Cross primer amplification primer group for detecting bean pod mottle virus, kit and application

A cross-primer amplification and mottled virus technology, applied in the biological field, can solve the problems of difficulty in realizing asymptomatic plant virus detection, difficult to meet the rapid detection of port quarantine, inability to identify virus types, etc., and achieve intuitive and accurate reaction results and easy reaction results. , the effect of rapid interpretation

Inactive Publication Date: 2021-04-30
CHINA JILIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The detection limit of the ELISA method is low, and it is difficult to detect a small amount of viruses in asymptomatic plants, and the ELISA method is often unable to identify the virus species, with poor specificity
The PCR detection method is widely used in the field of plant virus detection, but it requires complex thermal cycle instruments, has defects such as long reaction time, and is difficult to meet the needs of rapid detection of port quarantine

Method used

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  • Cross primer amplification primer group for detecting bean pod mottle virus, kit and application
  • Cross primer amplification primer group for detecting bean pod mottle virus, kit and application
  • Cross primer amplification primer group for detecting bean pod mottle virus, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1: Design and synthesis of cross-primer constant temperature amplification primers

[0052] One: Sequence acquisition

[0053] 1. Extraction of RNA

[0054] Take 50 mg each of tobacco leaves infected with BPMV, SBMV, ToRSV, ArMV and TRSV viruses, and extract the total RNA of the BPMV virus sample and negative control with reference to the operating instructions of the plant total RNA extraction kit.

[0055] 2. cDNA synthesis

[0056] Take 3 μl of each of the five kinds of virus total RNA extracted above, and use the two-step method kit (primescript TM II 1ststrand cdna synthesis kit, 6210A) reverse transcribed the cDNA of 5 kinds of viruses.

[0057] 2. Design, synthesis and screening of CP gene primers

[0058] Download the genome sequence of bean pod mottle virus from GenBank (GenBank: GQ996950), and use Geneious Primer to design multiple groups of bean pod mottle virus based on the principle of primer design with 40% to 50% GC content and 50% to 60% TM v...

Embodiment 2

[0069] Example 2: Application of cross-primed constant temperature amplification primers in the detection of bean pod mottle virus

[0070] 1. Primer screening

[0071] Using the total RNA of bean pod mottle virus as a template, real-time fluorescent cross primers were used for constant temperature amplification to screen and detect five primer sets for bean pod mottle virus. The results are shown in Figure (1): through real-time fluorescent cross primer amplification detection, determine two groups of primer sets S1 and S2 (table 1) for detecting the cross primer amplification of bean pod mottle virus, primer set S1 includes peripheral replacement Primers (BPBF and BPBR), two amplification primers (BPDR and BPMBR) and one cross primer (BPCPF); primer set S2 includes peripheral displacement primers (BPBF1 and BPMBR1), two amplification primers (BPDR1 and BPMDR1) and 1 cross primer (BPCPF1).

[0072] Table 1 The primer set of cross primer amplification reaction of vegetable pod...

Embodiment 3

[0103] Example 3 Cross-primer amplification detection system Blind sample detection BPMV blind sample detection

[0104] Seven blind samples of American soybeans intercepted and provided by Guangzhou Customs, China, and one healthy soybean purchased from the local market were taken to extract the total RNA of blind samples of American soybeans and normal soybeans, respectively. The total RNA of the American soybean blind sample was used as a template, the total RNA of healthy soybeans was used as a negative control, and the total RNA of the identified BPMV virus was used as a positive control. The two detection methods in Example 2 were used for detection.

[0105] 2. Real-time fluorescent cross-primer amplification detection blind sample

[0106] The results of real-time fluorescent cross-primer amplification detection of BPMV blind samples are shown in Figure (7): real-time amplification curves appearing with the total RNA of American soybean blind sample No. 1-7 as template...

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Abstract

The invention discloses a cross primer amplification primer group for detecting a bean pod mottle virus, a kit and the application. Aiming at the problems existing in the detection of the bean pod mottle virus, the invention relates to and screens a high-specificity cross primer amplification primer group for detecting the bean pod mottle virus, and further relates to a kit and a detection method. The kit and the method provided by the invention have good specificity, and have negative reaction to southern bean mosaic virus, tomato ringspot virus, arabis mosaic virus, tobacco ringspot virus and the like; the sensitivity is high, 5*10<-3>ng / mu l of the rapeseed pod mottle virus RNA template can be detected by a real-time fluorescence detection method at least, and 5*10 <-2> ng / mu l of the rapeseed pod mottle virus RNA template can be detected by a nucleic acid test strip detection method at least. The kit is very suitable for field detection of medical treatment and public health, food safety and import and export quarantine, and is easy for large-scale popularization and application.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a rapid detection kit for cross-primer amplification of soybean pod mottle virus, a soybean seed-borne quarantine virus, and its application [0002] technical background [0003] Bean pod mottle virus (BPMV) is a member of Comoviridae (Comoviridae, Comovirus). In 1948, bean pod mottle virus was first reported to be found on bean, and then spread to Soybean producing areas such as Canada, Brazil, Peru, Ecuador and Iran. The host of bean pod mottle virus is limited to legumes, and soybean and bean are its perennial hosts. Bean pod mottle virus will show chlorosis, mottle, and necrosis on soybeans Symptoms such as soybean damage the quality of soybeans and cause loss of soybean yield. Bean pod mottle virus is an important virus that harms soybeans and other leguminous plants. It is listed as a phytosanitary virus imported into China by my country. my country is the world's largest importer ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2537/1376C12Q2531/119C12Q2563/107C12Q2521/107
Inventor 杨倩倩王道俞晓平张蓬军孙凯刘光富尹传林
Owner CHINA JILIANG UNIV
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