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Kit for detecting bean pod mottle virus and detection process thereof

A detection kit and mottle virus technology, applied in the biological field, can solve problems such as no detection kits, and achieve the effects of stable results, economical use and strong specificity

Inactive Publication Date: 2010-06-09
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no report on the application of TC-RT-PCR (test tube capture RT-PCR) technology to detect BPMV so far, and there is no TC-RT-PCR detection kit specially used for detecting BPMV.

Method used

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  • Kit for detecting bean pod mottle virus and detection process thereof
  • Kit for detecting bean pod mottle virus and detection process thereof
  • Kit for detecting bean pod mottle virus and detection process thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: detection kit of bean pod mottle virus

[0039] A detection kit for bean pod mottle virus is characterized in that it consists of:

[0040] (1)1 # Solution, containing virus extraction reagent: PBS buffer solution, concentration 1×, pH7.4;

[0041] (2)2 # solution, containing the upstream primer of bean pod mottle virus: 5'-GGCAATTTTAGAGCAAGTGTTG-3', the concentration is 10 μmol / L;

[0042] (3)3 # solution, containing the downstream primer of bean pod mottle virus: 5'-GCATTTGGCATATTCATGAGAAT-3', the concentration is 10 μmol / L;

[0043] (4)4 # Solution, containing RT Buffer, concentration 5×;

[0044] (5)5 # Solution, containing RNase inhibitor, concentration 40U / μL;

[0045] (6)6 # Solution, containing reverse transcriptase, concentration 200U / μL;

[0046] (7)7 # Solution, containing dNTPs, concentration 10mmol / L;

[0047] (8)8 # Solution, containing PCR Buffer, concentration 10×;

[0048] (9)9 # solution, containing Mgcl 2 , concentration ...

Embodiment 2

[0055] Example 2 Detection method for bean pod mottle virus

[0056] The detection kit of above-mentioned bean pod mottle virus is used for detecting the detection method of bean pod mottle virus (BPMV), comprising:

[0057] 1) Weigh 0.05g of soybean diseased leaves, add virus extraction reagent 1 according to the weight-to-volume ratio of the sample to the extraction buffer as 1:10 (g / ml) # After fully grinding, centrifuge at 10,000 g for 10 min at 4°C, and take the supernatant as the virus extract;

[0058] 2) Take 50 μL of diseased soybean leaf supernatant (virus extract) to coat the PCR tube, and use 50 μL of 11 # Solution, 50μL 12 # The solution was used as positive control and negative control to coat PCR tubes respectively. Incubate each tube at 37°C for 3h, 13 # Liquid wash tube 3 times and 14 # The reverse transcription reaction was performed after the tube was washed twice; the reverse transcription reaction procedure was: add 1 μL 3 # solution and 9 μL 14 # A...

Embodiment 3

[0062] Example 3 Detection of imported diseased soybean seeds intercepted by ports in the United States

[0063] The difference from Example 2 is that the detection object is soybean seeds. After soaking the soybean seeds in water for 2 hours, peel off the seed coat as a sample, and then add the virus extraction reagent 1 according to the weight-to-volume ratio of the sample to the extraction buffer at 1:10 (g / ml). # solution, after fully grinding, 10000g centrifugal 10min under 4 ℃, get supernatant; All the other steps are with embodiment 2, the electrophoresis detection result that contains bean pod mottle virus (BPMV) sample is to appear bright DNA band at 542bp place ( figure 2 ), otherwise None.

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Abstract

The invention relates to a detection reagent kit for bean pod mottle viruses and a detection method thereof, which are specially used for the detection of the bean pod mottle viruses. The detection reagent kit comprises a primer, virus extraction reagent, RT Buffer, an RNase inhibiting factor, inverse transcriptase, dNTPs, PCR Buffer, Mgcl2, Taq DNA polymerase, positive control, negative control,PBST buffer solution and RNase-free ddH2O. The method designs a specific primer according to conservative areas of bean pod mottle virus genes, utilizes a TC-RT-PCR technology to detect the bean pod mottle viruses, does not need fussy RNA extraction steps, directly makes reverse transcription and PCR to virus extract solution, and has simple and convenient operation, steady result, strong specificity, high sensitivity and relatively lower detection cost, so the method has higher utility value.

Description

technical field [0001] The invention relates to a detection kit for vegetable pod mottle virus and a detection method thereof, which belongs to the field of biotechnology and is specially used for the rapid detection of soybean pod mottle virus by port inspection and quarantine and agricultural plant inspection departments and the like. Background technique [0002] Bean Pod Mottle Virus (BPMV) belongs to the family Comoviridae and the genus Comovirus. The genome of the virus is a binary linear positive-sense single-stranded RNA. The length of RNA-1 is about 6kb, and that of RNA-2 is long. About 3.6kb. BPMV was first reported in 1948 on the common bean Tendergreen variety in Charleston, USA, and is now mainly distributed in North America, in Arkansas, North Carolina, Virginia, Kentucky, Mississippi, Louisiana, Ireland Soybean growing regions such as Iowa, Illinois and Indiana are widely distributed. In addition to the United States, there have been reports of BPMV on soybe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N27/447
Inventor 沈建国郭琼霞黄可辉虞赟王念武
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU