and HAstV/EAdV/RV positive controls, described the moon Property control be cell culture fluid, the positive control be the plasmid bacterial containing HAstV, EAdV and RV target gene fragment.

4. a kind of multiplex PCR according to claim 3 detects three kinds of diarrhea virus kits, it is characterised in that：It is described PCR reaction buffers include 5 × RT-PCR buffer and dNTPs, and the HAstV/EAdV/RV primed probes mixed liquor is HAstV/EAdV/RV, interior label primer and probe, the enzyme system include archaeal dna polymerase, reverse transcriptase, RNase inhibitor.

5. the detection method of three kinds of diarrhea viruses, it is characterised in that this method comprises the following steps：

1) PCR reaction solutions are prepared by following composition (n is reaction tube number)

PCR reaction buffers 1 be 16 μ l × n, HAstV/EAdV/RV primed probes mixed liquor be 2 μ l × n, enzyme system 2 be 2 μ l × n；(note：Ensure that PCR reaction buffers, HAstV/EAdV/RV primed probes mixed liquor fully dissolve and mixed before use, enzyme Tying up to before use needs centrifugation to ensure that all enzymes concentrate on bottom)

2) reaction system is dispensed into PCR reaction tubes by 20 μ l/ pipes, the reaction tube that will be equipped with PCR reaction solutions is moved at sample Manage area；

3) sample-adding (being carried out in sample process area), processed good sample, negative control, the positive are taken with the suction nozzle with filter core respectively Control each 5 μ l of supernatant are added separately in the PCR reaction tubes equipped with PCR reaction solutions, cover lid, and amplification inspection is moved to after the centrifugation several seconds Survey area；

4) fluorescent quantitative PCR (being carried out in augmentation detection area), PCR reaction tubes are put into fluorescent PCR amplification instrument and expanded Increase detection.

6. detection method according to claim 5, it is characterised in that：Step 1) the final concentration of primer be 0.2uM, the final concentration of probe is 0.1uM.

7. according to any described detection method of claim 5 or 6, it is characterised in that：Step 4) reaction condition include it is as follows A-g program：

a：50 DEG C keep 25min (reverse transcription)；

b：95 DEG C keep 5min (thermal starting)；

c：95 DEG C maintain 10s (denaturation)；

d：55 DEG C maintain 15s (annealing)；

e：72 DEG C maintain 30s (extension), carry out 5 circulations (pre- amplification)；

f：95 DEG C maintain 10s (denaturation)；

g：60 DEG C maintain 45s (annealing/extension gathers fluorescence signal), carry out 40 circulations.