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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)

An RT-PCR and detection method technology, applied in the field of virus detection in molecular biology, can solve the problems of affecting judgment results, reducing detection costs, and difficulty, and achieving the effects of strong detection specificity, simplified operation, and cost saving.

Inactive Publication Date: 2013-05-08
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
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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 proteins
[0005] The Chinese patents "A method for detecting chrysanthemum B virus" (application number: CN200810240415) and "a method for detecting chrysanthemum chlorotic mottle virus" (application number: CN200810240414) were applied by Beijing Academy of Agriculture and Forestry Sciences in 2008 ) disclosed a method for detecting CVB and CChMVd using molecular biology methods, but the detection using LAMP is prone to contamination and false positive results, and the requirements for primer design are very high
[0006] At present, there is no report on the technology of simultaneous detection of pathogens in chrysanthemum under the combined infection of CVB and CChMVd. If pathogens can be detected at the same time, the detection efficiency can be improved and the detection cost can be significantly reduced.

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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 detection method for chrysanthemum CVB and CChMVd viruses, which belongs to the field of virus detection in molecular biology. The method comprises: (1) sampling and extracting total RNA from chrysanthemum plants; Two pairs of specific primers were designed for CVB and CChMVd viruses: CVB-F, CVB-R; CChMVd-F, CChMVd-R; (3) two cDNAs were obtained by reverse transcription using random hexamers as primers; (4) using The cDNA was amplified by RT-PCR technology, and the amplified product was subjected to agarose gel electrophoresis to obtain a 665bp specific fragment indicating infection of CVB, and obtaining a 206bp specific fragment indicating infection of CChMVd. The detection method provided by the invention can simultaneously detect diseased strains co-infected by CVB and CChMVd, has strong detection specificity, high sensitivity, simple procedure and cost saving.

Description

Technical field [0001] The invention involves the field of virus testing of molecular biology, which specifically says a dual RT-PCR detection method that involves a chrysanthemum CVB and CCHMVD virus. Background technique [0002] chrysanthemum( DENDRANTHEMA MORIFOLIUM Tzvel. ( Chrysanthemum morifolium Ramat.) Is a perennial root herbaceous plant in chrysanthemums. It is one of the top ten traditional flowers in my country. One of the earliest ornamental plants in the world has high economic and ornamental value and occupies an important position in the flower industry.Chrysanthemums are infected with viruses in transplanting, cutting, and planting.Chrysanthemum Virus B (CVB), which makes the diseased chrysanthemum on the chrysanthemum leaves as mild flower leaf. For susceptible varieties, it can form obvious flower and lobe symptoms or necrotic spots, which will cause brown dead spots.CVB generally occurs on the chrysanthemum, with an incidence of 60 ~ 65%.Chrysanthemum Chlorit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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