CN102206709A - CSFV detection method utilizing realtime fluorescence quantitative RT-PCR - Google Patents
CSFV detection method utilizing realtime fluorescence quantitative RT-PCR Download PDFInfo
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- CN102206709A CN102206709A CN2011100849772A CN201110084977A CN102206709A CN 102206709 A CN102206709 A CN 102206709A CN 2011100849772 A CN2011100849772 A CN 2011100849772A CN 201110084977 A CN201110084977 A CN 201110084977A CN 102206709 A CN102206709 A CN 102206709A
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Abstract
The invention discloses a CSFV (classical swine fever virus) detection method utilizing realtime fluorescence quantitative RT-PCR. According to results of singularity and sensitivity teats of a method established in the invention, the method can well distinguish the CSFV from FMDV (Foot And Mouth Disease Virus), TGEV (Transmissible Gastroenteritis Virus), PRRSV (Porcine Respiratory and Reproductive Syndrome Virus) and PCVII( Porcine Circovirus II), and sensitivity can reach an order of magnitude of 10 copy number. In addition, whole reaction process of the method established by the invention is operated in a close pipe, so as to reduce solution to the system at most. Compared with a traditional method, the method in the invention has a substantially shortened detection time, on the premise of guaranteeing detection result. For the whole process, comprising steps of obtaining of sample to be checked, nucleic acid extraction, preparation of fluorescence quantitative RT-PCR reaction system, and appearance of reaction result, can be finished within 2.5 h.
Description
Technical field
The present invention relates to the detection technique field, particularly a kind of can be accurate, special, the method for rapid detection CSFV.
Background technology
(swine fever SF), claims hog cholera (hog cholera again to swine fever, HC), rinderpest, then be called as in Europe classic swine fever (classical swine fever, CSF), be a kind of important transmissible disease of pig industry in the world, once brought serious economy loss to pig industry.
Traditional diagnostic mode for CSF mainly contains animal experiment, immunofluorescent test, serum neutralization test, ELISA, monoclonal antibody test.Animal experiment is the responsive method that detects Pestivirus suis, but does not also present typical swine fever symptom even detection time is long and exist the popular Pestivirus suis mostly to be the inoculation of low toxicity power virus strain greatly at present; The immunofluorescent test method is comparatively special, but this technical requirements has fluorescence antibody, fluorescent microscope and proofer's higher detection technology in the actual detected application process; Serum neutralization test and ELISA test all are difficult to differentiate vaccine antibody and infect the antibody that the back produces except that detection time is long, be easy to occur false positive; The monoclonal antibody technique specificity is stronger, but operates the process complexity, and cost is higher, does not fit into the requirement of batch samples diagnosis.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of real-time fluorescence quantitative RT-PCR to detect the method for CSFV, be intended to solve existing problem in the prior art.
Technical scheme of the present invention is as follows:
A kind of Auele Specific Primer that is used for real-time fluorescence quantitative RT-PCR detection CSFV, wherein, the CSFV specific primer sequence is shown in SEQ NO.1-2.
A kind of specific probe that is used for real-time fluorescence quantitative RT-PCR detection CSFV, wherein, the CSFV specific probe sequence is shown in SEQ NO.3.
According to claim 2ly be used for the specific probe that real-time fluorescence quantitative RT-PCR detects CSFV, wherein, 5 of described CSFV specific probe ' end mark luminophore FAM, 3 ' hold the mark group B HQ1 that dies out.
A kind of method of using the real-time fluorescence quantitative RT-PCR detection CSFV of above-mentioned Auele Specific Primer and specific probe wherein, may further comprise the steps:
S100, The pretreatment: the sample of gathering is numbered respectively, adds the two anti-solution of an amount of penicillin and streptomycin in sample, supernatant is got in homogenate, and 4 ℃ are spent the night;
S200, RNA extract: to the sample extraction RNA that gathers;
The real-time fluorescence quantitative RT-PCR reaction of S300, CSFV: the RNA that is extracted with above-mentioned steps is a template, adds Auele Specific Primer and the specific probe of CSFV, uses the single stage method RT-PCR test kit of TAKARA company to carry out pcr amplification; S400, according to CSFV real-time fluorescence quantitative RT-PCR reaction result, identify in the sample whether contain CSFV.
5, real-time fluorescence quantitative RT-PCR according to claim 4 detects the method for CSFV, it is characterized in that, real-time fluorescence quantitative RT-PCR reaction system among the described step S300 is 25 μ L, and every Auele Specific Primer final concentration is 0.2 μ mol/L, and specific probe concentration is 0.1 μ mol/L;
Reaction conditions is 42 ℃ of 30min, 92 ℃ of 2min, 92 ℃ of 10s, 55 ℃ of 30s, 40 circulations.
Beneficial effect: the present invention has set up the fluorescence RT-PCR method of quick, sensitive, special detection CSFV on the basis of ABI 7500 fast real-time PCR instrument.Through method that the present invention set up having been carried out the checking of specificity, sensitivity, the result shows that present method can distinguish the same foot and mouth disease virus of CSFV, transmissible gastro-enteritis virus, pig breathing preferably with breeding difficulty syndrome virus and 2 type pig circular ring virus, and sensitivity can reach the order of magnitude of 10 copy numbers.In addition, the method entire reaction course that the present invention set up is the stopped pipe operation, has reduced the pollution of system to the full extent.The present invention compares Zi obtaining sample to be checked to extracting nucleic acid with traditional method on Diagnostic Time, preparation fluorescence quantitative RT-RCR reaction system, up to reaction result occurring, whole process can be finished in 2.5h, has shortened the check required time greatly on the basis that guarantees assay.
Description of drawings
Fig. 1 is the sensitivity test result curve figure that real-time fluorescence quantitative RT-PCR detects CSFV, and the extension rate of curve from left to right is successively: 10
6, 10
5, 10
4, 10
3, 10
2, 10.
Fig. 2 is the specific amplification fluorescence curve that real-time fluorescence quantitative RT-PCR detects CSFV.
Fig. 3 is the figure as a result that real-time fluorescence quantitative RT-PCR detects CSFV in the embodiment of the invention 1.
Embodiment
The invention provides the method that a kind of real time fluorescent quantitative quantitative RT-PCR detects CSFV, clearer, clear and definite for making purpose of the present invention, technical scheme and effect, below the present invention is described in more detail.
The purpose of this invention is to provide the method that a kind of real-time fluorescence quantitative RT-PCR detects Pestivirus suis.Can reach the order of magnitude of 10 copy numbers after the sensitivity empirical tests of the inventive method.Pestivirus suis and foot and mouth disease virus, transmissible gastro-enteritis virus, pig breathing can well be distinguished with breeding difficulty syndrome and 2 type pig circular ring virus simultaneously, prove that specificity is good.It is simple to operate to implement the inventive method on ABI 7500 fast real-time PCR instrument, contaminative is little, fast sensitive, can in 2.5h, finish to detecting the result from obtaining sample to be checked, under the prerequisite that guarantees the Pestivirus suis detection accuracy, shortened detection time greatly.
Of the present inventionly be used for real-time fluorescence quantitative RT-PCR to detect the establishment process of method of CSFV as follows:
1, Auele Specific Primer, probe design: in GenBank, download a plurality of CSFV gene orders, carry out sequential analysis, find conservative fragments to carry out Auele Specific Primer, specific probe design with Primer Express 2.0 with CLUSTAL X.
2, specific probe is synthetic: synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.
3, specific probe is handled: at 5 of specific probe ' end mark luminophore FAM, 3 ' hold the mark group B HQ1 that dies out.
4, the extraction of CSFV and contrast viral nucleic acid: get 150 μ L vaccines, the Mag-Bind viral DNA/RNA Kit that uses OMEGA company to produce carries out viral DNA/RNA and extracts, and method is carried out according to the test kit specification sheets.There is not the DNA/RNA that RNA enzyme water dissolution is extracted with 20 μ L.
5, RT-PCR amplification: to extract CSFV in the step 4 and to contrast viral nucleic acid is template, use the single stage method RT-PCR test kit of TaKaRa company to increase, the final concentration of every Auele Specific Primer is 0.2 μ mol/L, reaction conditions is: 50 ℃ of 30min, 94 ℃ of 2min, 94 ℃ of 10s, 58 ℃ of 30s, 72 ℃ of 30s, 72 ℃ of 7min are set at 35 circulations.Get 5 μ L after electrophoresis finishes and in 1.5% agarose gel electrophoresis, detect amplification.
6, the clone of goal gene, construction recombination plasmid:, adopt T-A clone scheme to be cloned into the pMD18-T carrier with detected purpose band glue recovery after the RT-PCR amplification in the step 5.Use the plasmid DNA extracting and purifying test kit extracting recombinant plasmid of QIAGEN company, operation steps is carried out to specifications, and recombinant plasmid send the order-checking of Guangzhou Ying Jun company, and order-checking identifies that the back is as the standard positive product.
7, the foundation of real-time fluorescent quantitative RT-PCR method:
(1) real-time fluorescence quantitative RT-PCR detects the sensitivity test of CSFV: the OD260 value of measuring reorganization T carrier on ultraviolet spectrophotometer, be converted into copy number according to molecular weight size and Avogadro constant number, 10 doubling dilution recombinant vectorss then, the minimum copy number of detected recombinant vectors promptly is the sensitivity of real-time fluorescence RT-PCR on the fluorescent PCR instrument.The sensitivity test that real-time fluorescence quantitative RT-PCR detects CSFV the results are shown in Figure 1, and the extension rate of curve from left to right is successively: 10
6, 10
5, 10
4, 10
3, 10
2, 10.
(2) real-time fluorescence quantitative RT-PCR detects the specificity test of CSFV: be the specificity fluorescent RT-PCR reaction that template is CSFV with CSFV, foot and mouth disease virus, transmissible gastro-enteritis virus, pig breathing with the nucleic acid of breeding difficulty syndrome and 2 type pig circular ring virus respectively, and the negative contrast of nucleic acid of extracting simultaneously with the health pig viscera tissue.Reaction system is used the single stage method RT-PCR test kit of TAKARA company, and every Auele Specific Primer final concentration is 0.2 μ mol/L, and concentration and probe concentration is 0.1 μ mol/L, adds DEPC H
2O supplies 25 μ L with each reaction.Reaction conditions is 42 ℃ of 30min, 92 ℃ of 2min, 92 ℃ of 10s, 55 ℃ of 30s, 40 circulations; The result of CSFV specificity test as shown in Figure 2, only CSFV has amplification curve.
(3) clinical sample detects: the fluorescent RT-PCR method for detecting of foundation detects the tissue sample of 15 censorships, carries out virus simultaneously and separates.
8, the method that is used to detect the fluorescence quantitative RT-RCR of CSFV is established.
The method concrete steps that real-time fluorescence quantitative RT-PCR provided by the present invention detects CSFV are as follows:
S100, The pretreatment: the sample of gathering numbered respectively be numbered, add the two anti-solution of an amount of penicillin and streptomycin in sample, supernatant is got in homogenate, and 4 ℃ are spent the night.
S200, RNA extract: use the sample extraction RNA of TRIzol reagent to gathering of Invitrogen company, operation steps is undertaken by the test kit specification sheets.
S300, the reaction of CSFV real-time fluorescence quantitative RT-PCR: the RNA that is extracted with above-mentioned steps is a template, use the single stage method RT-PCR test kit of TAKARA company to carry out pcr amplification, every Auele Specific Primer final concentration is 0.2 μ mol/L, and specific probe concentration is 0.1 μ mol/L, adds DEPC H
2O supplies 25 μ L with each reaction.Reaction conditions is 42 ℃ of 30min, 92 ℃ of 2min, 92 ℃ of 10s, 55 ℃ of 30s, 40 circulations.React employed specific primer sequence: F:5 '-GAGCTCCCTGGGTGGTCTAA-3 ', R:5 '-AGCATMTCGAGGTGGGCTTCT-3 '; Specific probe sequence P:FAM-TACAGGACAGTCGTCAGTAGTTC GAC-BHQ 1.
S400, according to CSFV real-time fluorescence RT-PCR reaction result, identify in the sample whether contain CSFV.
Embodiment 1
Animal Quarantine laboratory, Zhuhai Entry-Exit Inspection and Quarantine Bureau technique center is accepted a quarantine pork and is organized whether contain Pestivirus suis, reproductive and respiratory syndrome virus, 2 type PCV-II, parvovirus, suis 2 type business, totally 15 batch samples plan to use institute's fluorescence RT-PCR method of setting up detection among the present invention.Concrete implementation step is as follows:
1, The pretreatment: at first to sample number into spectrum 1-15.Deduct a small amount of tissue with the scissors behind the autoclaving, add the two anti-solution of an amount of penicillin and streptomycin, supernatant is got in homogenate, and 4 ℃ are spent the night.
2, viral RNA extracts: use the sample extraction RNA of TRIzol reagent to gathering of Invitrogen company, operation steps is undertaken by the test kit specification sheets.
3, CSFV real-time fluorescence quantitative RT-PCR reaction: the RNA that is extracted with above-mentioned steps is a template, use the single stage method RT-PCR test kit of TAKARA company, every Auele Specific Primer final concentration is 0.2 μ mol/L, and specific probe concentration is 0.1 μ mol/L, adds DEPC H
2O supplies 25 μ L with each reaction.Reaction conditions is 42 ℃ of 30min, 92 ℃ of 2min, 92 ℃ of 10s, 55 ℃ of 30s, 40 circulations.React employed specific primer sequence: F:5 '-GAGCTCCCTGGGTGGTCTAA-3 ', R:5 '-AGCATMTCGAGGTGGGCTTC T-3 '; Specific probe sequence P:FAM-TACAGGACAGTCGTCAGTAGTTCG AC-BHQ1.
4, result: as shown in Figure 3,2 parts of positives (2/15) occur in the reaction of CSFV real-time fluorescence quantitative RT-PCR.
5, virus is separated: according to the viral sub-argument operation of routine.
Conclusion: fluorescence RT-PCR detects the CSFV methods and results with traditional viral separating resulting unanimity (2/15) among the present invention.But but shortened detection time greatly, and the inventive method is simple to operate, contaminative is little, can carry out generaI investigation screening in batches again, is convenient to use in actual clinical.
Should be understood that application of the present invention is not limited to above-mentioned giving an example, for those of ordinary skills, can be improved according to the above description or conversion that all these improvement and conversion all should belong to the protection domain of claims of the present invention.
＜110〉Zhuhai Entry-Exit Inspection and Quarantine Bureau
＜120〉a kind of real-time fluorescence quantitative RT-PCR detects the method for CSFV
<130> 20080502
<160> 3
<170> PatentIn?version?3.5
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<211> 20
<212> DNA
<213> Artificial?Sequence
<220>
＜223〉CSFV Auele Specific Primer
<400> 1
gagctccctg?ggtggtctaa 20
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<211> 21
<212> DNA
<213> Artificial?Sequence
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＜223〉CSFV Auele Specific Primer
<400> 2
agcatmtcga?ggtgggcttc?t 21
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<213> Artificial?Sequence
<220>
＜223〉CSFV specific probe sequence
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Claims (5)
1. one kind is used for the Auele Specific Primer that real-time fluorescence quantitative RT-PCR detects CSFV, it is characterized in that the specific primer sequence of CSFV is shown in SEQ NO.1-2.
2. one kind is used for the specific probe that real-time fluorescence quantitative RT-PCR detects CSFV, it is characterized in that the specific probe sequence of CSFV is shown in SEQ NO.3.
3. according to claim 2ly be used for the specific probe that real-time fluorescence quantitative RT-PCR detects CSFV, it is characterized in that, the 5' end mark luminophore FAM of described CSFV specific probe, the 3' end mark group B HQ1 that dies out.
4. a method of using the real-time fluorescence quantitative RT-PCR detection CSFV of Auele Specific Primer as claimed in claim 1 and specific probe as claimed in claim 2 is characterized in that, may further comprise the steps:
S100, The pretreatment: the sample of gathering is numbered respectively, adds the two anti-solution of an amount of penicillin and streptomycin in sample, supernatant is got in homogenate, and 4 ℃ are spent the night;
S200, RNA extract: to the sample extraction RNA that gathers;
S300, the reaction of CSFV real-time fluorescence quantitative RT-PCR: the RNA that is extracted with above-mentioned steps is a template, adds CSFV Auele Specific Primer and specific probe, uses the single stage method RT-PCR test kit of TAKARA company to carry out pcr amplification;
S400, according to CSFV real-time fluorescence quantitative RT-PCR reaction result, identify in the sample whether contain CSFV.
5. real-time fluorescence quantitative RT-PCR according to claim 4 detects the method for CSFV, it is characterized in that, real-time fluorescence quantitative RT-PCR reaction system among the described step S300 is 25 μ L, and every Auele Specific Primer final concentration is 0.2 μ mol/L, and specific probe concentration is 0.1 μ mol/L;
Reaction conditions is 42 ℃ of 30min, 92 ℃ of 2min, 92 ℃ of 10s, 55 ℃ of 30s, 40 circulations.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103088158A (en) * | 2013-01-11 | 2013-05-08 | 金宇保灵生物药品有限公司 | Method for quantitative determination of hog cholera lapinized virus by real-time fluorescence quantification PCR (polymer chain reaction) technology |
| CN103627821A (en) * | 2013-11-29 | 2014-03-12 | 华南农业大学 | RT-LAMP nucleic acid test strip kit for detecting transmissible gastroenteritis virus of swine and application thereof |
| CN109706270A (en) * | 2019-02-19 | 2019-05-03 | 珠海出入境检验检疫局检验检疫技术中心 | One breeder Ankara disease detection method |
| CN113999939A (en) * | 2021-12-30 | 2022-02-01 | 北京纳百生物科技有限公司 | Primer and kit for fluorescent quantitative RT-PCR detection of classical swine fever virus |
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| CN1405328A (en) * | 2002-11-08 | 2003-03-26 | 武汉大学 | Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use |
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2011
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1405328A (en) * | 2002-11-08 | 2003-03-26 | 武汉大学 | Fluorescent quantitative PCR kit for rapid and quantitative detecting hog choleravirus and hog cholera lapinized vaccine and its use |
| CN101928782A (en) * | 2009-11-19 | 2010-12-29 | 金陵科技学院 | Kit for simultaneously detecting classical strains and variant strains of porcine reproductive and respiratory syndrome virus and detection method thereof |
Non-Patent Citations (1)
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| 温国元 等: "应用荧光定量PCR技术快速定量检测猪瘟病毒", 《武汉大学学报（理学版）》, vol. 50, no. 6, 31 December 2004 (2004-12-31), pages 746 - 750 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103088158A (en) * | 2013-01-11 | 2013-05-08 | 金宇保灵生物药品有限公司 | Method for quantitative determination of hog cholera lapinized virus by real-time fluorescence quantification PCR (polymer chain reaction) technology |
| CN103627821A (en) * | 2013-11-29 | 2014-03-12 | 华南农业大学 | RT-LAMP nucleic acid test strip kit for detecting transmissible gastroenteritis virus of swine and application thereof |
| CN103627821B (en) * | 2013-11-29 | 2015-04-29 | 华南农业大学 | RT-LAMP nucleic acid test strip kit for detecting transmissible gastroenteritis virus of swine and application thereof |
| CN109706270A (en) * | 2019-02-19 | 2019-05-03 | 珠海出入境检验检疫局检验检疫技术中心 | One breeder Ankara disease detection method |
| CN113999939A (en) * | 2021-12-30 | 2022-02-01 | 北京纳百生物科技有限公司 | Primer and kit for fluorescent quantitative RT-PCR detection of classical swine fever virus |
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