CN104651538A - Primer group and kit for simultaneously detecting four diarrhea viruses - Google Patents
Primer group and kit for simultaneously detecting four diarrhea viruses Download PDFInfo
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Abstract
The invention discloses a primer group which is based on a high-resolution melting curve analysis technique and used for simultaneously detecting four diarrhea viruses, and a quadruple detection kit containing primers. The kit is capable of specifically detecting four diarrhea viruses, that is, rotavirus, astrovirus, norovirus and sapovirus. Through comparative analysis on respective homologies of the four viral genomes, respective conserved sequence design primers with high homologies can be found, the four diarrhea viruses can be simultaneously detected by using the high-resolution melting curve analysis technique, the primer group has the characteristics of being simple, convenient, rapid, cheap, safe, high in sensitivity and high in specificity, and powerful technical support can be provided for prevention and control and clinical diagnosis of dysentery.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of based on high resolving power melting curve analysis technology (High Resolution MeltingAnalysis, HRMA) diarrhea virus quick detection kit, and use in described test kit for four kinds of diarrhea viruses (rotavirus, Astrovirus, norovirus, letter are as virus), there are specific four pairs of primers.
Background technology
Dysentery (diarrhea diseases) is the important public hygiene problem having a strong impact on human health, developing country as sub-, non-, draw area particularly serious.The World Health Organization (WHO) statistic data shows, the 5 years old Infants Below in the whole world, and have 1,000,000,000 to suffer from diarrhoea every year, wherein 500 die ten thousand deaths and die.In China, dysentery is the second frequently-occurring disease being only second to respiratory tract disease, be also Infant and child deaths two serious diseases because of one of.Viral diarrhea occupies important ratio in diarrhoea, rotavirus (Rotavirus, RV), Astrovirus (Astrovirus, AV), norovirus (Norovirus, and prick if virus (Sapovirus, SLV) etc. is main pathogenic agent NV).They cause distributing and breaking out of acute gastroenteritis by fecal-oral route in children and adult, even be very popular, break out greatly at the norovirus of Britain's appearance at the beginning of 2008, about have weekly 100,000 people to infect this virus, had a strong impact on the health of people and the development of society.Therefore, a set of easy fast, the diarrhea virus detection technique of high specificity, strong technical support can be provided for the prevention and control of dysentery and clinical diagnosis.
At present, for the detection of diarrhea virus, classical method has electron microscopic observation, Viral isolation, nucleic acid hybridization, enzyme linked immunological etc.Electron microscopic observation not only susceptibility is low, and expensive, and equipment and technical qualification require very high, cannot be applied to routine clinical detection and large-scale epidemiology survey; The defect of Viral isolation method is complex operation, consuming time very long, usually needs the time of about one week just can observe cytopathic effect inhibition assay, is unsuitable for rapid detection and the process of the clinical urgency state of an illness; Nucleic acid hybridization and enzyme linked immunosorbent detection are because of the restriction of technology itself, and its sensitivity is all lower, and loss is high.Comparatively modern method comprises common nucleic acid amplification technique (PCR) and real-time fluorescence nucleic acid amplification technologies (Real time PCR) etc.Round pcr is end-point method analysis, and need the gel electrophoresis analysis result after the cyclic amplification of heating and cooling repeatedly and PCR, consuming time longer, easily produce Residual contamination, the dyestuff (EB etc.) of use has carinogenicity, and its detection sensitivity still has much room for improvement.Real-time fluorescence nucleic acid amplification technologies without the need to electrophoretic analysis, but needs to use specific probe costly, and cost is higher, is unfavorable for extensive use.
In recent years, a kind of brand-new high resolving power melting curve analysis technology (HRMA) is developed.HRMA employs the saturated fluorescence dyestuff of latest generation, and as LCGreen I or EvaGreen etc., after Standard PCR loop ends, design temperature rises to 95 DEG C gradually from 65 DEG C.In the process, amplicon unwinds gradually, and when arriving melting temperature (Tm) (Tm), DNA chain separates completely.At the initial stage that HRMA analyzes, fluorescence intensity is very high, and along with temperature raises, double-stranded DNA reduces gradually, and fluorescence intensity also just have dropped.The whole process of change in fluorescence recorded by fluorescent PCR instrument, and by the mapping to data, just generate high resolving power melting curve, its precision can reach 0.01 DEG C.Then by realizing high-throughput gene mutation analysis, gene type, single nucleotide polymorphism analysis, pathogen detection etc. to the analysis of these curves.For traditional round pcr or real-time fluorescence PCR technology, operation steps simplifies greatly, and without the need to using expensive probe, time and cost also reduce much, and sample directly carries out HRMA analysis after pcr amplification, without the need to transfer, really achieve stopped pipe operation, reduce the risk of pollution.
HRMA analytical technology at present for diarrhea virus foundation is few, what can search only has 2 sections of reports, what comprise that the people such as Etsuko Tajiri-Utagawa sets up analyzes for the HRMA detecting norovirus, and the HRMA detected for Astrovirus that the people such as Akihiko Hata sets up analyzes.But to there is no for rotavirus and letter as the HRMA analytical technology of virus, more do not detect the report of these four kinds viral HRMA technological development simultaneously.
Summary of the invention
In order to solve the problem, the invention provides a collection of primer four kinds of diarrhea viruses to specific detection effect; On this basis, provide a kind of based on HRMA technology, utilize above-mentioned primer to realize four kinds of diarrhea viruses simultaneously, fast, the test kit that detects of high specific, high sensitivity.
Technical scheme of the present invention is:
One group is detected the primer sets of four kinds of diarrhea viruses based on high resolving power melting curve analysis technology simultaneously, comprises,
For detecting the primer of rotavirus:
Forward F1:5 '-GATACNTTRCCTGAYGGAGA-3 ' (SEQ ID NO.1),
Reverse R1:5 '-CATCYTGAAAKATDGCATCACA-3 ' (SEQ ID NO.2);
For detecting the primer of Astrovirus:
Forward F2:5 '-AAAAYGTDCGAHTGAAGGA-3 ' (SEQ ID NO.3),
Reverse R2:5 '-ABACAGTTCTYAACCAATG-3 ' (SEQ ID NO.4);
For detecting the primer of norovirus:
Forward F3:5 '-AGRGTBGTCGTHTGGGACGA-3 ' (SEQ ID NO.5),
Reverse R3:5 '-TGNGGGGTGTTNCCRTTCTT-3 ' (SEQ ID NO.6);
For detecting the primer of letter as virus:
Forward F4:5 '-ACTGTNMAYGGKTTCCATGT-3 ' (SEQ ID NO.7),
Reverse R4:5 '-CAGGRGANCGGTGTGTRCCAAT-3 ' (SEQ ID NO.8);
The viral nucleic acid variant sites of described primer uses degeneracy base, and R is A/G, K be G/T, M be A/C, W be A/T, Y be C/T, S be C/G, V be A/C/G, B be C/G/T, H be A/T/C, D be A/G/T, N is A/G/T/C.
Content of the present invention also comprises one group is detected four kinds of diarrhea viruses simultaneously four link detection reagent kits based on high resolving power melting curve analysis technology, containing above-mentioned primer sets.
Further, four described link detection reagent kits comprise 2X amplification reaction solution, 5X enzyme mixation, positive reference substance, negative controls and blank product.
Further, described 2X amplification reaction solution comprises: primer sets described in Tris-HCl, KCl, MgCl2, DTT, spermidine, Tween-20, bovine serum albumin, dNTP, fluorescence dye and claim 1.
Further, described fluorescence dye is EvaGreen.
Further, described 5X enzyme mixation comprises ThermoScript II, RNases inhibitor and HS-Taq enzyme.
Further, described positive reference substance is respectively rotavirus, Astrovirus, norovirus and the letter RNA fragment as viral in-vitro transcription.
Further, described negative controls is stroke-physiological saline solution.
Further, described blank is the ultrapure water of the nuclease free of DEPC process.
In the present invention, a kind of technical scheme adopted based on the diarrhea virus detection test kit of HRMA technology is, four described link detection reagent kit amplification reaction solution components are as shown in table 1 below:
Table 1 2X amplified reaction fluid component
Enzyme mixation component is as shown in table 2 below:
Table 2 5X enzyme mixation component
The present invention by rotavirus, norovirus, Astrovirus and letter as virus four kinds of viral genome divide other sequence analysis analysis, find the conserved sequence design primer of very high homology, and utilize high resolving power melting curve analysis technology, detect four kinds of diarrhea viruses simultaneously, there is the feature of easy quick, cheap, safe, highly sensitive, high specificity, strong technical support can be provided for the prevention and control of dysentery and clinical diagnosis.
In order to understand better and implement, describe the present invention in detail below in conjunction with accompanying drawing.
Accompanying drawing explanation
Fig. 1 is embodiment three medium sensitivity analytical results.A-D is respectively the sensitivity as Virus Sample adopts during HRMA test kit detection of the present invention of rotavirus, norovirus, Astrovirus and letter, and E-H is respectively rotavirus, norovirus, Astrovirus and letter as sensitivity when Virus Sample adopts conventional RT-PCR to detect.Sample 1:1 × 10
8copy; 2:1 × 10
7copy; 3:1 × 10
6copy; 4:1 × 10
5copy; 5:1 × 10
4copy; 6:1 × 10
3copy; 7:1 × 10
2copy; 8:1 × 10
1copy; 9:1 × 10
0copy; 10:NTC (negative control).M：100bp DNAMarker。
Fig. 2 is specificity analyses result in embodiment four.Wherein sample 1: rotavirus, 2: Astrovirus, 3: norovirus, 4: letter as virus, 5: enterovirus EV71,6: coxsackie virus A 16-type, 7: vibrio cholerae, 8: Salmonellas, 9 is negative control.
Fig. 3 is clinical sample detected result in embodiment five.Figure A is HRMA detected result, and figure B is conventional RT-PCR detected result.M：50bp DNAMarker。
Embodiment
Below by concrete diarrhea virus testing process, the present invention will be described.
Embodiment one: specific primer design
First as far as possible many rotavirus, Astrovirus, norovirus and letter is downloaded as the genomic dna sequence of virus from the gene pool of U.S. NCBI, then molecular biology software Bioedit is utilized to carry out homology compare of analysis to the sequence downloaded, find the candidate regions of conserved sequence as primer and probe design of very high homology, carry out design of primers in conjunction with software PrimerSelect simultaneously.Core design thought is: consider the principle of universality (as Tm value, 3 ' end free energy, GC content, avoiding occurring internal secondary structure and forming dimer etc.) should followed specificity and versatility (in the process of viral nucleic acid variant sites degeneracy base), the design of primers of diarrhea virus detection, and influencing each other during multiplex amplification reaction between each primer.Carry out screening verification by clinical sample again after returning for the primer synthesis designed, last the present invention optimizes following primer:
Rotavirus forward primer F1:5 '-GATACNTTRCCTGAYGGAGA-3 ' (it is 502-521 in the position of GenBank reference sequences KF907285), reverse primer R1:5 '-CATCYTGAAAKATDGCATCACA-3 ' (it is 667-688 in the position of GenBank reference sequences KF907285);
Astrovirus forward primer F2:5 '-AAAAYGTDCGAHTGAAGGA-3 ' (it is 492-510 in the position of GenBank reference sequences L23513), reverse primer R2:5 '-ABACAGTTCTYAACCAATG-3 ' (it is 785-803 in the position of GenBank reference sequences L23513);
Norovirus forward primer F3:5 '-AGRGTBGTCGTHTGGGACGA-3 ' (it is 1620-1639 in the position of GenBank reference sequences AB447442), reverse primer R3:5 '-TGNGGGGTGTTNCCRTTCTT-3 ' (it is 1977-1996 in the position of GenBank reference sequences AB447442);
Letter as viral forward primer F4:5 '-ACTGTNMAYGGKTTCCATGT-3 ' (it is 3460-3479 in the position of GenBank reference sequences NC_006269), reverse primer R4:5 '-CAGGRGANCGGTGTGTRCCAAT-3 ' (it is 3913-3934 in the position of GenBank reference sequences NC_006269).
The viral nucleic acid variant sites of described primer uses degeneracy base, and R is A/G, K be G/T, M be A/C, W be A/T, Y be C/T, S be C/G, V be A/C/G, B be C/G/T, H be A/T/C, D be A/G/T, N is A/G/T/C.
Embodiment two: based on test kit composition and the detection method of HRMA technology for detection four kinds of diarrhea viruses
1. the composition (being stored in-20 DEG C) of test kit
(1) 2X amplification reaction solution: its component is: 100mM Tris-HCl (pH8.3), 100mM KCl, 20mM MgCl2,20mM DTT, 1mM Spermidine, 0.1%Tween-20,0.2mg/L BSA, 0.4mM dNTP, 2.4 μMs of EvaGreen, 1.2 μMs of primers F, 1,1.2 μMs of primer R1,0.4 μM of primers F, 2,0.4 μM of primer R2,1.6 μMs of primers F, 3,1.6 μMs of primer R3,1.6 μMs of primers F, 4,1.6 μMs of primer R4, wherein primers F 1 is sequence numbering is the nucleotide sequence shown in SEQ ID NO:1, primer R1 is sequence numbering is the nucleotide sequence shown in SEQ ID NO:2, primers F 2 is sequence numberings is the nucleotide sequence shown in SEQ ID NO:3, primer R2 is sequence numbering is the nucleotide sequence shown in SEQ ID NO:4, primers F 3 is sequence numberings is the nucleotide sequence shown in SEQ ID NO:5, primer R3 is sequence numbering is the nucleotide sequence shown in SEQ ID NO:6, primers F 4 is sequence numberings is the nucleotide sequence shown in SEQ ID NO:7, primer R4 is sequence numbering is the nucleotide sequence shown in SEQ ID NO:8,
(2) 5X enzyme mixation: its component is: 1.5U/ μ LAMV ThermoScript II, 5U/ μ L RibonucleaseInhibitor, 5U/ μ L HS-Taq enzyme;
(3) positive control: be respectively rotavirus, Astrovirus, norovirus and the letter RNA fragment as viral in-vitro transcription;
(4) negative control: be stroke-physiological saline solution, when nucleic acid extraction and sample simultaneously parallel extraction to use as negative control;
(5) blank: be the ultrapure water of the nuclease free of DEPC process.
2. the preparation of positive reference substance
The each portion of Stochastic choice rotavirus, Astrovirus, norovirus and letter diarrhoea sample as clinical in virus infection, extracts viral RNA.In the outside design primer amplified one fragment gene fragment of the primer that this test kit is invented, and carry out sequencing.In sequencing result and GenBank, 10 above-mentioned four kinds of virus gene sequences of Stochastic choice carry out homology compare of analysis, ensure that its homology reaches more than 95%.Then utilize t7 rna polymerase to carry out in-vitro transcription and obtain single stranded RNA, transcribe rear DNase I and digest the DNA molecular removed wherein, then utilize QIAGEN RNeasy MiniElute Cleanup kit to carry out column purification.Utilize micro-ultraviolet spectrophotometer to measure the concentration of RNA after purifying, calculate copy number by its Molecular weights.In-80 DEG C of preservations after packing, as the positive control of test kit.
3. the detection method of test kit is as follows:
(1) extract the RNA of sample, i.e. template ribonucleic acid: test kit of the present invention does not provide RNA sample extraction reagent, user can select suitable commercial kit to extract viral nucleic acid according to sample type;
(2) reaction solution is prepared in accordance with the following methods: reaction volume is 25 μ L; Get 12.5 μ L 2X amplification reaction solutions, 5 μ L 5X enzyme mixations, 2.5 μ L ultrapure waters add 0.2ml optics PCR reaction tubes, then add 5 μ L sample rnas or positive control, negative control and blank, the brief centrifugation several seconds after vibration mixing;
(3) upper machine testing: reaction tubes is placed on fluorescent PCR instrument and carries out increasing and detecting.Reaction conditions is: 42 DEG C 10 minutes; 94 DEG C 2 minutes; 40 circulation 94 DEG C 30 seconds-56 DEG C 30 seconds-72 DEG C 30 seconds; 55 DEG C are progressively risen to 95 DEG C, and each rising 0.1 DEG C also maintains 2 seconds, persistent collection fluorescent signal (FAM passage).
(4) result judges: be first that quality control system judges, namely the melting curve of positive control must be uniformly distributed and present the inverse sigmoid curve of standard, and negative control and blank are without typical reverse-s shape melting curve, otherwise systems axiol-ogy is invalid.When systems axiol-ogy is effective, judge sample results to be checked, when the melting curve of sample to be checked is judged as positive sample with during positive control consistent, when sample to be checked is judged as negative sample without typical reverse-s shape melting curve or with during negative control and blank consistent.
(5) precaution: experiment whole process all should use powder-free gloves.Crossed contamination in experiment, in the process adding template, should first add blank and negative control, next adds sample to be checked, finally adds positive control.
Embodiment three: based on the sensitivity analysis of the test kit of HRMA technology for detection four kinds of diarrhea viruses
1. method:
(1) sample process: using the viral RNA of above-mentioned in-vitro transcription as the template detected, micro-ultraviolet spectrophotometer is utilized to measure the concentration of RNA after purifying, the copy number of Initial R NA template is calculated by Molecular weights, then 10 times of gradient dilutions are taken turns doing to a position copy number, amount to 9 gradients, copy number is: 1 × 10
8to 1 × 10
0.
(2) parallel control experiment: select HRMA detection kit of the present invention and conventional RT-PCR technology (basic agent purchased from precious biotechnology (Dalian) company limited, article No. DRR055A) to carry out augmentation detection to above-mentioned 9 gradient dilution samples and 1 negative control respectively.
HRMA detects: adopt detection method described in embodiment two to carry out reaction solution preparation, be then placed in by reaction tubes on fluorescent PCR instrument and carry out increasing and detecting.Reaction conditions is: 42 DEG C 10 minutes; 94 DEG C 2 minutes; 40 circulation 94 DEG C 30 seconds-56 DEG C 30 seconds-72 DEG C 30 seconds; 55 DEG C are progressively risen to 95 DEG C, and each rising 0.1 DEG C also maintains 2 seconds, persistent collection fluorescent signal (FAM passage).React rear observation fluorescence curve collection of illustrative plates, analyze the template copy numbers minimum that test kit of the present invention can detect.
Conventional RT-PCR detects: containing 2 × One Step RT-PCRBuffer 10 μ L in 20 μ L RT-PCR reaction systems, 20 μMs of forward primers and reverse primer each 0.8 μ L, PrimeScript One StepEnzyme Mix 0.8 μ L.The reaction conditions of RT-PCR is: 50 DEG C of 30min; 94 DEG C of 3min; (94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 30sec) × 35 circulations, 72 DEG C of 7min.Amplified fragments size is respectively rotavirus 186bp, Astrovirus 311bp, norovirus 376bp, and letter is as virus-4 74bp.About 150 minutes working times of machine on whole RT-PCR.After RT-PCR reaction terminates, get 5 μ L products and carry out electrophoresis in the sepharose of 2.0%, electrophoresis time is 40 minutes, and electrophoresis terminates rear gel imaging system observations and takes pictures.
2. result: the detected result of parallel control experiment shows, the Monitoring lower-cut of HRMA test kit of the present invention is respectively: rotavirus 1 × 10
0copy (Figure 1A); Astrovirus 1 × 10
2copy (Figure 1B); Norovirus 1 × 10
0copy (Fig. 1 C); Letter is as virus 1 × 10
3copy (Fig. 1 D).The detection minimum of conventional RT-PCR reaction is then respectively 1 × 10
3copy (Fig. 1 E), 1 × 10
3copy (Fig. 1 F), 1 × 10
3copy (Fig. 1 G), 1 × 10
4copy (Fig. 1 H).Therefore, the remolding sensitivity conventional RT-PCR of HRMA test kit of the present invention high 1000 times (for rotavirus and norovirus) or 10 times (for Astrovirus and letter as virus).Visible, HRMA detection kit of the present invention is safely, efficiently, highly sensitive, can play a significant role in quick Emergent detection and trace sample detect.
Embodiment four: based on the specificity analyses of the test kit of HRMA technology for detection four kinds of diarrhea viruses
1. sample: the positive clinical sample selecting one group of related diseases substance of suffering from diarrhoea, number consecutively, is respectively 1: rotavirus, and 2: Astrovirus, 3: norovirus, 4: letter as virus, 5: enterovirus EV71,6: coxsackie virus A 16-type, 7: vibrio cholerae, 8: Salmonellas; 9 is negative control.
2. method: use HRMA detection kit of the present invention, adopts method described in embodiment two originally to detect above 9 increments, observes this test kit and whether nonspecific detected result can occur.
3. result: the fluorescence pattern according to the amplification of HRMA detection kit carries out high resolving power melting curve analysis, test kit of the present invention is only positive to the detected result of 1-4 sample, the detection of other pathogenic agent and negative control is feminine gender, proves that the method has good specificity.The results are shown in Figure 2.
Embodiment five: based on the test kit of HRMA technology for detection four kinds of diarrhea viruses to the detection of clinical sample
1. sample: all derive from the stool sample that this laboratory is preserved.Wherein Stochastic choice 10 parts from 300 parts of diarrheic stools samples, selects 1 part, detects simultaneously from 30 parts of normal fecal stools samples.
2. method: adopt double-blind method that 11 of Stochastic choice parts of samples are upset numbering, then carry out nucleic acid extraction, adopt HRMA detection kit of the present invention and conventional RT-PCR technology to detect respectively, the coincidence rate of last comparison and detection result.
3. detecting step:
(1) viral nucleic acid extracts: picking 100mg stool sample vibrates mixing in 800 μ L physiological saline, centrifugal 5 minutes of 8000rpm, get 200 μ L supernatants to extract for RNA, (article No.: 57704), operates by test kit specification sheets to adopt the test kit QIAamp MinElute Virus SpinKit of German QIAGEN company;
(2) HRMA detects: operate by the method for embodiment two;
(3) conventional RT-PCR detects: operate by the method for embodiment three.
4. detected result: utilize HRMA detection kit provided by the invention can detect whole 10 parts of virus-positive samples, completely the same with the detected result of conventional RT-PCR, the coincidence rate of two kinds of methods is 100%, and detected result is shown in Fig. 3 and table 3.
Table 3 11 parts of clinical sample detected results
Note: R: rotavirus; A: Astrovirus; N: norovirus; S: letter is as virus.
The present invention is by dividing other sequence analysis analysis to four kinds of viral genome, find the conserved sequence design primer of very high homology, and utilize high resolving power melting curve analysis technology, detect four kinds of diarrhea viruses simultaneously, there is the feature of easy quick, cheap, safe, highly sensitive, high specificity, strong technical support can be provided for the prevention and control of dysentery and clinical diagnosis, conventional sense and the control and prevention of disease field at clinical and port can be widely used in.
It should be noted that, above-mentionedly only to describe the present invention with preferred embodiment, interest field of the present invention can not be limited at this point, therefore when not departing from inventive concept, the equivalence that the content of all utilizations specification sheets of the present invention and accompanying drawing part is carried out changes, and all should be included in right of the present invention.
Claims (9)
1. one group is detected the primer sets of four kinds of diarrhea viruses simultaneously based on high resolving power melting curve analysis technology, comprises,
For detecting the primer of rotavirus:
Forward F1:5 '-GATACNTTRCCTGAYGGAGA-3 ' (SEQ ID NO.1),
Reverse R1:5 '-CATCYTGAAAKATDGCATCACA-3 ' (SEQ ID NO.2);
For detecting the primer of Astrovirus:
Forward F2:5 '-AAAAYGTDCGAHTGAAGGA-3 ' (SEQ ID NO.3),
Reverse R2:5 '-ABACAGTTCTYAACCAATG-3 ' (SEQ ID NO.4);
For detecting the primer of norovirus:
Forward F3:5 '-AGRGTBGTCGTHTGGGACGA-3 ' (SEQ ID NO.5),
Reverse R3:5 '-TGNGGGGTGTTNCCRTTCTT-3 ' (SEQ ID NO.6);
For detecting the primer of letter as virus:
Forward F4:5 '-ACTGTNMAYGGKTTCCATGT-3 ' (SEQ ID NO.7),
Reverse R4:5 '-CAGGRGANCGGTGTGTRCCAAT-3 ' (SEQ ID NO.8);
The viral nucleic acid variant sites of described primer uses degeneracy base, and R is A/G, K be G/T, M be A/C, W be A/T, Y be C/T, S be C/G, V be A/C/G, B be C/G/T, H be A/T/C, D be A/G/T, N is A/G/T/C.
2. one group is detected four link detection reagent kits of four kinds of diarrhea viruses, containing primer sets according to claim 1 simultaneously based on high resolving power melting curve analysis technology.
3. four link detection reagent kits according to claim 2, is characterized in that: described test kit comprises 2X amplification reaction solution, 5X enzyme mixation, positive reference substance, negative controls and blank product.
4. four link detection reagent kits according to claim 3, is characterized in that: described 2X amplification reaction solution comprises: Tris-HCl, KCl, MgCl
2, DTT, spermidine, Tween-20, bovine serum albumin, dNTP, primer sets described in fluorescence dye and claim 1.
5. four link detection reagent kits according to claim 4, is characterized in that: described fluorescence dye is EvaGreen.
6. four link detection reagent kits according to claim 3, is characterized in that: described 5X enzyme mixation comprises ThermoScript II, RNases inhibitor and HS-Taq enzyme.
7. four link detection reagent kits according to claim 3, is characterized in that: described positive reference substance is respectively rotavirus, Astrovirus, norovirus and the letter RNA fragment as viral in-vitro transcription.
8. four link detection reagent kits according to claim 3, is characterized in that: described negative controls is stroke-physiological saline solution.
9. test kit according to claim 3, is characterized in that: described blank is the ultrapure water of the nuclease free of DEPC process.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838827A (en) * | 2016-05-24 | 2016-08-10 | 北京市疾病预防控制中心 | Virus genome primer and method for detecting virus genome through same |
| CN107090518A (en) * | 2017-04-05 | 2017-08-25 | 苏州协云基因科技有限公司 | The multiple RT PCR Polymorphism chip inspecting reagent units of the related pathogen of diarrhoea |
| CN110592278A (en) * | 2019-09-04 | 2019-12-20 | 广西大学 | Multiplex RT-PCR kit for PRoV, PoSaV and PAStV |
-
2015
- 2015-03-11 CN CN201510106726.8A patent/CN104651538A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838827A (en) * | 2016-05-24 | 2016-08-10 | 北京市疾病预防控制中心 | Virus genome primer and method for detecting virus genome through same |
| CN107090518A (en) * | 2017-04-05 | 2017-08-25 | 苏州协云基因科技有限公司 | The multiple RT PCR Polymorphism chip inspecting reagent units of the related pathogen of diarrhoea |
| WO2018184531A1 (en) * | 2017-04-05 | 2018-10-11 | 苏州协云基因科技有限公司 | Detection kit for diarrhea-related pathogens combining multiple rt-pcr with gene chip |
| CN107090518B (en) * | 2017-04-05 | 2020-03-20 | 苏州协云基因科技有限公司 | Diarrhea-related pathogen multiple RT-PCR combined gene chip detection kit |
| CN110592278A (en) * | 2019-09-04 | 2019-12-20 | 广西大学 | Multiplex RT-PCR kit for PRoV, PoSaV and PAStV |
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