Method and a special kit for detecting bean pod mottle virus

A kit and detection location technology, applied in the field of detection of bean pod mottle virus, can solve the problems of limited widespread use, high detection cost, expensive detection reagents, etc., and achieve the effects of wide practicability, accurate and reliable detection results, and simple operation.

Inactive Publication Date: 2010-10-20
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

After ordinary PCR amplification, electrophoresis and ethidium bromide staining are required to determine the presence or absence of PCR products and the size of the fragments. Experimenters have to be exposed to carcinogenic ethidium bromide, and improper handling may easily cause environmental pollution
[0004] RTF-PCR and biochip detection methods have high requirements for instruments and software, expensive detection reagents, and high detection costs, which are difficult for ordinary laboratories to bear, and objectively limit the widespread use of this method

Method used

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  • Method and a special kit for detecting bean pod mottle virus
  • Method and a special kit for detecting bean pod mottle virus
  • Method and a special kit for detecting bean pod mottle virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1, composition and preparation of kit

[0042] Mouse anti-fluorescein monoclonal antibody (Antti-FI monoclonal antibody) was purchased from Sigma-Aldrich Company, the product catalog number is F5636; mouse anti-digoxigenin monoclonal antibody (Anti-DI monoclonal antibody) was purchased from Sigma-Aldrich Company, the catalog number is D8156; the goat anti-rabbit antibody was purchased from Beijing Xinjingke Biotechnology Co., Ltd., the catalog number is A30608;

[0043] Rabbit anti-biotin polyclonal antibody was purchased from Beijing Xinjingke Biotechnology Co., Ltd., the product catalog number is A30622;

[0044] 1. The composition of the kit:

[0045] (1) Colloidal gold chromatography test paper;

[0046] Colloidal gold chromatography test paper is composed of sample absorbent pad, reaction membrane and water absorbent pad connected in sequence; the sample absorbent pad overlaps with the reaction membrane by 2mm, and the reaction membrane and water absor...

Embodiment 2

[0062] Embodiment 2, detect whether bean pod mottle virus (Bean pod mottle virus, BPMV) is contained in soybean diseased leaf

[0063] The schematic diagram of the detection principle is as figure 1 shown.

[0064] 1. Amplify the specific conserved fragment of the virus from the sample to be tested

[0065] BI: biotin; FI: fluorescein; DI: digoxin.

[0066] Common primers: BPMVf: 5’-ATA GTT CCA TTA GAG GGC GTG-3’; BPMVr: 5’-AGTGGA CCA TGT GAG AAA C-3’.

[0067] Modified primer pair 1: BPMVf-BI: 5’-BI-ATA GTT CCA TTA GAG GGC GTG-3’; BPMVr-FI: 5’-FI-AGT GGA CCA TGT GAG AAA C-3’.

[0068] Modified primer pair 2: BPMVf-BI: 5’-BI-ATA GTT CCA TTA GAG GGC GTG-3’; BPMVr-DI: 5’-DI-AGT GGA CCA TGT GAG AAA C-3’.

[0069] Bean pod mottle virus (Bean pod mottle virus) (purchased from ATCC, No. PV-934) was rubbed and inoculated onto soybean leaves;

[0070] Extraction of total RNA from diseased soybean leaves

[0071] Take 0.1g of fresh diseased leaves, add liquid nitrogen to grind fi...

Embodiment 3

[0103] Embodiment 3, the detection result of the chromatographic reaction membrane of different flow rates

[0104] Using Whatman's nitrocellulose membranes (SP, FP, RP membranes) with different flow rates, for the detection of PCR amplification products with BI and FI modified primers, the SP membrane is the slowest (capillary rising speed is 160-220s / 4cm), The FP membrane (with a capillary rise speed of 110-150s / 4cm) is in the middle, and the RP membrane (with a capillary rise speed of 85-115s / 4cm) has the fastest flow rate.

[0105] SP film test results (see Figure 4 ); FP film detection results (see Figure 5 ); RP film detection results (see Image 6 ). In the figure, the PCR products modified by adding FI and BI in sequence from left to right: 4 μL, 2 μL, 1 μL. The upper end is point C; the lower end is point T.

[0106] Using these three kinds of chromatographic membranes with different flow rates, when the amount of PCR product is 4 μL and 2 μL, it can be detecte...

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Abstract

The invention discloses a method and a special kit for detecting a bean pod mottle virus. The kit comprises colloidal gold chromatography test paper, a colloidal gold-marking antibody and a specific primer for amplifying a substance to be tested. In the invention, the high sensitivity of nucleic acid amplification and the advantages of the rapidness, the simplicity and the convenience and the visibility of the colloidal gold chromatography method are combined, the PCR (polymerase chain reaction) specific primer is modified, and then the antibody of a specific modifier is marked by colloidal gold to finally realize the specificity detection of an amplified target nucleic acid fragment. The method has wide practicability. Proved by an experimental result, the method has accurate and reliable detection result. The method saves time and labor and has simple operation.

Description

technical field [0001] The invention relates to a method and a special kit for detecting the bean pod mottle virus. Background technique [0002] Bean pod mottle virus (BPMV) is an imported plant quarantine pest in my country, distributed in the United States, Canada, Brazil, Peru, Ecuador, Iran and other countries. The virus primarily affects plants such as soybeans, kidney beans and cowpeas, causing yield loss and reduced quality. The detection of the virus mainly includes double-antibody sandwich ELISA, colloidal gold immunochromatography (GICA), RT-PCR, real-time fluorescent PCR (RTF-PCR) and biochip and other methods. The basic principle of the first two methods is the specific reaction of antigen and antibody, and the antibody of plant virus is used to detect the antigen of plant virus coat protein. When the virus concentration in the plant is low enough to exceed the detection sensitivity of serological methods, or when there is no antibody to a certain virus, or wh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/558
Inventor 魏梅生张永江李桂芬
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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