CN1811388A

CN1811388A - Viral hemorrhagic viruse fluorescence quantitative detecting method for rabbit

Info

Publication number: CN1811388A
Application number: CN 200610042409
Authority: CN
Inventors: 田夫林; 陈静; 陈书民; 孙圣福; 张秀娥; 王贵升; 马慧玲; 李运兰; 兰邹然; 于青海; 张栋
Original assignee: 田夫林
Priority date: 2006-02-23
Filing date: 2006-02-23
Publication date: 2006-08-02

Abstract

The present invention relates to a rabbit hemorrhagic disease virus fluorescent quantitative detection method. Said invented technical scheme is characterized by adopting Taqman MGB probe, comparing rabbit hemorrhagic disease virus VP 60 gene, designing a pair of conservative specific primers and a specific MGB probe, and utilizing fluorescent signal to judge rabbit hemorrhagic disease virus.

Description

Viral hemorrhagic viruse fluorescence quantitative detecting method for rabbit

Technical field: the present invention relates to animal epidemic diagnosis detecting method technical field, is viral hemorrhagic viruse fluorescence quantitative detecting method for rabbit more specifically.

Background technology: rabbit hemorrhagic disease virus has the effect of the blood red blood cell of aggegation people O type, and can be suppressed by corresponding antibody.But because toxic amount is lower in the sick rabbit tissue, be not enough to produce the blood clotting phenomenon, thereby the diagnosis of rabbit hemorrhagic disease virus need carry out the rabbit body earlier and inoculate, do hemagglutination test with the higher liver organization of toxic amount again.So not only wasted time and energy but also delayed treatment time.Conventional PCR method has realized diagnosing fast, delicately the purpose of rabbit hemorrhagic disease virus substantially, but traditional molecular diagnosis method exist complex operation, time-consuming, easy pollution, non-specific height and quantitatively difficulty, be prone to defective such as false positive.

Summary of the invention:, the objective of the invention is to invent a kind of viral hemorrhagic viruse fluorescence quantitative detecting method for rabbit for overcoming above-mentioned shortcoming.Its technical scheme is: 1, design is synthetic and filter out a pair of Auele Specific Primer and specific probe: ForwardPrimer (P1): ACTGGTCGCGGCTGTGAT; Reverse Primer (P2): CCTCCAACCCTGGTCCAAT, fluorescence probe are FAM 5 ' CCACCAGGCATCGA-3 ' MGB; 2, the extraction of RNA (ribonucleic acid) (RNA): in 250 μ l virus concentrate, add 750 μ l lysates, jolting 15s, room temperature is placed 5min; Add 200 μ l chloroforms, jolting 15s; Room temperature is placed 10min; 4 ℃, 10, the centrifugal 15min of 000rpm gets the upper strata water and add the equal-volume isopropyl alcohol in another centrifuge tube, and room temperature is placed 10min behind the mixing; 4 ℃, 12, the centrifugal 12min of 000rpm, precipitated rna; Carefully abandon clean supernatant, with 75% ice-cold ethanol 1ml washing precipitation, behind the suspendible 12, the centrifugal 20min of 000rpm carefully abandons air-dry RNA precipitation in the clean supernatant super-clean bench; Handle water 10 μ l dissolution precipitations with DEPC, and add 1.5 μ l RNA enzyme inhibitors, 5 μ l/ manage packing, frozen or reverse transcription immediately; 3, reverse transcription: will add 2 μ l reverse transcription damping fluids, 0.5 μ l deoxyribonucleotide mixed liquor in each sample of carrying RNA; 0.5 μ l random primer; 0.25 μ l reverse transcriptase adds DEPC water to 10 μ l; 42 ℃ 10～15 minutes, 95 ℃ 2 minutes, reverse transcription becomes cDNA; 4, PCR (PCR): add 25 μ L reaction buffers in each reaction tube, specificity upstream primer and downstream primer respectively add 1ul, probe 2ul; Positive control adds positive plasmid 4ul; ROX confidential reference items 1ul; CDNA adds 4ul; Negative control replaces with deionized water; Add sterile purified water to 50ul; 5, response parameter: 95 ℃ of 10s; 95 ℃ of 5s, 60 ℃ of 31s, 72 ℃ of 10s, 40 circulations; 72 ℃ of 2min; Read fluorescence signal at 60 ℃ of annealing steps of each circulation; Reaction tube packed into start reaction in the quantitative PCR instrument; 6, the result judges: the CT value of positive control should be less than 28, and negative control CT value should be greater than 35; The CT value is smaller or equal to 30, and the tangible amplification of appearance line, shows to have RHDV in the sample, is judged to the positive; The CT value is considered as suspicious between 30-35, answers revision test, if still be judged to feminine gender in this scope.The advantage of this invention is: successfully Auele Specific Primer and probe have been screened in design, have optimized reaction conditions, have successfully drawn typical curve; Utilize this fluorescence quantitative detecting method to solve the diagnosis difficult problem of rabbit hemorrhagic disease, this method can real-time quantitative detects rabbit hemorrhagic disease virus content in the clinical pathological material of disease, and is applicable to the fast detecting of outlet rabbit product.

Embodiment:

One, material:

1. lysate (trizol purchases in reagent company): contain guanidinium isothiocyanate and phenol etc.

2. PCR kit for fluorescence quantitative is purchased the precious biotech firm (Code No:DRR051A) in Dalian: include reverse transcription damping fluid (containing magnesium chloride, Tris-hydrochloric acid, potassium chloride); Four kinds of deoxyribonucleotide mixed liquors; Random primer; Reverse transcriptase; Reaction buffer (containing magnesium chloride, Tris-hydrochloric acid, potassium chloride, archaeal dna polymerase); The RNA enzyme inhibitor; 1%DEPC (burnt ethylene carbonate) water; The ROX reference.

3. reactant liquor (containing Auele Specific Primer 10pmol/ul and probe 5pmol/ul);

Forward?Primer(P1)：ACTGGTCGCGGCTGTGAT，

Reverse?Primer(P2)：CCTCCAACCCTGGTCCAAT，

Fluorescence probe is FAM 5 ' CCACCAGGCATCGA-3 ' MGB,

4. positive plasmid;

5. chloroform; Isopropyl alcohol; 75% cold ethanol; Fluorescence reaction pipe and lid; 1%DEPC (burnt ethylene carbonate) water.

Two, method of operating:

1. the extraction of RNA (ribonucleic acid) (PNA): in 250 μ l virus concentrate, add 750 μ l lysates, jolting 15s, room temperature is placed 5min; Add 200 μ l chloroforms, jolting 15s.Room temperature is placed 10min; 4 ℃, 10, the centrifugal 15min of 000rpm gets the upper strata water and add the equal-volume isopropyl alcohol in another centrifuge tube, and room temperature is placed 10min behind the mixing; 4 ℃, 12, the centrifugal 12min of 000rpm, precipitated rna; Carefully abandon clean supernatant, with 75% ice-cold ethanol 1ml washing precipitation, behind the suspendible 12, the centrifugal 20min of 000rpm carefully abandons air-dry RNA precipitation in the clean supernatant super-clean bench; Handle water 10 μ l dissolution precipitations with DEPC, and add 1.5 μ l RNA enzyme inhibitors, 5 μ l/ manage packing, frozen or reverse transcription immediately.

2. reverse transcription: will add 2 μ l reverse transcription damping fluids, 0.5 μ l deoxyribonucleotide mixed liquor in each sample of carrying RNA; 0.5 μ l random primer; 0.25 μ l reverse transcriptase adds DEPC water to 10 μ l; 42 ℃ 10～15 minutes, 95 ℃ 2 minutes, reverse transcription becomes cDNA.

3. PCR (PCR): each reaction tube, that is: add 25 μ L reaction buffers in a part, specificity upstream primer and downstream primer respectively add 1ul, probe 2ul; Positive control adds positive plasmid 4ul; ROX confidential reference items 1ul; CDNA adds 4ul; Negative control replaces with deionized water; Add sterile purified water to 50ul.

4. response parameter: 95 ℃ of 10s; 95 ℃ of 5s, 60 ℃ of 31s, 72 ℃ of 10s, 40 circulations; 72 ℃ of 2min.Read fluorescence signal at 60 ℃ of annealing steps of each circulation.Reaction tube packed into start reaction in the quantitative PCR instrument.

5. the result judges: the CT value of positive control should be less than 28, and negative control CT value should be greater than 35;

The CT value is smaller or equal to 30, and the tangible amplification of appearance line, shows to have RHDV in the sample, is judged to the positive;

The CT value is considered as suspicious between 30-35, answers revision test, if still be judged to feminine gender in this scope.

Claims

1, viral hemorrhagic viruse fluorescence quantitative detecting method for rabbit is characterized in that: 1, a pair of Auele Specific Primer of design and probe: Forward Primer (P1): ACTGGTCGCGGCTGTGAT; Reverse Primer (P2): CCTCCAACCCTGGTCCAAT, fluorescence probe are FAM 5 ' CCACCAGGCATCGA-3 ' MGB; 2, the extraction of RNA (ribonucleic acid) (RNA): in 250 μ l virus concentrate, add 750 μ l lysates, jolting 15s, room temperature is placed 5min; Add 200 μ l chloroforms, jolting 15s; Room temperature is placed 10min; 4 ℃, 10, the centrifugal 15min of 000rpm gets the upper strata water and add the equal-volume isopropyl alcohol in another centrifuge tube, and room temperature is placed 10min behind the mixing; 4 ℃, 12, the centrifugal 12min of 000rpm, precipitated rna; Carefully abandon clean supernatant, with 75% ice-cold ethanol 1ml washing precipitation, behind the suspendible 12, the centrifugal 20min of 000rpm carefully abandons air-dry RNA precipitation in the clean supernatant super-clean bench; Handle water 10 μ l dissolution precipitations with DEPC, and add 1.5 μ l RNA enzyme inhibitors, 5 μ l/ manage packing, frozen or reverse transcription immediately; 3, reverse transcription: will add 2 μ l reverse transcription damping fluids, 0.5 μ l deoxyribonucleotide mixed liquor in each sample of carrying RNA; 0.5 μ l random primer; 0.25 μ l reverse transcriptase adds DEPC water to 10 μ l; 42 ℃ 10～15 minutes, 95 ℃ 2 minutes, reverse transcription becomes cDNA; 4, PCR (PCR): add 25 μ L reaction buffers in each reaction tube, specificity upstream primer and downstream primer respectively add 1ul, probe 2ul; Positive control adds positive plasmid 4ul; ROX confidential reference items 1ul; CDNA adds 4ul; Negative control replaces with deionized water; Add sterile purified water to 50ul; 5, response parameter: 95 ℃ of 10s; 95 ℃ of 5s, 60 ℃ of 31s, 72 ℃ of 10s, 40 circulations; 72 ℃ of 2min; Read fluorescence signal at 60 ℃ of annealing steps of each circulation; Reaction tube packed into start reaction in the quantitative PCR instrument; 6, the result judges: the CT value of positive control should be less than 28, and negative control CT value should be greater than 35; The CT value is smaller or equal to 30, and the tangible amplification of appearance line, shows to have RHDV in the sample, is judged to the positive; The CT value is considered as suspicious between 30-35, answers revision test, if still be judged to feminine gender in this scope.