CN101831507A - Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I - Google Patents
Real-time fluorescence quantitative PCR (polymerase chain reaction) detection method of grass carp reovirus SYBR Green I Download PDFInfo
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Abstract
The invention provides a real-time fluorescence quantitative PCR detection method of grass carp reovirus SYBR Green I, at least comprising the following steps of: extracting the total RNA of the grass carp reovirus, designing a primer, carrying out RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification on the coat protein gene of the grass carp reovirus, cloning and identifying the coat protein gene of the grass carp reovirus, diluting a standard template and establishing a fluorescence quantitative PCR standard curve. The detection method has low requirements on the concentration of the template and high detection sensitivity and can carry out quantitative analysis. The detection method can detect 72 samples at a time; a result can be observed immediately once the PCR reaction is finished without the operations of electrophoresis, dyeing and the like, and therefore, the detection method is more convenient and rapid than conventional RT-PCR methods.
Description
Technical field
The present invention relates to the external molecular diagnostic techniques of biological medicine field, specially refer to GCRV SYBR GreenI real-time fluorescence quantitative PCR detection method.
Background technology
Hemorrhagic disease of grass carp (Hemorrhagedisease of grasscarp) is a kind of serious communicable disease that infects grass carp.Epidemic season is long, and M ﹠ M is all higher, often causes the death of large quantities of grass carp fry; The full age black carp also was injured in 1 year, and 2 ages, above grass carp also suffered from this disease sometimes.(Grass Carp Reovir μ s GCRV), is the strongest virus of virulence in the virus of Reoviridae, Aquareovirus to the cause of disease of hemorrhagic disease of grass carp for GCRV.The reovirus genes of grass carps group is made up of 11 segmented dsRNA, and total molecular weight is 15.46 * 10
6Dalton, 12 polypeptide of having encoded studies show that, each genomic fragment is corresponding one by one substantially with the relation of coded polypeptide.
Detection method to GCRV comprises at present: immunological method such as staphylococcal protein a coagglutination test (SPA-COA) method, fluorescent-antibody technique (FA), enzyme linked immunosorbent assay (ELISA) method and dot-ELISA (Dot-ELISA) method; Molecular biology method such as reverse transcriptase polymerase chain reaction (RT-PCR).These methods or complex operation, the cycle is long, perhaps can only carry out qualitative detection and can not quantitative analysis.The fluorescent quantitative PCR technique that latest developments are got up, highly sensitive with it, advantage such as specificity is good, quick, accurate is used widely at the aspects such as qualitative and detection by quantitative of pathogenic agent.
Summary of the invention
The object of the present invention is to provide a kind of SYBR GreenI real-time fluorescence quantitative PCR detection method that is used for the hemorrhagic disease of grass carp gene diagnosis of the suspicious ill grass carp liver,spleen,kidney tissue of GCRV.
For achieving the above object, concrete technical scheme of the present invention is:
GCRV SYBR GreenI real-time fluorescence quantitative PCR detection method comprises at least: clone and evaluation, the dilution of standard substance template and the foundation of quantitative fluorescent PCR reaction normal curve of the total RNA of extracting GCRV, design of primers, GCRV outer capsid protein gene RT-PCR amplification, GCRV outer capsid protein gene.
The concrete steps of GCRV SYBR GreenI real-time fluorescence quantitative PCR detection method are as follows:
1, adopt lysate and RNA extract to handle the total RNA of extractive GCRV;
2, design of primers: according to two pairs of Auele Specific Primers of hemorrhagic disease of grass carp among the GenBank-873 outer capsid albumen (VP7) gene order (AF403396) design, expanding fragment length is the fragment of 100bp, and designed primer is as follows:
Upstream primer: 5 '-GGAATTCACCACGATGCCACTTCAC-3 '
Downstream primer: 5 '-CGGGATCCCGGTGCTTAATCGGATGG-3 ';
3, GCRV outer capsid albumen (VP7) gene RT-PCR amplification:
Get 1～10 μ g virus total RNA, add upstream primer, carry out reverse transcription, negate transcription product cDNA adds reaction buffer, dNTPs, upstream and downstream primer, sterilization distilled water, the Taq archaeal dna polymerase, with the laggard performing PCR amplification of above-mentioned reaction system mixing, the PCR product carries out 1.0% agarose gel electrophoresis analysis, and reclaims product;
4, the clone and the evaluation of GCRV outer capsid albumen (VP7) gene:
GCRV outer capsid albumen (VP7) gene amplification product spends the night in 14～25 ℃ after glue reclaims purifying and is connected with carrier, with connecting product transformed competence colibacillus cell, picking colony is inoculated in the LB liquid nutrient medium that contains penbritin from the LB agar plate, 28～37 ℃ of concussion overnight incubation, use plasmid DNA to extract test kit in a small amount and extract plasmid, recombinant plasmid is used downstream primer carry out the PCR evaluation;
5, the dilution of standard substance template:
Measure the absorbancy of positive recombinant plasmid respectively with ultraviolet spectrophotometer, and calculate the DNA concentration and the purity of recombinant plasmid, recombinant plasmid is carried out 10 times of gradient dilutions, with 1 * 10 according to formula in 260 and 280 nanometers
2～1 * 10
8Copy number/microlitre is used for the optimization of quantitative fluorescent PCR reaction conditions, the foundation of typical curve as the standard substance template, and used formula is as follows:
Molecule copy number (individual/milliliter)=DNA mass concentration/dna molecular amount
Dna molecular amount=DNA base number * 324.5
DNA mass concentration=260 nanometer absorbancy * 50 mcg/ml * extension rate * 6.02 * 10
23
6, the foundation of quantitative fluorescent PCR reaction normal curve:
Respectively with 10 times of standard plasmid DNA (1 * 10 that copy serial dilutions
2～1 * 10
8Copy number/microlitre) carry out fluorescent quantitative PCR for the standard substance template, each extent of dilution is done two multiple holes, measures the detection sensitivity of this method, the repeatability of analysis experiment.
The reaction conditions of pcr amplification is in above-mentioned steps 3 and step 5:
(1), 94 ℃ of pre-sex change are 5 minutes;
(2), 94 ℃ of sex change 15 seconds, 50 ℃ of annealing 15 seconds, 72 ℃ were extended 20 seconds, were a circulation, carried out 30 circulations altogether;
(3), extended 10 minutes at 72 ℃.
Positively effect of the present invention is:
1, this detection method requires broad for template concentrations, and the detection sensitivity height can carry out quantitative analysis;
2, this detection method once can detect 72 each sample simultaneously;
3, after the PCR reaction finished, observations did not need operations such as electrophoresis, dyeing immediately.
Description of drawings
The canonical plotting of Fig. 1, the reaction of GCRV quantitative RT-PCR.
Embodiment
Further specify the specific embodiment of the present invention below in conjunction with embodiment:
Embodiment one
1, adopt lysate and RNA extract to handle the total RNA of extractive GCRV;
2, design of primers:
According to two pairs of Auele Specific Primers of hemorrhagic disease of grass carp among the GenBank-873 outer capsid albumen (VP7) gene order (AF403396) design, expanding fragment length is 98bp, and designed primer is as follows:
Upstream primer: 5 '-GGAATTCACCACGATGCCACTTCAC-3 '
Downstream primer: 5 '-CGGGATCCCGGTGCTTAATCGGATGG-3 ';
3, GCRV outer capsid albumen (VP7) gene RT-PCR amplification:
Get 1 μ g virus total RNA, add the 20pmol upstream primer, according to the Improm-II
TMReverseTranscription System specification sheets carries out reverse transcription.Get 8 microlitre reverse transcription product cDNA, add 10 * Taq reaction buffer, 5 microlitres, 2.5 1/ liter of dNTPs 4 microlitres, 50 micromole of mmole/rise, each 1 microlitre of upstream and downstream, sterilization distilled water 30.5 microlitres, 5U/ microlitre Taq archaeal dna polymerase 0.5 microlitre, with the amplification of the laggard performing PCR of above-mentioned reaction system mixing, reaction parameter: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 15 seconds, 50 ℃ of annealing 15 seconds, 72 ℃ are extended 20 seconds is a circulation, carries out 30 circulations altogether; Last 72 ℃ were extended 10 minutes.Set up the negative control of no template simultaneously, reaction is got 5 microlitre PCR products and is carried out electrophoresis detection with 1% sepharose after finishing.
4, the clone and the evaluation of GCRV outer capsid albumen (VP7) gene:
GCRV outer capsid albumen (VP7) gene amplification product spends the night in 16 ℃ after glue reclaims purifying and is connected with carrier, with connecting product Transformed E .coliDH5a competent cell, picking colony is inoculated in 5 milliliters of LB liquid nutrient mediums that contain 50 mcg/ml penbritins (Amp+) from the LB agar plate, 37 ℃ of concussion overnight incubation, use plasmid DNA to extract test kit in a small amount and extract plasmid, recombinant plasmid is used downstream primer carry out the PCR evaluation.
5, the dilution of standard substance template:
Measure the absorbancy of positive recombinant plasmid respectively with ultraviolet spectrophotometer, and calculate the DNA concentration and the purity of recombinant plasmid according to formula in 260 and 280 nanometers.Recombinant plasmid is carried out 10 times of gradient dilutions, with 1 * 10
2, 1 * 10
3, 1 * 10
4, 1 * 10
5, 1 * 10
6, 1 * 10
7, 1 * 10
8Copy number/microlitre is used for the optimization of quantitative fluorescent PCR reaction conditions, the foundation of typical curve as the standard substance template, and used formula is as follows:
Molecule copy number (individual/milliliter)=DNA mass concentration/dna molecular amount
Dna molecular amount=DNA base number * 324.5
DNA mass concentration=260 nanometer absorbancy * 50 mcg/ml * extension rate * 6.02 * 10
23
6, the foundation of quantitative fluorescent PCR reaction normal curve:
Respectively with 10 times of standard plasmid DNA (1 * 10 that copy serial dilutions
2～1 * 10
8Copy number/microlitre) carry out fluorescent quantitative PCR for the standard substance template, the reaction conditions of PCR is identical with step 3, and each extent of dilution is done two multiple holes, measures the detection sensitivity of this method, the repeatability of analysis experiment.
Embodiment two
1, adopt lysate and RNA extract to handle the total RNA of extractive GCRV;
2, design of primers:
According to two pairs of Auele Specific Primers of hemorrhagic disease of grass carp among the GenBank-873 outer capsid albumen (VP7) gene order (AF403396) design, expanding fragment length is 98bp, and designed primer is as follows:
Upstream primer: 5 '-GGAATTCACCACGATGCCACTTCAC-3 '
Downstream primer: 5 '-CGGGATCCCGGTGCTTAATCGGATGG-3 ';
3, GCRV outer capsid albumen (VP7) gene RT-PCR amplification:
Get 5 μ g virus total RNA, add the 20pmol upstream primer, according to the Improm-II
TMReverseTranscription System specification sheets carries out reverse transcription.Get 8 microlitre reverse transcription product cDNA, add 10 * Taq reaction buffer, 5 microlitres, 2.5 1/ liter of dNTPs 4 microlitres, 50 micromole of mmole/rise, each 1 microlitre of upstream and downstream, sterilization distilled water 30.5 microlitres, 5U/ microlitre Taq archaeal dna polymerase 0.5 microlitre, with the amplification of the laggard performing PCR of above-mentioned reaction system mixing, reaction parameter: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 15 seconds, 50 ℃ of annealing 15 seconds, 72 ℃ are extended 20 seconds is a circulation, carries out 30 circulations altogether; Last 72 ℃ were extended 10 minutes.Set up the negative control of no template simultaneously, reaction is got 5 microlitre PCR products and is carried out electrophoresis detection with 1% sepharose after finishing.
4, the clone and the evaluation of GCRV outer capsid albumen (VP7) gene:
GCRV outer capsid albumen (VP7) gene amplification product spends the night in 16 ℃ after glue reclaims purifying and is connected with carrier, with connecting product Transformed E .coliDH5a competent cell, picking colony is inoculated in 5 milliliters of LB liquid nutrient mediums that contain 50 mcg/ml penbritins (Amp+) from the LB agar plate, 37 ℃ of concussion overnight incubation, use plasmid DNA to extract test kit in a small amount and extract plasmid, recombinant plasmid is used downstream primer carry out the PCR evaluation.
5, the dilution of standard substance template:
Measure the absorbancy of positive recombinant plasmid respectively with ultraviolet spectrophotometer, and calculate the DNA concentration and the purity of recombinant plasmid according to formula in 260 and 280 nanometers.Recombinant plasmid is carried out 10 times of gradient dilutions, with 1 * 10
2, 1 * 10
3, 1 * 10
4, 1 * 10
5, 1 * 10
6, 1 * 10
7, 1 * 10
8Copy number/microlitre is used for the optimization of quantitative fluorescent PCR reaction conditions, the foundation of typical curve as the standard substance template, and used formula is as follows:
Molecule copy number (individual/milliliter)=DNA mass concentration/dna molecular amount
Dna molecular amount=DNA base number * 324.5
DNA mass concentration=260 nanometer absorbancy * 50 mcg/ml * extension rate * 6.02 * 10
23
6, the foundation of quantitative fluorescent PCR reaction normal curve:
Respectively with 10 times of standard plasmid DNA (1 * 10 that copy serial dilutions
2～1 * 10
8Copy number/microlitre) carry out fluorescent quantitative PCR for the standard substance template, the reaction conditions of PCR is identical with step 3, and each extent of dilution is done two multiple holes, measures the detection sensitivity of this method, the repeatability of analysis experiment.
Embodiment three
1, adopt lysate and RNA extract to handle the total RNA of extractive GCRV;
2, design of primers:
According to two pairs of Auele Specific Primers of hemorrhagic disease of grass carp among the GenBank-873 outer capsid albumen (VP7) gene order (AF403396) design, expanding fragment length is 98bp, and designed primer is as follows:
Upstream primer: 5 '-GGAATTCACCACGATGCCACTTCAC-3 '
Downstream primer: 5 '-CGGGATCCCGGTGCTTAATCGGATGG-3 ';
3, GCRV outer capsid albumen (VP7) gene RT-PCR amplification:
Get 10 μ g virus total RNA, add the 20pmol upstream primer, according to the Improm-II
TMReverseTranscription System specification sheets carries out reverse transcription.Get 8 microlitre reverse transcription product cDNA, add 10 * Taq reaction buffer, 5 microlitres, 2.5 1/ liter of dNTPs 4 microlitres, 50 micromole of mmole/rise, each 1 microlitre of upstream and downstream, sterilization distilled water 30.5 microlitres, 5U/ microlitre Taq archaeal dna polymerase 0.5 microlitre, with the amplification of the laggard performing PCR of above-mentioned reaction system mixing, reaction parameter: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 15 seconds, 50 ℃ of annealing 15 seconds, 72 ℃ are extended 20 seconds is a circulation, carries out 30 circulations altogether; Last 72 ℃ were extended 10 minutes.Set up the negative control of no template simultaneously, reaction is got 5 microlitre PCR products and is carried out electrophoresis detection with 1% sepharose after finishing.
4, the clone and the evaluation of GCRV outer capsid albumen (VP7) gene:
GCRV outer capsid albumen (VP7) gene amplification product spends the night in 16 ℃ after glue reclaims purifying and is connected with carrier, with connecting product Transformed E .coliDH5a competent cell, picking colony is inoculated in 5 milliliters of LB liquid nutrient mediums that contain 50 mcg/ml penbritins (Amp+) from the LB agar plate, 37 ℃ of concussion overnight incubation, use plasmid DNA to extract test kit in a small amount and extract plasmid, recombinant plasmid is used downstream primer carry out the PCR evaluation.
5, the dilution of standard substance template:
Measure the absorbancy of positive recombinant plasmid respectively with ultraviolet spectrophotometer, and calculate the DNA concentration and the purity of recombinant plasmid according to formula in 260 and 280 nanometers.Recombinant plasmid is carried out 10 times of gradient dilutions, with 1 * 10
2, 1 * 10
3, 1 * 10
4, 1 * 10
5, 1 * 10
6, 1 * 10
7, 1 * 10
8Copy number/microlitre is used for the optimization of quantitative fluorescent PCR reaction conditions, the foundation of typical curve as the standard substance template, and used formula is as follows:
Molecule copy number (individual/milliliter)=DNA mass concentration/dna molecular amount
Dna molecular amount=DNA base number * 324.5
DNA mass concentration=260 nanometer absorbancy * 50 mcg/ml * extension rate * 6.02 * 10
23
6, the foundation of quantitative fluorescent PCR reaction normal curve:
Respectively with 10 times of standard plasmid DNA (1 * 10 that copy serial dilutions
2～1 * 10
8Copy number/microlitre) carry out fluorescent quantitative PCR for the standard substance template, the reaction conditions of PCR is identical with step 3, and each extent of dilution is done two multiple holes, measures the detection sensitivity of this method, the repeatability of analysis experiment.
Example detection result: recombinant plasmid is carried out 10 times of serial dilutions, each extent of dilution do two parallel, carry out quantitative RT-PCR.By mapping as can be seen, plasmid concentration is 1 * 10
8～1 * 10
2Individual copy, quantitative RT-PCR has " S " type amplification curve in the scope of 7 orders of magnitude, the exponential growth phase of promptly having reflected PCR, the linear growth phase and plateau three phases.Detection sensitivity is 100 virus particle.Further analysing amplified curve finds that the curve of exponential growth phase is parallel, has reflected that the amplification efficiency of PCR is close; And the Ct between the different extent of dilution differs evenly, meets linear relationship strict between the Ct value of quantitative PCR and the initial copy number.Each dilution two repetition, its amplification curve coincide together substantially, has reflected that the collimation of experiment is good.
Utilize the STATISTICA of statistical software 6.0, the Ct value in the experiment is carried out statistical study.Analytical results shows the Ct value variation coefficient (CV) of 7 different dilution gradient standard plasmid DNA between 0.08%～3.26%, difference all not remarkable (P＞0.05) between the identical extent of dilution standard plasmid Ct value.
According to the dependency of plasmid copy number and Ct value, obtain typical curve (as Fig. 1) by SDS (Sequence Detection System) 2.1 softwares.X-coordinate is represented plasmid copy number (x), and ordinate zou is the Ct value, the copy number of the digitized representation template on the point.Copy number (x) with the pass of Ct is: Ct=-2.982lgX+31.038; Coefficient R
2=0.99102.Through analysis, can find that the DNA melting temperature (Tm) (Tm) of amplified production is worth very homogeneous, the appearance of specific amplification products and primer dimer nothing but to melting curve.
The above only is a non-limiting embodiment of the present invention; for the person of ordinary skill of the art; not breaking away from the invention design and not making under the prerequisite of creative work, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (3)
1. GCRV SYBR Green I real-time fluorescence quantitative PCR detection method, it is characterized in that: GCRV SYBR Green I real-time fluorescence quantitative PCR detection method comprises at least: clone and evaluation, the dilution of standard substance template and the foundation of quantitative fluorescent PCR reaction normal curve of the total RNA of extracting GCRV, design of primers, GCRV outer capsid protein gene RT-PCR amplification, GCRV outer capsid protein gene.
2. GCRV SYBR Green I real-time fluorescence quantitative PCR detection method according to claim 1 is characterized in that: the concrete steps of GCRV SYBR Green I real-time fluorescence quantitative PCR detection method are as follows:
(1), adopt lysate and RNA extract to handle the total RNA of extractive GCRV;
(2), design of primers: according to two pairs of Auele Specific Primers of hemorrhagic disease of grass carp among the GenBank-873 outer capsid albumen (VP7) gene order (AF403396) design, expanding fragment length is the fragment of 90～200bp, and designed primer is as follows:
Upstream primer: 5 '-GGAATTCACCACGATGCCACTTCAC-3 '
Downstream primer: 5 '-CGGGATCCCGGTGCTTAATCGGATGG-3 ';
(3), GCRV outer capsid albumen (VP7) gene RT-PCR amplification:
Get 1～10 μ g virus total RNA, add upstream primer, carry out reverse transcription, negate transcription product cDNA adds reaction buffer, dNTPs, upstream and downstream primer, sterilization distilled water, the Taq archaeal dna polymerase, with the laggard performing PCR amplification of above-mentioned reaction system mixing, the PCR product carries out 1.0% agarose gel electrophoresis analysis, and reclaims product;
(4), the clone and the evaluation of GCRV outer capsid albumen (VP7) gene:
GCRV outer capsid albumen (VP7) gene amplification product spends the night in 14～25 ℃ after glue reclaims purifying and is connected with carrier, with connecting product transformed competence colibacillus cell, picking colony is inoculated in the LB liquid nutrient medium that contains penbritin from the LB agar plate, 28～37 ℃ of concussion overnight incubation, use plasmid DNA to extract test kit in a small amount and extract plasmid, recombinant plasmid is used downstream primer carry out the PCR evaluation;
(5), the dilution of standard substance template:
Measure the absorbancy of positive recombinant plasmid respectively with ultraviolet spectrophotometer, and calculate the DNA concentration and the purity of recombinant plasmid, recombinant plasmid is carried out 10 times of gradient dilutions, with 1 * 10 according to formula in 260 and 280 nanometers
2～1 * 10
8Copy number/microlitre is used for the optimization of quantitative fluorescent PCR reaction conditions, the foundation of typical curve as the standard substance template, and used formula is as follows:
Molecule copy number (individual/milliliter)=DNA mass concentration/dna molecular amount
Dna molecular amount=DNA base number * 324.5
DNA mass concentration=260 nanometer absorbancy * 50 mcg/ml * extension rate * 6.02 * 10
23
(6), the foundation of quantitative fluorescent PCR reaction normal curve:
Respectively with 10 times of standard plasmid DNA (1 * 10 that copy serial dilutions
2～1 * 10
8Copy number/microlitre) carry out fluorescent quantitative PCR for the standard substance template, each extent of dilution is done two multiple holes, measures the detection sensitivity of this method, the repeatability of analysis experiment.
3. according to claim 1 and 2 described GCRV SYBR Green I real-time fluorescence quantitative PCR detection method and concrete steps, it is characterized in that: the reaction conditions of pcr amplification is in above-mentioned steps (3) and the step (5):
(1), 94 ℃ of pre-sex change are 5 minutes;
(2), 94 ℃ of sex change 15 seconds, 50 ℃ of annealing 15 seconds, 72 ℃ were extended 20 seconds, were a circulation, carried out 30 circulations altogether;
(3), extended 10 minutes at 72 ℃.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967524A (en) * | 2010-09-26 | 2011-02-09 | 中国人民解放军军事医学科学院微生物流行病研究所 | Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR |
| CN102787176A (en) * | 2012-08-09 | 2012-11-21 | 江苏省农业科学院 | Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants |
| CN106198479A (en) * | 2016-08-26 | 2016-12-07 | 周辉 | The SYBR Green I immue quantitative detection reagent box of a kind of detection by quantitative DNA content and detection method thereof |
| CN106811550A (en) * | 2017-03-11 | 2017-06-09 | 中国水产科学研究院珠江水产研究所 | A kind of type vaccine strain of GCRV II and street strain's diagnostic primerses and kit and diagnosis detecting method containing it |
| CN108411031A (en) * | 2017-02-09 | 2018-08-17 | 转导精进（武汉）生物技术有限公司 | A kind of detection kit and detection method of grass carp hemorrhage virus |
| CN111363850A (en) * | 2020-04-15 | 2020-07-03 | 西南民族大学 | Dye fluorescent quantitative primer and kit for detecting orthoreovirus |
| CN112048575A (en) * | 2020-10-26 | 2020-12-08 | 深圳市莱孚生物科技有限公司 | Primer, probe and detection method for identifying grass carp hemorrhagic fever virus |
| CN113817875A (en) * | 2021-11-16 | 2021-12-21 | 河南省农业科学院烟草研究所 | Fluorescent quantitative PCR method for detecting tobacco etch virus |
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2010
- 2010-05-10 CN CN 201010173229 patent/CN101831507A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101967524A (en) * | 2010-09-26 | 2011-02-09 | 中国人民解放军军事医学科学院微生物流行病研究所 | Kit for detecting eastern equine encephalitis virus and west equine encephalitis virus by real-time fluorescence quantitative RT-PCR |
| CN102787176A (en) * | 2012-08-09 | 2012-11-21 | 江苏省农业科学院 | Method for real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) quantitative detection of virus RNA (ribose nucleic acid) copy number in SRBSV (southern rice black-streaked dwarf virus) plants |
| CN106198479A (en) * | 2016-08-26 | 2016-12-07 | 周辉 | The SYBR Green I immue quantitative detection reagent box of a kind of detection by quantitative DNA content and detection method thereof |
| CN108411031A (en) * | 2017-02-09 | 2018-08-17 | 转导精进（武汉）生物技术有限公司 | A kind of detection kit and detection method of grass carp hemorrhage virus |
| CN106811550A (en) * | 2017-03-11 | 2017-06-09 | 中国水产科学研究院珠江水产研究所 | A kind of type vaccine strain of GCRV II and street strain's diagnostic primerses and kit and diagnosis detecting method containing it |
| CN111363850A (en) * | 2020-04-15 | 2020-07-03 | 西南民族大学 | Dye fluorescent quantitative primer and kit for detecting orthoreovirus |
| CN112048575A (en) * | 2020-10-26 | 2020-12-08 | 深圳市莱孚生物科技有限公司 | Primer, probe and detection method for identifying grass carp hemorrhagic fever virus |
| CN113817875A (en) * | 2021-11-16 | 2021-12-21 | 河南省农业科学院烟草研究所 | Fluorescent quantitative PCR method for detecting tobacco etch virus |
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Application publication date: 20100915 |