CN102242225B - Double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid) - Google Patents
Double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid) Download PDFInfo
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Abstract
The invention discloses a double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid), belonging to the virus detection field of molecular biology. The method comprises the following steps of: (1) sampling from chrysanthemum plants, and extracting total RNA (ribonucleic acid); (2) respectively designing two pairs of specific primers for CVB and CChMVd: CVB-F and CVB-R; CChMVd-F and CChMVd-R; (3) carrying out reverse transcription by taking a random hexamer as a primer to obtain two kinds of cDNA (complementary deoxyribonucleic acid); and (4) amplifying the cDNA by utilizing the RT-PCR technology, and carrying out agarose gel electrophoresis on the amplified products to obtain a 665bp specific fragment for expressing infection of CVB and obtain a 206bp specific fragment for expressing infection of CChMVd. The detection method provided by the invention can be used for simultaneously detecting diseased plants which are compositely infected by CVB and CChMVd, and has the advantages of strong detection specificity, high sensitivity, simple process and cost saving.
Description
Technical field
The present invention relates to molecular biological field of virus detection, relate in particular to the duplex RT-PCR detection method of a kind of chrysanthemum CVB and CChMVd virus.
Background technology
Chrysanthemum (
Dendranthema morifoliumTzvel. (
Chrysanthemum morifoliumRamat.)) being composite family Chrysanthemum perennial root herbaceous plant, is China's ten great tradition famous flowers, and one of ornamental plant of cultivating the earliest in the world has very high economy and ornamental value, occupies consequence in the industry of flowers and plants.Chrysanthemum is easily infected virus in transplanting, cuttage and planting process.Chrysanthemum B virus (Chrysanthemum virus B, CVB) makes ill chrysanthemum show as light-duty floral leaf disease on Folium chrysanthemi, and for the susceptible kind, can form obvious flower leaf paresthesia or necrotic plaque, and serious meeting produces the withered spot of brown.CVB generally occurs in viewing and admiring on chrysanthemum, sickness rate 60 ~ 65%.Chrysanthemum chlorotic mottle virus (Chrysanthemum chloritic mottleviroid, CChMVd) is the another kind of important pathogen of chrysanthemum of causing harm, and causes the chrysanthemum piebaldism.The plant that is injured presents mottling usually on leaf, rear complete chlorisis.Some kind shows as growth retardation and downgrades, and takes root bad.
Detecting plant virus can detect and identify with the negative staining electron microscope color method in the past, but negative staining Electronic Speculum method is to the tyro, difficulty is larger, not only easily is subject to the interference of smudge cells and affects judged result, and can not identify and hide and Combined Infection virus.
Development along with modern plants virus and viroid detection means, using at present is euzymelinked immunosorbent assay (ELISA) (ELISA) the most widely, formed abroad commercialization production, but every kind of virus all needs special enzyme labelling specific antibody, the labeling process more complicated, price comparison is expensive, sometimes also can produce nonspecific color interference, and can not identify the viroid that does not contain coat protein.
The Beijing City Agriculture and Forestry Institute is in Chinese patent " a kind of method for detection of the chrysanthemum B virus " (application number: CN200810240415) with " a kind of method for detection of chrysanthemum chlorotic mottle virus " (application number: CN200810240414) disclose the method that adopts molecular biology method detection CVB and CChMVd of application in 2008, but adopting LAMP to detect is easily polluted, false positive results appears, and very high to the requirement of design of primers.
The technology that can detect simultaneously cause of disease at present for chrysanthemum under CVB and CChMVd Combined Infection has no report, if can detect simultaneously cause of disease, can improve detection efficiency, significantly reduces testing cost.
Summary of the invention
Goal of the invention: the purpose of this invention is to provide the duplex RT-PCR detection method of a kind of chrysanthemum CVB and CChMVd virus, can detect simultaneously CVB and CChMVd.
Technical scheme: for achieving the above object, the duplex RT-PCR detection method of a kind of chrysanthemum CVB of the present invention and CChMVd virus comprises the steps:
(1) take a sample and extract total RNA from the chrysanthemum plant;
(2) respectively for CVB and two couples of Auele Specific Primer: CVB-F of CChMVd virus design, CVB-R; CChMVd-F, CChMVd-R;
(3) carry out reverse transcription take random hexamer as primer and obtain two kinds of cDNA;
(4) utilize the described cDNA of RT-PCR technology amplification, the product that increases is carried out agarose gel electrophoresis, obtain the 665bp specific fragment and represent to infect CVB, obtain the 206bp specific fragment and represent to infect CChMVd.
The described sampling of step (1) comprises flower, upper blade, the lower blade of chrysanthemum plant, after sampling, sample is preserved with liquid nitrogen freezing.The RNA that the total RNA employing of described extraction Takara company provides extracts test kit, and the total RNA that obtains is stored in-70 ℃ of Ultralow Temperature Freezers.
The described two pairs of Auele Specific Primers of step (2) are respectively:
CVB-F：5’-ACCGAATTCTTAGTCACAATGCCTCCC-3’，
CVB-R：5’-TCCGAGCTCATAGAGACGGCATACCTT-3’；
CChMVd-F：5’-CAGTTTCGGCTTGTGCGGGAGT-3’，
CChMVd-R：5’-TCCGAGGAGAATATCCAACGAG-3’。
The described RT-PCR reaction system 25 μ l of step (4) comprise 0.5 μ l CVB-F, 0.5 μ l CVB-R, 0.5 μ l CChMVd-F, 0.5 μ l CChMVd-R.Specifically, described RT-PCR reaction also adds in the reaction system of 25 μ l: 1 μ l dNTPs(2.5mmol/L), and 1 μ l cDNA, 2.5 μ l 10 * PCR Buffer, 0.2 μ l Taq enzyme, 1.5 μ l Mg
2+
The described RT-PCR reaction of step (4) is set as 94 ℃ of denaturation 5 min, 94 ℃ of sex change 45s, and 52 ℃ of annealing 45s, 72 ℃ are extended 1min circulation 33 times, and 72 ℃ are extended 10min.The product of described amplification obtains the CVB specific fragment of 665bp and the CChMVd specific fragment of 206bp by 2% agarose gel electrophoresis.
The present invention can increase in same annealing temperature because the Tm value of CVB and two kinds of primers of CChMVd approaches, and has simplified operation; And specific fragment length is respectively 665bp and 206bp, and significant difference can easily separate two kinds of ribbon area by agarose gel electrophoresis.
Beneficial effect: the duplex RT-PCR detection method of a kind of chrysanthemum CVB of the present invention and CChMVd virus can detect simultaneously by the diseased plant of CVB and CChMVd Combined Infection, and detection specificity is strong, and is highly sensitive, and operation is simple, saves cost.
Description of drawings
Fig. 1 is disease leaf sample graph;
Fig. 2 is the gel electrophoresis figure of embodiment 1 dual RT-PCR, 1:CChMVd; 2:CVB; 3: flower; 4: upper leaf; 5: the bottom leaf;
Fig. 3 is the gel electrophoresis figure of CVB in test example 1 substance RT-PCR, 1: the bottom leaf; 2: upper leaf; 3: flower;
Fig. 4 is the gel electrophoresis figure of CChMVd in test example 1 substance RT-PCR, 1: flower; 2: leaf;
Fig. 5 is the PCR gel electrophoresis figure after the dilution of cDNA template different ratios in test example 2,1: primary template; 2: dilution 10
-13: dilution 10
-24: dilution 10
-3
Embodiment
Below in conjunction with accompanying drawing, the present invention is further described.
Get field performance floral leaf, chrysanthemum plant mottled, chlorisis is material, the plant of getting is carried out total RNA extract, and the RNA extraction test kit that total RNA adopts Takara company to provide is provided, the RNA that obtains is stored in the Ultralow Temperature Freezer of-70 ℃.
The Auele Specific Primer of design CVB and CChMVd:
CVB-F：5’-ACCGAATTCTTAGTCACAATGCCTCCC-3’，
CVB-R：5’-TCCGAGCTCATAGAGACGGCATACCTT-3’；
CChMVd-F：5’-CAGTTTCGGCTTGTGCGGGAGT-3’，
CChMVd-R：5’-TCCGAGGAGAATATCCAACGAG-3’；
Carry out reverse transcription take random hexamer as primer, obtain two kinds of cDNA of CVB and CChMVd.The reaction system of 20 μ l is: add 1 μ l template ribonucleic acid in the Microtube pipe, 2 μ l Random Primers(25 μ M), 9 μ l RNase free H
2O after 70 ℃ of insulation 10min rapidly at chilling 2min on ice, make the denaturing soln of template ribonucleic acid be gathered in the bottom of Microtube pipe after the centrifugal several seconds, add 4 μ l 5 * M-MLV Buffer again in the denaturing soln of 12 μ l, 1 μ l dNTP Mixture(10mM), 0.5 μ l RNase Inhibitor(40U/ μ l), 0.8 the RTase M-MLV(200U/ μ l of μ L), 1.7 μ l RNase free H
2O is in 30 ℃ of reaction 10min, and then at 42 ℃ of insulation 60min, cooled on ice after 70 ℃ of insulation 15min obtains two kinds of cDNA of CVB and CChMVd.
Dual RT-PCR reaction system 25 μ l comprise 0.5 μ l CVB-F, 0.5 μ l CVB-R, 0.5 μ l CChMVd-F, 0.5 μ l CChMVd-R, 1 μ l dNTPs(2.5mmol/L), 1 μ l cDNA, 2.5 μ l 10 * PCR Buffer, 0.2 μ l Taq enzyme, 1.5 μ l Mg
2+The RT-PCR reaction is set as: 94 ℃ of denaturation 5 min, and 94 ℃ of sex change 45s, 52 ℃ of annealing 45s, 72 ℃ are extended 1min circulation 33 times, and 72 ℃ are extended 10min.
Amplified production is with 2% agarose gel electrophoresis analysis, and as shown in Figure 2, CChMVd has specific fragment at 206bp, and CVB has specific fragment at 665bp.And can detect simultaneously whether by CVB and CChMVd Combined Infection at flower, upper leaf and the bottom leaf of diseased plant.
Get field performance floral leaf, chrysanthemum plant mottled, chlorisis is material, the plant of getting is carried out total RNA extract, and the RNA extraction test kit that total RNA adopts Takara company to provide is provided, the RNA that obtains is stored in the Ultralow Temperature Freezer of-70 ℃.
The Auele Specific Primer of design CVB and CChMVd:
CVB-F：5’-ACCGAATTCTTAGTCACAATGCCTCCC-3’，
CVB-R：5’-TCCGAGCTCATAGAGACGGCATACCTT-3’；
CChMVd-F：5’-CAGTTTCGGCTTGTGCGGGAGT-3’，
CChMVd-R：5’-TCCGAGGAGAATATCCAACGAG-3’；
Carry out reverse transcription take random hexamer as primer, obtain two kinds of cDNA of CVB and CChMVd.The reaction system of 20 μ l is: add 1 μ l template ribonucleic acid in the Microtube pipe, 2 μ l Random Primers(25 μ M), 9 μ l RNase free H
2O after 70 ℃ of insulation 10min rapidly at chilling 2min on ice, make the denaturing soln of template ribonucleic acid be gathered in the bottom of Microtube pipe after the centrifugal several seconds, add 4 μ l 5 * M-MLV Buffer again in the denaturing soln of 12 μ l, 1 μ l dNTP Mixture(10mM), 0.5 μ l RNase Inhibitor(40U/ μ l), 0.8 the RTase M-MLV(200U/ μ l of μ L), 1.7 μ l RNase free H
2O is in 30 ℃ of reaction 10min, and then at 42 ℃ of insulation 60min, cooled on ice after 70 ℃ of insulation 15min obtains two kinds of cDNA of CVB and CChMVd.
Dual RT-PCR reaction system 25 μ l comprise 0.5 μ l CVB-F, 0.5 μ l CVB-R, 0.5 μ l CChMVd-F, 0.5 μ l CChMVd-R, 2 μ l dNTPs(2.5mmol/L), 1 μ l cDNA, 2.5 μ l 10 * PCR Buffer, 0.2 μ l Taq enzyme, 1.5 μ l Mg2
+The RT-PCR reaction is set as: 94 ℃ of denaturation 5 min, and 94 ℃ of sex change 43s, 52 ℃ of annealing 40s, 72 ℃ are extended 1min circulation 35 times, and 72 ℃ are extended 10min.
Get field performance floral leaf, chrysanthemum plant mottled, chlorisis is material, the plant of getting is carried out total RNA extract, and the RNA extraction test kit that total RNA adopts Takara company to provide is provided, the RNA that obtains is stored in the Ultralow Temperature Freezer of-70 ℃.
The Auele Specific Primer of design CVB and CChMVd:
CVB-F：5’-ACCGAATTCTTAGTCACAATGCCTCCC-3’，
CVB-R：5’-TCCGAGCTCATAGAGACGGCATACCTT-3’；
CChMVd-F：5’-CAGTTTCGGCTTGTGCGGGAGT-3’，
CChMVd-R：5’-TCCGAGGAGAATATCCAACGAG-3’；
Carry out reverse transcription take random hexamer as primer, obtain two kinds of cDNA of CVB and CChMVd.The reaction system of 20 μ l is: add 1 μ l template ribonucleic acid in the Microtube pipe, 2 μ l Random Primers(5 μ M), 9 μ l RNase free H
2O after 70 ℃ of insulation 10min rapidly at chilling 2min on ice, make the denaturing soln of template ribonucleic acid be gathered in the bottom of Microtube pipe after the centrifugal several seconds, add 4 μ l 5 * M-MLV Buffer again in the denaturing soln of 12 μ l, 1 μ l dNTP Mixture(10mM), 0.5 μ l RNase Inhibitor(40U/ μ l), 0.8 the RTase M-MLV(200U/ μ l of μ L), 1.7 μ l RNase free H
2O is in 30 ℃ of reaction 10min, and then at 42 ℃ of insulation 60min, cooled on ice after 70 ℃ of insulation 15min obtains two kinds of cDNA of CVB and CChMVd.
Dual RT-PCR reaction system 25 μ l comprise 0.5 μ l CVB-F, 0.5 μ l CVB-R, 0.5 μ l CChMVd-F, 0.5 μ l CChMVd-R, 2 μ l dNTPs(2.5mmol/L), 1 μ l cDNA, 2.5 μ l 10 * PCR Buffer, 0.2 μ l Taq enzyme, 1.5 μ l Mg2
+The RT-PCR reaction is set as: 94 ℃ of denaturation 5 min, and 94 ℃ of sex change 45s, 52 ℃ of annealing 47s, 72 ℃ are extended 1min circulation 30 times, and 72 ℃ are extended 10min.
Test example 1
Get field performance floral leaf, chrysanthemum plant mottled, chlorisis is material, the plant of getting is carried out total RNA extract, the RNA that obtains is stored in the Ultralow Temperature Freezer of-70 ℃.
The Auele Specific Primer of design CVB and CChMVd:
CVB-F：5’-ACCGAATTCTTAGTCACAATGCCTCCC-3’，
CVB-R：5’-TCCGAGCTCATAGAGACGGCATACCTT-3’；
CChMVd-F：5’-CAGTTTCGGCTTGTGCGGGAGT-3’，
CChMVd-R：5’-TCCGAGGAGAATATCCAACGAG-3’；
Carry out reverse transcription take random hexamer as primer, obtain the cDNA of the first chain.The reaction system of 20 μ l is: add 1 μ l template ribonucleic acid in the Microtube pipe, 2 μ l Random Primers(5 μ M), 9 μ l RNase free H
2O after 70 ℃ of insulation 10min rapidly at chilling 2min on ice, make the denaturing soln of template ribonucleic acid be gathered in the bottom of Microtube pipe after the centrifugal several seconds, add 4 μ l 5 * M-MLV Buffer again in the denaturing soln of 12 μ l, 1 μ l dNTP Mixture(10mM), 0.5 μ l RNase Inhibitor(40U/ μ l), 0.8 the RTase M-MLV(200U/ μ l of μ L), 1.7 μ l RNase free H
2O is in 30 ℃ of reaction 10min, and then at 42 ℃ of insulation 60min, cooled on ice after 70 ℃ of insulation 15min obtains two kinds of cDNA of CVB and CChMVd.
Respectively with two kinds of cDNA of RT-PCR amplification CVB and CChMVd:
The 25 μ l reaction systems of CVB are: 2.5 μ l 10 * PCR Buffer, 2.0 μ l dNTPs(2.5mmol/L), 1.5 μ l Mg
2+, 1 μ L CVB-R, 1 μ l CVB-F, 1 μ l cDNA, 0.2 μ l Taq enzyme is supplied 25 μ l with deionized water.The amplification condition of PCR system is: 94 ℃ of denaturation 5min, and 94 ℃ of sex change 45s, 55 ℃ of annealing 45s, 72 ℃ are extended 1min circulation 30 times, and 72 ℃ are extended 10min.Amplified production is with 2% detected through gel electrophoresis analysis.Obtain expecting that length is the specific fragment of 665bp, as shown in Figure 3;
The 25 μ l reaction systems of CChMVd are: 2.5 μ l 10 * PCR Buffer, 2.0 μ l dNTPs(2.5mmol/L), 1.5 μ l Mg
2+, 1 μ l CChMVd-R, 1 μ l CChMVd-F, 1 μ l cDNA, 0.2 μ l Taq enzyme, deionized water is supplied 25 μ l.The amplification condition of PCR system is: 94 ℃ of denaturation 5 min, and 94 ℃ of sex change 45s, 50 ℃ of annealing 30s, 72 ℃ are extended 1min circulation 30 times, and 72 ℃ are extended 10min.The PCR product that obtains obtains expecting that length is the specific fragment of 206bp, as shown in Figure 4 with 2% agarose gel electrophoresis analysis.
Respectively resulting two kinds of RT-PCR products are cut glue and reclaimed the purpose band, be connected to pMD19-T Simple carrier, transform the TOP10 competent cell, cultivate 15h for 37 ℃, according to blue hickie screening, the picking white colony is cultivated, positive identification is carried out in the PCR reaction, the sample order-checking is completed by Beijing six directions China large Gene science company, carries out sequence homology analysis with BLAST software, and result is as follows:
The coat protein expanding fragment length of CVB is 665bp, with (GenBank:AM493895.2) such as Singh. and Jabeen(GenBank:AJ876635.1) the corresponding nucleotide homology of isolate be 86% and 84%; And the sequence homology of CChMVd and Flores (GenBank:AJ878089.1) and Yamamoto(GenBank:AB181858.1) homology of the corresponding nucleotide of isolate up to 97%.Thereby determined the kind of two-strain, simultaneously proved that also the gene order of CVB and CChMVd virus has conservative property, also had suitability widely according to the primer of its CP sequences Design.
Test example 2
The process of this test example is identical with embodiment 1, but with the cDNA template respectively according to 10
-1, 10
-2, 10
-3Carry out the different ratios dilution.Detect the sensitivity that the method detects CVB and CChMVd simultaneously, result as shown in Figure 5, as seen, when template concentrations dilutes 10
-3In time, still can detect at 206bp and 665bp, illustrates that the method detects simultaneously two-strain and has very high sensitivity.
The above is only the preferred embodiment of the present invention; be noted that for those skilled in the art; under the prerequisite that does not break away from the principle of the invention, can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (3)
1. the duplex RT-PCR detection method of a chrysanthemum CVB and CChMVd virus, is characterized in that, comprises the steps:
(1) take a sample and extract total RNA from the chrysanthemum plant;
(2) respectively for CVB and two couples of Auele Specific Primer: CVB-F of CChMVd virus design, CVB-R; CChMVd-F, CChMVd-R;
Wherein:
CVB-F：5’-ACCGAATTCTTAGTCACAATGCCTCCC-3’，
CVB-R：5’-TCCGAGCTCATAGAGACGGCATACCTT-3’；
CChMVd-F：5’-CAGTTTCGGCTTGTGCGGGAGT-3’，
CChMVd-R：5’-TCCGAGGAGAATATCCAACGAG-3’；
(3) carry out reverse transcription take random hexamer as primer and obtain two kinds of cDNA;
(4) utilize the described cDNA of RT-PCR technology amplification, the product that increases is carried out agarose gel electrophoresis, obtain the 665bp specific fragment and represent to infect CVB, obtain the 206bp specific fragment and represent to infect CChMVd.
2. the duplex RT-PCR detection method of chrysanthemum CVB according to claim 1 and CChMVd virus, it is characterized in that: the described RT-PCR reaction system 25 μ l of step (4) comprise 0.5 μ l CVB-F, 0.5 μ l CVB-R, 0.5 μ l CChMVd-F, 0.5 μ l CChMVd-R.
3. the duplex RT-PCR detection method of chrysanthemum CVB according to claim 1 and CChMVd virus, it is characterized in that: the described RT-PCR reaction of step (4) is set as 94 ℃ of denaturation 5 min, 94 ℃ of sex change 43 ~ 47s, 52 ℃ of annealing 40 ~ 45s, 72 ℃ are extended 1min circulation 30 ~ 35 times, and 72 ℃ are extended 10min.
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| CN104673938A (en) * | 2015-03-06 | 2015-06-03 | 南京农业大学 | High-sensitivity primers and method for detecting CVB (chrysanthemum virus B), as well as application |
| CN112695139A (en) * | 2021-02-11 | 2021-04-23 | 滁州职业技术学院 | Detection method for simultaneously detecting multiple chrysanthemum common viruses/viroids |
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Non-Patent Citations (4)
| Title |
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| Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue;M. Hosokawa 等;《Journal of Virological Methods》;20061231;第131卷;28–33 * |
| M. Hosokawa 等.Direct RT-PCR method for detecting two chrysanthemum viroids using minimal amounts of plant tissue.《Journal of Virological Methods》.2006,第131卷28–33. |
| 菊花CVB、TAV和CChMVd病毒的RT-PCR检测及匍匐小菊叶盘再生体系的建立;颜琛娜;《中国优秀硕士学位论文全文数据库》;20090815(第8期);全文 * |
| 颜琛娜.菊花CVB、TAV和CChMVd病毒的RT-PCR检测及匍匐小菊叶盘再生体系的建立.《中国优秀硕士学位论文全文数据库》.2009,(第8期), |
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