CN102242224B - Method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction) - Google Patents
Method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction) Download PDFInfo
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Abstract
The invention discloses a method for synchronously detecting three kinds of viruses on tobacco through triple one-step method RT-PCR (Reverse Transcription Polymerase Chain Reaction). The method is characterized by being used for synchronously detecting TMV (Tobacco Mosaic Virus), PVY (Potato Virus Y) and CMV (Cucumber Mosaic Virus), the method comprises the following steps of screening and comparing conservation areas in a virus gene group, artificially screening and synthesizing specificity primers suitable for composite RT-PCR of three kinds of virus diseases; analyzing to obtain TMV, PVY and CMV infection tobacco leaf samples; and integrating enzymes and a buffer solution required by a reverse transcription and a PCR (Polymerase Chain Reaction), and establishing a method for compositely detecting the TMV, PCY and CMV infection of tobacco leaves based on one-step method RT-PCR through optimization of reaction conditions. The triple one-step method RT-PCR utilized by the invention has the advantages of simple and convenient operation, time and labor saving, reliable result and low cost; simultaneously, the probability of cross-contamination of samples in an operation process can be effectively reduced, and the monitoring on the TMV, PVY and CMV in tobacco agricultural production has wide application prospect.
Description
Technical field
The invention belongs to viral diseases of plants detection technique field, provide a kind of triple single stage method RT-PCR detect simultaneously the tobacco mosaic viruses disease (
Tobacco mosaic virus, TMV), tobacco potato Y virus (
Potato Y virus, PVY) and tobacco cucumber mosaic virus (
Cucumber mosaic virus, method CMV) is specifically related to the effective foundation of synthetic and this detection method that grows tobacco virus-specific nucleotide primer sequence for three.
Background technology
Tobacco mosaic viruses (
Tobacco mosaic virus, TMV), marmor upsilon (
Potato virus Y, PVY), cucumber mosaic virus (
Cucumber mosaic virus, CMV) host range is extensive, diversified economy crop and the garden crops such as main harm tobacco, tomato, cucumber, potato.Its regional distribution is extremely wide, belongs to worldwide virus disease, and the crop production of different continents is the virus disease that causes of Ceng Yinqi and be subject to heavy losses all.
More than three kinds of virus diseases be that important virus disease general, that harm is serious occurs on the tobacco, alleviate tobacco agriculture produce in the key of virus disease harm be that spread is cultivated and used virus-free strong sprout, enhances field management and take effectively viral prevention and control technical measures.Viral detection monitoring is an important step in the prevention and control technical measures, has the advantages that for tobacco virus harm is large, popular extensively, prevent and treat difficulty, and it is very necessary to set up as early as possible as early as possible quick, accurate and efficient detection technique.The method of at present land for growing field crops tobacco virus evaluation mainly contains conventional Biological Detection, immunology and molecular biology method, but all has in various degree defective and deficiency.Conventional Biological Detection wastes time and energy, and detection of plasma and molecular biology for detection are to preparing restriction and the somewhat expensive that the viral sample material requirements is higher, be subject to experiment condition.Usually in the molecular Biological Detection process of actual virus, TMV, PVY, CMV etc. identify that separately repetitive operation is more, and the cost of wasting time and energy is higher.Multiple RT-PCR (Multiplex reverse transcription polymerase chain reaction) is the primer that adds in a reverse transcription (RT) reaction more than 1 pair, at first the purpose viral nucleic acid is carried out reverse transcription, then take synthetic a plurality of reverse transcription products (cDNA) of this stage as the polymerase chain reaction template of (PCR), the corresponding above virus causing disease authentication information of index amplification acquisition.Because tobacco virus mostly is mixed infection, Symptoms is complicated, it is larger to utilize many kinds of single RT-PCR virus to carry out the workload that system detects, compare with the single RT-PCR of routine, multiple RT-PCR can detect simultaneously multiple virus or distinguish the different strains of virus, can improve significantly detection efficiency, reduce testing cost, thereby the superiority of multiple RT-PCR in tobacco virus detects is more obvious.The key that detects simultaneously the dual of two or more viruses or multiple RT-PCR technology is to select suitable primer, under the prerequisite that guarantees primer specificity, avoids mutual interference the between the primer.
Multiple RT-PCR is divided into two operation stepss and carries out in actual application, at first the purpose viral nucleic acid is carried out reverse transcription (RT), then carries out the index amplification take reverse transcription product (cDNA) as template and obtains corresponding virus causing disease authentication information.Two reactions steps and stage corresponding two different reaction systems, the influence factor of whole test is more, has also strengthened that test is polluted and the probability of artificial incorrect operation simultaneously.
Summary of the invention
It is not enough and triple single stage method RT-PCR that provide detect the method for three kinds of important viruses on the tobaccos that purpose of the present invention just is being based on above-mentioned prior art.Design respectively synthetic a pair of Auele Specific Primer according to TMV, PVY, CMV genome sequence conservative region, integrate reverse transcription and PCR and react required enzyme and damping fluid, by reaction condition optimization, successfully set up based on single stage method RT-PCR and detected simultaneously the method that tobacco leaf infects TMV, PVY, CMV.The foundation of this technological method will provide for the accurate detection of TMV, PVY, CMV in the tobacco agriculture production process a kind of sensitivity, quick, special and high-throughout method.
The present invention seeks to be achieved through the following technical solutions: triple single stage method RT-PCR detect the method for tobacco mosaic viruses, marmor upsilon and cucumber mosaic virus.The method comprises screens conservative region in the comparison viral genome, and the synthetic screening is applicable to three kinds of compound RT-PCR Auele Specific Primers of virus disease; Analysis obtains TMV, PVY, CMV infects the tobacco leaf sample; Integrate reverse transcription and PCR and react required enzyme and damping fluid, by reaction condition optimization, set up the method that infects TMV, PVY, CMV based on single stage method RT-PCR compound detection tobacco leaf.
Concrete steps are as follows:
(1) three kind of virus-specific nucleotide primer design is with synthetic
Use DNAMAN6.0 software, TMV, PVY, each strain of CMV virus, each region disconnecting thing whole genome sequence that GenBank is included compare, determine the relative conservative region in the virus sequence, in selected conservative region, use Oligo 6.0 software design primers, and guarantee primer specificity by the BLAST analysis.Because multiplex PCR requires each primer and the right annealing temperature of primer to approach as far as possible, the size of amplicon also should be as far as possible close in the situation that agarose gel electrophoresis can significantly be distinguished simultaneously, so finally selected TMV, PVY, each pair of primers of CMV spend the RNase water dissolution to working concentration 10 μ M behind the synthetic.
TMV: upstream primer (TF1) 5 '-TGTGTGCAAAACTTACTTCCC-3 ';
Downstream primer (TR1) 5 '-AGGACAAAACATTTGCGTATG-3 ';
PVY: upstream primer (PF1) 5 '-ACTGTGATGAATGGGCTTATG-3 ';
Downstream primer (PR1) 5 '-GGCATATATGGTTCCTTTTTG-3 ';
CMV: upstream primer (C3F1) 5 '-GGCTTTCCAAGGTACCAGTAG-3 ';
Downstream primer (C3R1) 5 '-ATCGAGCTTGCCAATTACTAC-3 '.
(2) extraction of the total RNA of tobacco
At first serological diagnostic kit detect to be analyzed the virus infection tobacco sample, the tobacco sample that finds TMV, PVY, CMV to infect by serological method.Get classical symptom tobacco leaf 50mg, grinding powder in the liquid nitrogen extracts the total RNA of tobacco, and extract product is dissolved in the distilled water of 50 μ l without the pollution of RNA enzyme, and-20 ℃ of storages are for subsequent use.
(3) triple single stage method RT-PCR detect the foundation of three kinds of viral reaction conditionss simultaneously
The system factor is groped and adjusted to this test repeatedly by a plurality of gradient trial tests, finally obtained to make the top condition of selected 3 pairs of primers normal reaction in same RT-PCR reaction tubes.Reaction system through optimization: 10 * PCR Buffer, 7.5 μ l; Mg
2+(25mM) 6 μ l; DNTP(2.5mM each) 4 μ l; Taq enzyme 4U; AMV ThermoScript II 5U; Ribonuclease Inhibitor 10U; Each 2 μ l of TMV upstream and downstream primer, each 2 μ l of CMV upstream and downstream primer, each 3 μ l of PVY upstream and downstream primer; Each 500ng of RNA; Go RNase water to complement to 50 μ l.Reaction conditions: 42 ℃ of reverse transcription 30min, 95 ℃ of heat inactivation 5min; 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 70sec, 30 circulations; 72 ℃ are replenished extension 10min, and amplified production is got 5 ~ 10 μ l electrophoresis detection in 1% sepharose.The enzyme that this experiment RT-PCR uses is all available from TaKaRa company.
In the present invention, use Agdia company's T MV, PVY, CMV diagnostic kit PathoScreen in the step (2)
RTMV, PathoScreen
RPVY, PathoScreen
RCMV detect to analyze viral tobacco sample, the tobacco sample that finds TMV, PVY, CMV to infect by serological method; Adopt RNeasy Plant Mini test kit to extract the total RNA of tobacco.
Triple single stage method RT-PCR reaction of the present invention, have easy and simple to handle, time saving and energy saving, reliable results, advantage with low cost, simultaneously can effectively reduce the possibility that causes the sample crossed contamination in the operating process, the monitoring of TMV, PVY, CMV is with a wide range of applications in producing for tobacco agriculture.
The present invention changes the defective in the multiple RT-PCR actual application, solved long, the problem such as sensitivity is low of tobacco virus detection method cycle, utilizing single RT-PCR to detect on the basis of TMV, PVY, CMV, design respectively synthetic Auele Specific Primer according to TMV, PVY, CMV genome sequence conservative region, integrate reverse transcription and PCR and react required enzyme and damping fluid, by reaction condition optimization, simultaneously triple single stage method RT-PCR methods of three kinds of virus diseases of rapid detection of a cover have successfully been set up.Compare with multiple RT-PCR than single RT-PCR, triple single stage method RT-PCR detection methods of setting up can a step detect TMV, PVY, CMV virus causing disease simultaneously, detect easier, can reduce pollute and reaction in influence factor, and greatly shortened detection time.
Description of drawings
PVY, the TMV of the triple single stage method RT-PCR reaction systems of Fig. 1, the electrophoresis detection result of CMV viral sample
Wherein: M is DNA marker, and the 1-4 swimming lane is respectively single PVY single stage method RT-PCR product, single TMV single stage method RT-PCR product, single CMV single stage method RT-PCR product, triple single stage method RT-PCR product.
Embodiment
The present invention is described further below with reference to embodiment, but does not limit the present invention.
Embodiment 1:
(1) uses DNAMAN6.0 software, TMV, PVY, each strain of CMV virus, each region disconnecting thing whole genome sequence that GenBank is included compare, determine the relative conservative region in the virus sequence, in selected conservative region, use Oligo 6.0 software design primers, and guarantee primer specificity by the BLAST analysis.Because multiplex PCR requires each primer and the right annealing temperature of primer to approach as far as possible, the size of amplicon also should be as far as possible close in the situation that agarose gel electrophoresis can significantly be distinguished simultaneously, so finally selected TMV, PVY, each pair of primers of CMV spend the RNase water dissolution to working concentration 10 μ M behind the synthetic.
TMV: upstream primer (TF1) 5 '-TGTGTGCAAAACTTACTTCCC-3 ';
Downstream primer (TR1) 5 '-AGGACAAAACATTTGCGTATG-3 ';
PVY: upstream primer (PF1) 5 '-ACTGTGATGAATGGGCTTATG-3 ';
Downstream primer (PR1) 5 '-GGCATATATGGTTCCTTTTTG-3 ';
CMV: upstream primer (C3F1) 5 '-GGCTTTCCAAGGTACCAGTAG-3 ';
Downstream primer (C3R1) 5 '-ATCGAGCTTGCCAATTACTAC-3 '.
(2) serological diagnostic kit PathoScreen at first
RTMV, PathoScreen
RPVY, PathoScreen
RCMV detect to analyze the virus infection tobacco sample that cigarette district, land for growing field crops gathers, the tobacco sample that finds TMV, PVY, CMV to infect by serological method.Get classical symptom tobacco leaf 50mg, grinding powder in the liquid nitrogen adopts the Shanghai RNeasy Plant Mini of bio-engineering corporation test kit to extract the total RNA of tobacco, and extract product is dissolved in the distilled water of 50 μ l without the pollution of RNA enzyme, and-20 ℃ of storages are for subsequent use.
(3) foundation of triple single stage method RT-PCR reaction conditionss
Reaction system through optimization: 10 * PCR Buffer, 7.5 μ l; Mg
2+(25mM) 6 μ l; DNTP(2.5mM each) 4 μ l; Taq enzyme 4U; AMV ThermoScript II 5U; Ribonuclease Inhibitor 10U; Each 2 μ l of TMV upstream and downstream primer, each 2 μ l of CMV upstream and downstream primer, each 3 μ l of PVY upstream and downstream primer; Each 500ng of RNA; Go RNase water to complement to 50 μ l.Reaction conditions: 42 ℃ of reverse transcription 30min, 95 ℃ of heat inactivation 5min; 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 70sec, 30 circulations; 72 ℃ are replenished extension 10min, and amplified production is got 5-10 μ l electrophoresis detection in 1% sepharose.
Embodiment 2:
(1) uses DNAMAN6.0 software, TMV, PVY, each strain of CMV virus, each region disconnecting thing whole genome sequence that GenBank is included compare, determine the relative conservative region in the virus sequence, in selected conservative region, use Oligo 6.0 software design primers, and guarantee primer specificity by the BLAST analysis.Because multiplex PCR requires each primer and the right annealing temperature of primer to approach as far as possible, the size of amplicon also should be as far as possible close in the situation that agarose gel electrophoresis can significantly be distinguished simultaneously, so finally selected TMV, PVY, each pair of primers of CMV (primer is with embodiment 1) spend the RNase water dissolution to working concentration 10 μ M behind the synthetic.
(2) TMV, the PVY, the CMV virus causing disease that utilize this laboratory to preserve are inoculated separately respectively 6-8 leaf phase tobacco (non-homophyletic tobacco), treat to gather when viral Symptoms is obvious the morbidity tobacco sample, get three kinds of classical symptom morbidity balanced mix 60mg, grinding powder in the liquid nitrogen, adopt the Shanghai RNeasy Plant Mini of bio-engineering corporation test kit to extract the total RNA of tobacco, extract product is dissolved in the distilled water of 50 μ l without the pollution of RNA enzyme, and-20 ℃ of storages are for subsequent use.
(3) foundation of triple single stage method RT-PCR reaction conditionss
Reaction system through optimization: 10 * PCR Buffer, 7.5 μ l; Mg
2+(25mM) 6 μ l; DNTP(2.5mM each) 4 μ l; Taq enzyme 4U; AMV ThermoScript II 5U; Ribonuclease Inhibitor 10U; Each 2 μ l of TMV upstream and downstream primer, each 2 μ l of CMV upstream and downstream primer, each 3 μ l of PVY upstream and downstream primer; Each 500ng of RNA; Go RNase water to complement to 50 μ l.Reaction conditions: 42 ℃ of reverse transcription 30min, 95 ℃ of heat inactivation 5min; 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 70sec, 30 circulations; 72 ℃ are replenished extension 10min, and amplified production is got 5-10 μ l electrophoresis detection in 1% sepharose.
Embodiment 3:
(1) uses DNAMAN6.0 software, TMV, PVY, each strain of CMV virus, each region disconnecting thing whole genome sequence that GenBank is included compare, determine the relative conservative region in the virus sequence, in selected conservative region, use Oligo 6.0 software design primers, and guarantee primer specificity by the BLAST analysis.Because multiplex PCR requires each primer and the right annealing temperature of primer to approach as far as possible, the size of amplicon also should be as far as possible close in the situation that agarose gel electrophoresis can significantly be distinguished simultaneously, so finally selected TMV, PVY, each pair of primers of CMV (primer is with embodiment 1) spend the RNase water dissolution to working concentration 10 μ M behind the synthetic.
(2) TMV, the PVY, the CMV virus causing disease that utilize this laboratory to preserve are successively inoculated 8-10 leaf phase tobacco (homophyletic tobacco), treat to gather when viral Symptoms is obvious the morbidity tobacco sample, get classical symptom morbidity tobacco leaf 50mg, grinding powder in the liquid nitrogen, adopt the Shanghai RNeasy Plant Mini of bio-engineering corporation test kit to extract the total RNA of tobacco, extract product is dissolved in the distilled water of 50 μ l without the pollution of RNA enzyme, and-20 ℃ of storages are for subsequent use.
(3) foundation of triple single stage method RT-PCR reaction conditionss
Reaction system through optimization: 10 * PCR Buffer, 7.5 μ l; Mg
2+(25mM) 6 μ l; DNTP(2.5mM each) 4 μ l; Taq enzyme 4U; AMV ThermoScript II 5U; Ribonuclease Inhibitor 10U; Each 2 μ l of TMV upstream and downstream primer, each 2 μ l of CMV upstream and downstream primer, each 3 μ l of PVY upstream and downstream primer; Each 500ng of RNA; Go RNase water to complement to 50 μ l.Reaction conditions: 42 ℃ of reverse transcription 30min, 95 ℃ of heat inactivation 5min; 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 70sec, 30 circulations; 72 ℃ are replenished extension 10min, and amplified production is got 5-10 μ l electrophoresis detection in 1% sepharose.
Sequence table
＜110〉Zhengzhou Tobacco Research Institute of CNTC
＜120〉triple single stage method RT-PCR detect the method for three kinds of viruses on the tobacco simultaneously
<160>6
<210>1
<211>21
<212>DNA
＜213〉artificial sequence
<222>（1）..(21)
<400>1
tgtgtgcaaa acttacttcc c
<210>2
<211>21
<212>DNA
＜213〉artificial sequence
<222>（1）..(21)
<400>2
aggacaaaac atttgcgtat g
<210>3
<211>21
<212>DNA
＜213〉artificial sequence
<222>（1）..(21)
<400>3
actgtgatga atgggcttat g
<210>4
<211>21
<212>DNA
＜213〉artificial sequence
<222>（1）..(21)
<400>4
ggcatatatg gttccttttt g
<210>5
<211>21
<212>DNA
＜213〉artificial sequence
<222>（1）..(21)
<400>5
ggctttccaa ggtaccagta g
<210>6
<211>21
<212>DNA
＜213〉artificial sequence
<222>（1）..(21)
<400>6
atcgagcttg ccaattacta c
Claims (1)
1. triple single stage method RT-PCR detect the method for three kinds of viruses on the tobacco simultaneously, it is characterized in that: be the method that detects simultaneously tobacco mosaic viruses, marmor upsilon and cucumber mosaic virus, the method comprises screens conservative region in the comparison viral genome, and artificial screening is synthetic to be applicable to three kinds of compound RT-PCR Auele Specific Primers of virus disease; Analysis obtains TMV, PVY, CMV infects the tobacco leaf sample; Integrate reverse transcription and PCR and react required enzyme and damping fluid, by reaction condition optimization, set up the method that infects TMV, PVY, CMV based on single stage method RT-PCR compound detection tobacco leaf, concrete steps are as follows:
(1) three kind of virus-specific nucleotide primer design is with synthetic
Use DNAMAN6.0 software, TMV, PVY, each strain of CMV virus, each region disconnecting thing whole genome sequence that GenBank is included compare, determine the relative conservative region in the virus sequence, in selected conservative region, use Oligo 6.0 software design primers, and guarantee primer specificity by the BLAST analysis; Because multiplex PCR requires each primer and the right annealing temperature of primer to approach as far as possible, the size of amplicon also should be as far as possible close in the situation that agarose gel electrophoresis can significantly be distinguished simultaneously, so finally selected TMV, PVY, each pair of primers of CMV spend the RNase water dissolution to working concentration 10 μ M behind the synthetic;
TMV: upstream primer TF1 5 '-TGTGTGCAAAACTTACTTCCC-3 '
Downstream primer TR1 5 '-AGGACAAAACATTTGCGTATG-3 '
PVY: upstream primer PF1 5 '-ACTGTGATGAATGGGCTTATG-3 '
Downstream primer PR1 5 '-GGCATATATGGTTCCTTTTTG-3 '
CMV: upstream primer C3F1 5 '-GGCTTTCCAAGGTACCAGTAG-3 '
Downstream primer C3R1 5 '-ATCGAGCTTGCCAATTACTAC-3 '
(2) extraction of the total RNA of tobacco
At first serological diagnostic kit detects and analyzes the virus infection tobacco sample, the tobacco sample that finds TMV, PVY, CMV to infect by serological method, get classical symptom tobacco leaf 50mg, grinding powder in the liquid nitrogen, extract the total RNA of tobacco, extract product is dissolved in the distilled water of 50 μ l without the pollution of RNA enzyme, and-20 ℃ of storages are for subsequent use;
(3) triple single stage method RT-PCR detect the foundation of three kinds of viral reaction conditionss simultaneously
Through the reaction system of optimizing be: 10 * PCR Buffer, 7.5 μ l; 25mM Mg
2+6 μ l; DNTP 2.5mM each 4 μ l; Taq enzyme 4U; AMV ThermoScript II 5U; Ribonuclease Inhibitor 10U; Each 2 μ l of TMV upstream and downstream primer, each 2 μ l of CMV upstream and downstream primer, each 3 μ l of PVY upstream and downstream primer; Each 500ng of RNA; Go RNase water to complement to 50 μ l; Reaction conditions: 42 ℃ of reverse transcription 30min, 95 ℃ of heat inactivation 5min; 94 ℃ of sex change 30sec, 52 ℃ of annealing 30sec, 72 ℃ are extended 70sec, 30 circulations; 72 ℃ are replenished extension 10min, and amplified production is got 5-10 μ l electrophoresis detection in 1% sepharose.
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| CN103740865B (en) * | 2014-01-25 | 2015-04-01 | 云南省烟草农业科学研究院 | Method for rapidly and sensitively detecting tobacco mosaic virus |
| CN104357580A (en) * | 2014-10-09 | 2015-02-18 | 江苏省农业科学院 | Multiplex RT-PCR (reverse transcription-polymerase chain reaction) method for detecting various viruses of cucurbit plant with two-step method as well as special primer group for method |
| CN104651530A (en) * | 2014-12-19 | 2015-05-27 | 贵州省生物技术研究所 | Method for capturing RT-PCR by multiple tubes |
| CN105039594A (en) * | 2015-07-24 | 2015-11-11 | 湖南农业大学 | Triple RT-PCR method capable of detecting three viruses causing potato tuber necrosis simultaneously and primer combination thereof |
| CN108531654A (en) * | 2018-04-27 | 2018-09-14 | 王涛 | A kind of kit and its detection method for Potyvirus antidiastole in tobacco arteries and veins pinta |
| CN110066888A (en) * | 2019-04-12 | 2019-07-30 | 华南农业大学 | A kind of triple real time fluorescent quantitative RT-qPCR methods of synchronous detection TMV, CMV and PVY |
| CN110923365A (en) * | 2019-12-27 | 2020-03-27 | 江苏省农业科学院 | Multiple RT-PCR detection method for taro virus |
| CN111961674B (en) * | 2020-08-31 | 2022-05-06 | 中国烟草总公司郑州烟草研究院 | Tobacco PVY early warning gene Ntab0224260 and application thereof |
| CN114703318A (en) * | 2021-10-13 | 2022-07-05 | 贵州省烟草公司贵阳市公司 | Flue-cured tobacco aphid-borne virus multiplex composite PCR detection method |
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| CN101294963A (en) * | 2007-04-26 | 2008-10-29 | 福建农林大学 | Immune colloidal gold reagent for detecting orchid virus series and its preparation |
| CN101556282B (en) * | 2008-04-09 | 2012-12-26 | 河南农业大学 | Field rapid detection test strip for tobacco compound virus and preparation method thereof |
| CN101875981B (en) * | 2010-07-21 | 2013-01-09 | 陕西省烟草研究所 | Primer group and kit for synchronously detecting multiple tobacco viruses |
| CN101875982B (en) * | 2010-07-21 | 2012-09-12 | 陕西省烟草研究所 | Method for synchronously detecting multiple tobacco viruses |
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