Double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid)

A technology of RT-PCR and detection method, which is applied in the field of virus detection in molecular biology, which can solve the problems of affecting judgment results, high price, and difficulty, and achieve the effects of strong detection specificity, high sensitivity, and cost saving

Inactive Publication Date: 2011-11-16
NANJING AGRICULTURAL UNIVERSITY
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AI Technical Summary

Problems solved by technology

Some varieties show stunted growth, poor rooting
[0003] In the past, the negative staining method of electron microscopy can be used to detect and identify plant viruses. However, negative staining electron microscopy is more difficult for beginners. It is not only susceptible to the interference of broken cells and affects the judgment results, but also cannot identify latent and compound infections. Virus
[0004] With the development of modern plant virus and virus-like detection methods, enzyme-linked immunosorbent assay (ELISA) is currently the most widely used method, which has been commercially produced abroad, but each virus requires specific enzyme-labeled specific antibodies. More complex, more expensive, sometimes produces non-specific color interference, and cannot identify viroids without coat p

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  • Double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid)
  • Double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid)
  • Double RT-PCR (reverse transcription-polymerase chain reaction) detection method of CVB (chrysanthemum virus B) and CChMVd (chrysanthemum chlorotic mottle viroid)

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The chrysanthemum plants showing mottled leaves, mottled and chlorotic in the field were taken as materials, and the total RNA was extracted from the taken plants. The total RNA was extracted using the RNA extraction kit provided by Takara Company, and the obtained RNA was stored in an ultra-low temperature freezer at -70°C .

[0031] Design specific primers for CVB and CChMVd:

[0032] CVB-F: 5'-ACCGAATTCTTAGTCACAATGCCTCCC-3',

[0033] CVB-R: 5'-TCCGAGCTCATAGAGACGGCATACCTT-3';

[0034] CChMVd-F: 5'-CAGTTTCGGCTTGTGCGGGAGT-3',

[0035] CChMVd-R: 5'-TCCGAGGAGAATATCCAACGAG-3';

[0036] Two cDNAs of CVB and CChMVd were obtained by reverse transcription using random hexamers as primers. The 20μl reaction system is: add 1μl template RNA, 2μl Random Primers (25μM), 9μl RNase free H in a Microtube tube 2 O was incubated at 70°C for 10 minutes, then rapidly cooled on ice for 2 minutes, centrifuged for a few seconds, and the denatured solution of template RNA was collected a...

Embodiment 2

[0040] The chrysanthemum plants showing mottled leaves, mottled and chlorotic in the field were taken as materials, and the total RNA was extracted from the taken plants. The total RNA was extracted using the RNA extraction kit provided by Takara Company, and the obtained RNA was stored in an ultra-low temperature freezer at -70°C .

[0041] Design specific primers for CVB and CChMVd:

[0042] CVB-F: 5'-ACCGAATTCTTAGTCACAATGCCTCCC-3',

[0043] CVB-R: 5'-TCCGAGCTCATAGAGACGGCATACCTT-3';

[0044] CChMVd-F: 5'-CAGTTTCGGCTTGTGCGGGAGT-3',

[0045] CChMVd-R: 5'-TCCGAGGAGAATATCCAACGAG-3';

[0046] Two cDNAs of CVB and CChMVd were obtained by reverse transcription using random hexamers as primers. The 20μl reaction system is: add 1μl template RNA, 2μl Random Primers (25μM), 9μl RNase free H in a Microtube tube 2 O was incubated at 70°C for 10 minutes, then rapidly cooled on ice for 2 minutes, centrifuged for a few seconds, and the denatured solution of template RNA was collected at ...

Embodiment 3

[0049] The chrysanthemum plants showing mottled leaves, mottled and chlorotic in the field were taken as materials, and the total RNA was extracted from the taken plants. The total RNA was extracted using the RNA extraction kit provided by Takara Company, and the obtained RNA was stored in an ultra-low temperature freezer at -70°C .

[0050] Design specific primers for CVB and CChMVd:

[0051] CVB-F: 5'-ACCGAATTCTTAGTCACAATGCCTCCC-3',

[0052] CVB-R: 5'-TCCGAGCTCATAGAGACGGCATACCTT-3';

[0053] CChMVd-F: 5'-CAGTTTCGGCTTGTGCGGGAGT-3',

[0054] CChMVd-R: 5'-TCCGAGGAGAATATCCAACGAG-3';

[0055] Two cDNAs of CVB and CChMVd were obtained by reverse transcription using random hexamers as primers. The 20μl reaction system is: add 1μl template RNA, 2μl Random Primers (5μM), 9μl RNase free H in a Microtube tube 2 O was incubated at 70°C for 10 minutes, then rapidly cooled on ice for 2 minutes, centrifuged for a few seconds, and the denatured solution of template RNA was collected at...

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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 [0001] The invention relates to the field of virus detection in molecular biology, in particular to a double RT-PCR detection method for chrysanthemum CVB and CChMVd viruses. Background technique [0002] chrysanthemum( Dendranthema morifolium Tzvel.( Chrysanthemum morifolium Ramat.)) is a perennial herbaceous plant of the genus Chrysanthemum in the family Asteraceae. It is one of the top ten traditional famous flowers in my country and one of the earliest cultivated ornamental plants in the world. It has high economic and ornamental value and occupies an important position in the flower industry. Chrysanthemums are susceptible to viruses during transplanting, cutting, and planting. Chrysanthemum virus B (Chrysanthemum virus B, CVB) causes diseased chrysanthemums to show mild mosaic disease on chrysanthemum leaves, while for susceptible varieties, it can form obvious mosaic symptoms or necrotic spots, and severe brown spots will occur. CVB commonly occu...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/94
Inventor 陈发棣尤燕平陈素梅蒋甲福房伟民管志勇
Owner NANJING AGRICULTURAL UNIVERSITY
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