CN102534049B - Gene diagnostic reagent kit and detection method of mikania micrantha wilt virus - Google Patents
Gene diagnostic reagent kit and detection method of mikania micrantha wilt virus Download PDFInfo
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- CN102534049B CN102534049B CN 201210027972 CN201210027972A CN102534049B CN 102534049 B CN102534049 B CN 102534049B CN 201210027972 CN201210027972 CN 201210027972 CN 201210027972 A CN201210027972 A CN 201210027972A CN 102534049 B CN102534049 B CN 102534049B
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Abstract
The invention discloses a gene diagnostic reagent kit and a detection method of mikania micrantha wilt virus. The reagent kit comprises two pairs of specificity primers which are respectively MMWV RNA1 F: 5'-TGAGGAATTGGGAAGGTC; MMWV RNA1 R: 5'-CGACTCGGCGTCATAAAC; MMWV RNA2 F: 5'-GGAATCCCTGAAACTATGC and MMWV RNA2 R: 5'-TGCTCCACCAATCACAACA. The detection method includes the following steps: (1) extracting RNA of a plant sample to be detected; (2) adding the two primers in the RNA and respectively obtaining two cDNA after polymerase chain reaction (PCR) amplification; and (3) respectively adding two primers into the cDNA and obtaining two amplification products after PCR amplification and conducting electrophoresis. If a first amplification product has a bright strip belt at 329bp and simultaneously a second amplification product has a bright strip belt at 364bp, MMWV is positive, and the fact that the plant sample to be detected carries the mikania micrantha wilt virus can be proved. The reagent kit and the method can amplify relative gene segments of the sample to be detected and carrying the MMWV virus in specificity mode, and a built PCR reaction system ensures quickness, stability and accuracy of the detection result.
Description
Technical field
The present invention relates to diagnostic kit and the detection method of plant virus, specifically for Mikania micrantha wilt gene diagnosis kit and the detection method of virus (Mikania micrantha wilt virus, MMWV).
Background technology
Mikania micrantha (Mikania micrantha H.B.K.) is the perennial vine of composite family (Compositae) Mikania (MikaniaWilld).Biotic intrusion to China South China, causes serious harm to ecotope at present.Mikania micrantha wilting virus belongs to Comoviridae (Comoviridae) fabavirus and belongs to (Fabavirus).Mikania micrantha the symptoms such as blade wilting, apical dieback, internode dwarfing occur after infecting this virus, can seriously suppress the growth of Mikania micrantha.This virus and broad bean wilt virus-1 (Broad bean wilt virus-1, BBWV-1) and broad bean wilt virus-2 (Broad bean wilt virus-2, BBWV-2) higher genetic homogeny is arranged, can infect the multiple important cash crop such as vegetable crop, leguminous crop, cause seriously financial loss.There is no effective detection method for Mikania micrantha wilting virus at present.
Therefore easy fast, high specificity, highly sensitive diagnostic kit and method thereof be the effective ways of Rapid﹠Early diagnosis Mikania micrantha wilting virus and this virus disseminating approach of effective monitoring.
Polymerase chain reaction (Polymerase Chain Reaction, PCR) is the strand primer that utilizes the DNA fragmentation both sides, in the technology of external rapid amplifying specific DNA fragment.The characteristics such as the method has fast, sensitivity and high specificity are widely applied in every scientific effort and production practice.
Summary of the invention
For the shortcoming and deficiency that overcome prior art, primary and foremost purpose of the present invention is to provide a kind of Mikania micrantha wilt disease virus gene diagnostic kit.
Another object of the present invention is to provide a kind of method of utilizing the mentioned reagent box to detect Mikania micrantha wilt disease virus gene.
Purpose of the present invention is achieved through the following technical solutions:
A kind of Mikania micrantha wilt disease virus gene diagnostic kit comprises two pairs of Auele Specific Primers, is respectively:
MMWV?RNA1?F：5’-TGAGGAATTGGGAAGGTC；
MMWV?RNA1?R：5’-CGACTCGGCGTCATAAAC；
MMWV?RNA2?F：5’-GGAATCCCTGAAACTATGC；
MMWV?RNA2?R：5’-TGCTCCACCAATCACAACA；
Above-mentioned two pairs of primers are with the design of the conservative region of MMWV RNA1 and MMWV RNA2, the genes involved fragment of the testing sample that carries MMWV virus of can increasing specifically;
Described Mikania micrantha wilt disease virus gene diagnostic kit also comprises the dNTP mixture, contains mg
2+10 times of reaction buffers and polysaccharase;
The preferred TaqE of described polysaccharase.
A kind of mentioned reagent box of using detects the viral method of Mikania micrantha wilting, comprises the following steps:
(1) extract the RNA of plant sample to be measured according to conventional universal method;
(2) add respectively primer MMWV RNA1 F and MMWVRNA1 R, primer MMWV RNA2 F and MMWV RNA2 R in the RNA that obtains toward step (1), obtain respectively MMWV RNA1 cDNA and MMWV RNA2 cDNA after pcr amplification;
(3) add primer MMWV RNA1 F coin MMWV RNA1R in MMWV RNA1 cDNA, carry out pcr amplification and obtain amplified production 1; Add primer MMWVRNA2 F and MMWV RNA2 R in MMWV RNA2 cDNA, carry out pcr amplification and obtain amplified production 2; Two kinds of amplified productions are carried out agarose gel electrophoresis, if amplified production 1 bright band occurs at 329bp, amplified production 2 bright band occurs at 364bp simultaneously, and MMWV is positive, illustrate that carrying Mikania micrantha in plant sample to be measured wilts viral; Otherwise MMWV is negative, shows that plant sample to be measured does not carry Mikania micrantha and wilts viral.
The present invention has following advantage and effect with respect to prior art:
Mikania micrantha of the present invention wilt gene diagnosis kit and the detection method of virus are to design take two pairs of Auele Specific Primers of the gene conservative region design of MMWVRNA1 and MMWV RNA2 as main body.Utilize two pairs of primers, the genes involved fragment of the testing sample that carries MMWV virus of can increasing specifically, the PCR reaction system of setting up guarantees rapidity, stability and the accuracy of detected result.Therefore use test kit of the present invention and detection method, can detect easy, fast, delicately the sample that infects MMWV, the tracking that can be used for this virus detects, the detection of potential host plant, avoid the spread and epidemic of virus, the ecomanagement of reinforcement to the Mikania micrantha biotic intrusion has broad application prospects.
Description of drawings
Fig. 1 is pcr amplification product electrophoresis result figure.
Embodiment
The present invention is described in further detail below in conjunction with embodiment and accompanying drawing, but embodiments of the present invention are not limited to this.
The gene diagnosis kit of Mikania micrantha wilting virus comprises following component:
1). template extract I, 1 bottle, in-built Trizol;
2). protein liquid removal II, 1 bottle, in-built chloroform;
3) .RNA precipitated liquid III, 1 bottle, in-built Virahol;
4). washings IV, 1 bottle, in-built 75% ethanol;
5). elutriant V, 1 bottle, in-built sterilization distilled water;
6). reaction solution VI, 1 bottle, in-built PCR reaction solution comprises ddH
2O, dNTP, contain mg
2+10 * Buffer, TaqE, primer MMWVRNA1F be: 5 '-TGAGGAATTGGGAAGGTC and MMWVRNA1R are: 5 '-CGACTCGGCGTCATAAAC;
7). reaction solution VII, 1 bottle, in-built PCR reaction solution comprises ddH
2O, dNTP, contain mg
2+10 * Buffer, TaqE, primer MMWVRNA2F be: 5 '-GGAATCCCTGAAACTATGC, MMWVRNA2R is: 5 '-TGCTCCACCAATCACAACA;
8). positive control solution VIII, 1 bottle, the plasmid DNA of in-built MMWV RNA1 and MMWV RNA2 genes involved fragment;
9). a cystose, the circular hole that is no less than above-mentioned bottle quantity is arranged on cystose, each bottle can be placed in respectively corresponding aperture;
10). box, can hold above-mentioned all reagent and material etc.
After positive control solution VIII in this test kit is pcr amplification MMWV RNA1 and MMWV RNA2, obtain respectively MMWV RNA1 cDNA (sequence is SQE ID NO.5) and MMWV RNA2cDNA (sequence is SQE ID NO.6), MMWV RNA1 cDNA and MMWV RNA2 cDNA are connected respectively on the pMD18-T carrier, change intestinal bacteria E.coli over to, the amoxicillin culture medium flat plate is cultivated, picking positive colony, sequencing analysis checking, extract its plasmid DNA, be dissolved in 50 μ l ddH
2Be positive control solution in O.
A kind of test kit of Application Example 1 detects the method for Mikania micrantha wilting virus, comprises the following steps:
1). get plant sample to be measured, add appropriate liquid nitrogen fully to be ground to Powdered;
2). ground powder is moved in the 2ml sterilization centrifuge tube of RNase-free, add 1ml template extract I liquid, abundant mixing, the standing 6min of room temperature;
3). add 200 μ l protein liquid removal II liquid, cover tightly centrifuge tube, the abundant mixing of concuss 30s, the standing 5min of room temperature;
4) .4 ℃, the centrifugal 12min of 12000r/min;
5). get supernatant liquor to the 2ml sterilization centrifuge tube of another RNase-free, add 500 μ l RNA precipitated liquid III liquid, mixing, place 10min under room temperature gently;
6) .4 ℃, the centrifugal 12min of 12000r/min;
7). abandon supernatant, add 1ml washings IV to rinse, the centrifugal 12min of 12000r/min rinses twice;
8). abandon supernatant, dry up on Bechtop or room temperature under air-dry 2min;
9). add 20 μ l elutriant V liquid resuspended to fully dissolving, obtain the RNA of plant sample to be measured;
10). add respectively RNA 1 μ l and the PCR reaction solution VII 9 μ l of the RNA 1 μ l of step (9) plant sample to be measured and PCR reaction solution VI 9 μ l, step (9) plant sample to be measured in two PCR pipes, the centrifugal several seconds after mixing, carry out pcr amplification, 37 ℃ of 20min, 98 ℃ of 5min; 4 ℃ of preservations; Obtain respectively MMWVRNA1 cDNA and MMWV RNA2 cDNA;
11). get the MMWV RNA1 cDNA that 2 μ l steps (10) obtain and mix with PCR reaction solution VI 18 μ l; Getting the MMWV RNA2 cDNA that 2 μ l steps (10) obtain mixes with PCR reaction solution VII 18 μ l; Carry out pcr amplification, 94 ℃ of 5min denaturations; 94 ℃ of 30sec, 57 ℃ of 30sec, 72 ℃ of 1min; 35 circulations, 72 ℃ of 10min, 4 ℃ of preservations.Obtain respectively amplified production 1 and 2 after amplification.
12). get respectively 5 μ l amplified productions 1, amplified production 2, add 1 μ l tetrabromophenol sulfonphthalein mixing, through 1.5% agarose gel electrophoresis 25～40min, (swimming lane 1 is amplified production 1 for electrophoresis result such as Fig. 1; Swimming lane 3 is amplified production 2; Swimming lane 2 and 4 is respectively corresponding negative control, namely do not add the amplified production of cDNA template) shown in, amplified production 1 bright band occurs at 329bp, simultaneously, amplified production 2 bright band occurs at 364bp, testing sample MMWV is positive, illustrates that carrying Mikania micrantha in plant sample to be measured wilts viral.
Above-described embodiment is the better embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, within being included in protection scope of the present invention.
Claims (2)
1. a Mikania micrantha wilt disease virus gene diagnostic kit, is characterized in that comprising two pairs of Auele Specific Primers, is respectively:
MMWV?RNA1?F：5’-TGAGGAATTGGGAAGGTC；
MMWV?RNA1?R：5’-CGACTCGGCGTCATAAAC；
MMWV?RNA2?F：5’-GGAATCCCTGAAACTATGC；
MMWV?RNA2?R：5’-TGCTCCACCAATCACAACA。
2. Mikania micrantha wilt disease virus gene diagnostic kit according to claim 1, it is characterized in that: described Mikania micrantha wilt disease virus gene diagnostic kit also comprises the dNTP mixture, contains mg
2+10 times of reaction buffers and polysaccharase.
3, Mikania micrantha wilt disease virus gene diagnostic kit according to claim 2, it is characterized in that: described polysaccharase is TaqE.
4, a kind of application rights requires the described test kit of 1-3 any one to detect the method for Mikania micrantha wilting virus, it is characterized in that comprising the following steps:
(1) extract the RNA of plant sample to be measured;
(2) add respectively primer MMWV RNA1 F and MMWV RNA1 R in the RNA that obtains toward step (1), primer MMWV RNA2 F and MMWV RNA2 R obtain respectively MMWV RNA1 cDNA and MMWV RNA2 cDNA after pcr amplification;
(3) add primer MMWV RNA1 F and MMWV RNA1 R in MMWV RNA1 cDNA, carry out pcr amplification and obtain amplified production 1; Add primer MMWV RNA2 F and MMWV RNA2 R in MMWV RNA2 cDNA, carry out pcr amplification and obtain amplified production 2; Two kinds of amplified productions are carried out agarose gel electrophoresis, if amplified production 1 bright band occurs at 329bp, amplified production 2 bright band occurs at 364bp simultaneously, and MMWV is positive, illustrate that carrying Mikania micrantha in plant sample to be measured wilts viral; Otherwise MMWV is negative, shows that plant sample to be measured does not carry Mikania micrantha and wilts viral;
Described MMWV refers to Mikania micrantha wilting virus.
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| "薇甘菊水杨酸羧甲基转移酶基因的分离鉴定及表达分析";王文天等;《热带亚热带植物学报》;20091231;第17卷(第5期);445-450 * |
| 王文天等."薇甘菊水杨酸羧甲基转移酶基因的分离鉴定及表达分析".《热带亚热带植物学报》.2009,第17卷(第5期),445-450. |
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