CN105603120A - GeXP multiple rapid detection primers for detection of bluetongue virus, bovine viral diarrhea virus and foot and mouth disease virus and detection method - Google Patents
GeXP multiple rapid detection primers for detection of bluetongue virus, bovine viral diarrhea virus and foot and mouth disease virus and detection method Download PDFInfo
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Abstract
GeXP multiple rapid detection primers for detection of bluetongue virus, bovine viral diarrhea virus and foot-and-mouth disease virus and a detection method are provided; the detection method for various cow diseases based on a GeXP multiple gene expression genetic analysis system is intended to be built up; the problems that when detection of the various cow diseases, a serological method has low sensitivity, conventional PCR only detects single pathogens and a gene chip method has relatively high cost are solved; three cow disease DNA positive sample standard plasmids are constructed; a reference obtained by 10-time continuous dilution of the plasmids DNA is used as a detection object, and the detection sensitivity is 10-100 copies; no cross reactions with other cow diseases happen, and no false negative results exist; the number of detected actual simples is 600, and monitoring screening requirements of various diseases of a lot of cows are met.
Description
Technical field
The present invention relates to biological technology application, in particular for three kinds of inward milk cow virosis time, detect and qualification.
Background technology
Aftosa (FootandMouthDisease, FMD), bovine viral diarrhoea (BovineViralDiarrhea, BVD) and blue tongue disease (Bluetongue, BT) be 3 kinds of viral diseases that the inward milk cow of national regulation needs quarantine procedures. In recent years, along with the lifting of its people to high-quality dairy produce demand, the milk cow quantity that enters the territory was soaring year by year, brought certain operating pressure and funds burden to animal epidemic testing laboratory, the inward port of milk cow. Therefore the high flux method for quick of, setting up the multiple milk cow epidemic disease of a kind of simultaneously antidiastole to alleviate milk cow enter the territory port testing laboratory work load, reduce detection funds and carry out the epidemiology survey that the milk cow that enters the territory carries epidemic disease significant.
At present, for the multiple viral disease of the milk cow that enters the territory, animal quarantine laboratory, port is generally applied viral separation, serological method and molecular biology method and is detected. Virus separates and cultivates is the goldstandard of diagnosis, but has the problems such as survey cycle length, complex operation; Virus neutralization tests needs specific standard serum or virus stain, has in actual applications certain limitation; The enzyme linked immunological kit prevailing price costliness of import; Regular-PCR, quantitative fluorescent PCR equimolecular biological method are all for single virus and detect, and cannot meet the detection demand of multiple cause of disease mixed infection antidiastole. Multiple PCR technique has been applied to the antidiastole of multiple cause of disease, but the high flux that cannot realize truly due to amplification preferences problem detects.
Summary of the invention
In recent years due to the increasing year by year of Imported Holstein quantity, the animal quarantine staff who bears the milk cow epidemic disease Detection task that enters the territory needs a kind of highly sensitive, detection method that specificity is good badly and reduces external animal epidemic and import the work requirements that risk and realization speed passage through customs into meet. At present, the applied serology in Dong Jian laboratory, port (ELISA) experiment mostly, every kind of detection kit can only be checked single animal epidemic, cannot differentiate the mixed infection of many animals epidemic disease, and the price of import detection kit costliness and bring very large economic pressures to testing laboratory. Therefore, set up a kind of high flux, fast, detection method that specificity is good imports risk into for reducing animal epidemic, the animal that accelerates to enter the territory is open to the custom, judicial convenience and reduce animal epidemic testing cost and all have important meaning gives a impetus to trade.
GeXP multiple gene expression analytical system combines multiple PCR technique and capillary electrophoresis technique, not only has advantages of that multiplex PCR amplification high flux detects, and has the advantages that Capillary Electrophoresis sensitivity is high. The technical characterstic of GeXP is to add universal primer sequence at 5 ' end of Auele Specific Primer, composition specific chimeric primer. In PCR course of reaction, specific chimeric primer first respectively with corresponding template combination, ensure the specificity of follow-up amplification, after multiple circulations, by the leading follow-up amplification of universal primer, can reduce like this interference between different primers in same reaction system, realize every pair of primer amplification efficiency and reach unanimity, thereby efficiently solve the amplification preferences problem of conventional multiple PCR technique.
This research is according to the gene conserved sequence of 3 kinds of inward milk cow quarantine virus, multipair Auele Specific Primer and universal primer are designed respectively, optimize reaction condition, by the detection validation to single viral template, hybrid virus template specificity and the accuracy of every pair of primer amplification in GeXP multiple RT-PCR system, set up the GeXP high throughput method that simultaneously detects 3 kinds of milk cow virosis, detection sensitivity at least can reach 102Copy/μ L. Molecular diagnosis and the epidemiology survey of milk cow epidemic disease overseas that the method can be the milk cow virosis that enters the territory provide technical support. From now on, also need a large amount of positive sample or virus stain to do further checking and optimization to this method.
Brief description of the drawings
Fig. 1-Fig. 4 GeXP multiple RT-PCR specificity the result (Fig. 1, BTV; Fig. 2, BVDV; Fig. 3, FMDV; Fig. 4,50bp-800bpMarker), Tu ZhongYZhou Wei relative fluorescence unit, the fluorescence intensity of expression amplified production, X-axis is the size of amplified production in Capillary Electrophoresis process.
Sensitivity result (Fig. 5,10 of Fig. 5-Figure 10 GeXP multiple RT-PCR to 3 kinds of viral template6Copies/ μ L; Fig. 6,105Copies/ μ L; Fig. 7,104Copies/ μ L; Fig. 8,103Copies/ μ L; Fig. 9,102Copies/ μ L; Figure 10,50bp-800bpMarker).
Detailed description of the invention
GeXP starts kit, DNAsizestandardKit, dissociating buffer, sample-loading buffer purchased from Beckman company of the U.S., one-step method RT-PCR kit, RNA extract kit, isogeneity kit purchased from German QIAGEN company, T7 in-vitro transcription kit, SpeI restriction endonuclease and RNA enzyme inhibitor are all purchased from the precious biotech firm in Dalian, and pGEX-T carrier is purchased from Promega company.
Bibliography is selected each viral gene conserved region and from GenBank database, is downloaded 3 kinds of viral gene orders, by DNASTAR software analysis sequence homology, and with GeXPeXpressProfiler tool design multiple specific primer. Connect respectively one section of non-homology sequence as universal primer (Tag) at forward and reverse Auele Specific Primer 5 ' end, composition specific chimeric primer. Primer is synthesized by Invitrogen company, HPLC purifying.
SEQIDNO:1-BT3:5 '-AGGTGACACTATAGAATAGTTCTCTAGTTGACCACC-3 ' 5 ' end cy5 mark;
SEQIDNO：2-BT4：5′-GTACGACTCACTATAGGGAAAGCCAGACTGTTTCCCGAT-3′；
SEQIDNO:3-BVDV3:5 '-AGGTGACACTATAGAATAAGCGAAGGCCGAAAAGAGGCTA-3 ' 5 ' end cy5 mark;
SEQIDNO：4-BVDV4：
5′-GTACGACTCACTATAGGGAAACTCCATGTGCCATGTACAGCAG-3′；
SEQIDNO:5-FMDV3:5 '-AGGTGACACTATAGAATAGCCTGGTCTTTCCAGGTCT-3 ' 5 ' end cy5 mark;
SEQIDNO：6-FMDV4：5′-GTACGACTCACTATAGGGACCAGTCCCCTTCTCAGATC-3′；
SEQIDNO:7-universal primer 1:5 '-AGGTGACACTATAGAATA-3 ' 5 ' end cy5 mark;
SEQIDNO:8-universal primer 2:5 '-GTACGACTCACTATAGGGA-3 ';
BT, the BVDV of deactivation, FMDV strain are this laboratory and preserve. The extraction of viral RNA is used the RNA of QIAGEN company to extract kit, and elution volume is 40 μ L, in-80 DEG C of preservations.
With not containing the Auele Specific Primer of the universal primer viral RNA that contains genes of interest region that increases respectively, PCR product after amplification is connected to pGEX-T carrier and carries out monoclonal structure plasmid, extract cloned plasmids, SpeI linearization for enzyme restriction, and with isogeneity kit to linearisation after plasmid carry out purifying, carry out in-vitro transcription with T7 transcript reagent box, utilize ultraviolet specrophotometer to measure RNA concentration and calculate the copy number of each viral RNA.
Universal primer is diluted to working concentration 10 μ mol/L, and Auele Specific Primer is diluted to working concentration 1 μ mol/L. Extract 3 kinds of viral RNAs template, prepare reaction system according to one-step method RT-PCR kit: 10 × RT-PCR buffer solution, 2.5 μ L, dNTP premixed liquid 2 μ L, RT-PCREnzymeMix1 μ L, RNA enzyme inhibitor 0.1 μ L, upstream and downstream chimeric primers (1 μ mol/L) is respectively got 1 μ L, and upstream and downstream universal primer (10 μ mol/L) is respectively got 1 μ L, template ribonucleic acid 2 μ L, use the not water containing nuclease to supply 25 μ L. After condition optimizing, finally determine that reaction condition is: 45 DEG C of 30min, 94 DEG C of 15min reverse transcriptions; Then: 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 10 circulations; 94 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 30s, 10 circulations; 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 30s, 20 circulations; 72 DEG C of 10min, 1 circulation. Product is carried out GeXPsystem capillary electrophoresis analysis.
Chimeric primers corresponding to each virus got respectively to equivalent to be mixed, be mixed with multiple mix primer, making each primer concentration is 10 μ mol/L, and final concentration in reaction system is 50nmol/L, in reaction system, other composition, with substance RT-PCR, is got the specificity of each primer in 3 kinds of inactivation of virus sample nucleic acid checking Multiple RT-PCR detec-tion systems.
By in-vitro transcription viral RNA gradient dilution to 105、104、103、102、101Copy/μ L, respectively gets 1 μ L as template, detects the sensitivity of multiple system.
Adjust each to primer concentration according to many primers single mode plate sensitivity test result. It is 10 that outer 3 kinds of virion transcribe rna isoconcentration is mixed to also gradient dilution6、105、104、103、102Copy/μ L is as simulation biased sample, and remaining reaction condition is constant, carries out the sensitivity analysis of many primers multi-template. Test non-on the same day in triplicate, record testing result and calculate the coefficient of variation CV of each genetic test.
Detailed description of the invention
Embodiment 1 primer checking
Substance RT-PCR product is through GeXP system capillary electrophoresis analysis, and each genes of interest amplified fragments size is respectively BTV:306～310bp, BVDV:347～352bp, and FMDV:365～370bp, amplified fragments size conforms to design.
Embodiment 2 Multiple detection Establishings and single template specificity the result
In many primers detection architecture, each reaction only amplifies the specific fragment of single viral template, no cross reaction, and prompting the method high specificity, can differentiate and distinguish each virus according to amplified fragments size, the results are shown in Figure 1.
Embodiment 3 Multiple detection system single mode plate sensitivity test results
Utilizing cloned plasmids in-vitro transcription RNA is template, and the detection lower limit of various viruses is respectively: FMDV is 10 copies/μ L, and BVDV is 100 copies/μ L.
Embodiment 4 Multiple detection system multi-template sensitivity results
Optimize after reaction condition, Multiple detection system can be 106、105、104、103、102Copy/μ L level detects the simulation hybrid template of 3 kinds of viral RNAs simultaneously, the results are shown in Figure 2. Test non-three repetitions on the same day, 104When copy/μ L, each viral gene coefficient of variation is between 1.2%～7.6%.
Claims (3)
1. one kind is detected aftosa (FootandMouthDisease, FMD), bovine viral diarrhoea (Bovine simultaneouslyViralDiarrhea, BVD), the combination of primers of blue tongue disease (Bluetongue, BT): it is characterized in that spyOpposite sex primer and universal primer sequence are as shown in SEQNO:1 to 8.
2. combination of primers claimed in claim 1 is at preparation aftosa (FootandMouthDisease, FMD), bovine viral abdomenRush down the application in (BovineViralDiarrhea, BVD), blue tongue disease (Bluetongue, BT) diagnostic reagent.
3. detect simultaneously aftosa (FootandMouthDisease, FMD), bovine viral diarrhoea (BovineViralDiarrhea,BVD), the kit of blue tongue disease (Bluetongue, BT), it is characterized in that its Auele Specific Primer and universal primer sequence are respectivelyFor: the primer shown in SEQNO:1-8; GeXP starts kit, dissociating buffer, sample-loading buffer, one-step method RT-PCRReagent, RNA extract reagent, isogeneity reagent;
Universal primer is diluted to working concentration 10 μ mol/L, and Auele Specific Primer is diluted to working concentration 1 μ mol/L, extracts 3 kinds of virusesRNA, as template, prepares reaction system according to one-step method RT-PCR kit: 10 × RT-PCR buffer solution, 2.5 μ L, dNTPPremixed liquid 2 μ L, RT-PCREnzymeMix1 μ L, RNA enzyme inhibitor 0.1 μ L, upstream and downstream chimeric primers (1 μ mol/L)Respectively get 1 μ L, upstream and downstream universal primer (10 μ mol/L) is respectively got 1 μ L, and template ribonucleic acid 2 μ L, by the water benefit that does not contain nucleaseFoot 25 μ L; After condition optimizing, finally determine that reaction condition is: 45 DEG C of 30min, 94 DEG C of 15min reverse transcriptions; Then:94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 10 circulations; 94 DEG C of 30s, 65 DEG C of 30s, 72 DEG C of 30s, 10 circulations; 94 DEG C30s, 50 DEG C of 30s, 72 DEG C of 30s, 20 circulations; 72 DEG C of 10min, 1 circulation, product is carried out GeXPsystem hairCons electrophoresis is analyzed.
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Cited By (4)
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| CN105969913A (en) * | 2016-07-19 | 2016-09-28 | 广西壮族自治区兽医研究所 | Primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses |
| CN106191309A (en) * | 2016-07-19 | 2016-12-07 | 广西壮族自治区兽医研究所 | A kind of primer combination simultaneously differentiating 8 kinds of cattle disease substances and GeXP detection method |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
| CN108588275A (en) * | 2018-03-23 | 2018-09-28 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method of BTV-10 types, 20 types, 23 type genotypings |
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| CN105969913A (en) * | 2016-07-19 | 2016-09-28 | 广西壮族自治区兽医研究所 | Primer combination and GeXP detection method for simultaneously identifying 5 bovine viral dermatitis viruses |
| CN106191309A (en) * | 2016-07-19 | 2016-12-07 | 广西壮族自治区兽医研究所 | A kind of primer combination simultaneously differentiating 8 kinds of cattle disease substances and GeXP detection method |
| CN108342510A (en) * | 2018-03-23 | 2018-07-31 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method that BTV-11 types, 17 types, 20 types, 23 types, 24 type genotypings differentiate |
| CN108588275A (en) * | 2018-03-23 | 2018-09-28 | 重庆出入境检验检疫局检验检疫技术中心 | The multiple RT-PCR kit and its detection method of BTV-10 types, 20 types, 23 type genotypings |
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