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NASBA primer, kit and method for detecting peach latent mosaic viroid

A virus-like and kit technology, applied in the field of plant pathogen detection, achieves the effects of high sensitivity, low mismatch rate and high accuracy

Inactive Publication Date: 2015-04-08
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004]Because the cell wall is rich in a large amount of polysaccharides and phenolic substances, the extraction of plant virus RNA has problems such as poor stability, low repeatability, and low efficiency. The self-degradation phenomenon of RNA itself during the process, the content of viral RNA is often low, which puts forward higher requirements for the sensitivity and stability of the detection method
At the same time, due to the inhibitory effect of polysaccharides and other substances on Taq polymerase, the application of RT-PCR technology in the detection of plant virus RNA with high polyphenol content such as grapes, strawberries, and grains is limited.

Method used

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  • NASBA primer, kit and method for detecting peach latent mosaic viroid
  • NASBA primer, kit and method for detecting peach latent mosaic viroid
  • NASBA primer, kit and method for detecting peach latent mosaic viroid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] 1 material

[0055] 1.1 Viruses

[0056] The peach latent mosaic virus was provided by the Chinese Academy of Inspection and Quarantine, and the peach latent mosaic virus ( Peach latent mosaic viroid , PLMVd), hop dwarf viroid ( Hop stunt vroid , HSVd), apple hydrangea virus ( Apple scar skid viroid , ASSVd), pear blister ulcer viroid ( Pear Blister Canker Viroid , PBCVd), apple stem pox virus ( Apple stem pitting virus , ASPV) are preserved by our laboratory.

[0057] 1.2 Reagents

[0058] Reverse transcription polymerase AMV, RNaseH, RNase inhibitor, T7 RNA polymerase, dNTP, ssRNA marker, and rNTP were all purchased from NEW ENGLAND BIOLAB; PCR amplification reagents, DNA markers, etc. were purchased from Beijing Tiangen Company;

[0059] 1.3 Primers

[0060] According to the full-length gene sequence of peach latent mosaic virus in the NCBI nucleic acid sequence database (accession number AY685181.1), the height of peach latent mosaic virus was analyzed b...

Embodiment 2

[0072] Embodiment 2 specificity experiment

[0073] 1. Extract the RNA of peach latent mosaic virus, hop dwarf virus, pear blister canker virus, peach latent mosaic virus and apple stempox virus, and use NASBA method for detection.

[0074] 2. Extraction of viral RNA

[0075] Take 0.1 g sample, add liquid nitrogen and grind it into powder, transfer the ground material into a 1.5 mL centrifuge tube quickly, add 1 mL Trizol Reagent, mix by inversion, 2°C~8°C, 12000 g, centrifuge for 10 min. Take the supernatant, keep it at 15°C~30°C for 5min; add 0.2 mL of chloroform, shake vigorously by hand (do not vortex) for 15s. 15°C~30°C, stand for 2min~3min; 2°C~8°C, 12000 g, centrifuge for 15min. Carefully pipette 600 μL of the upper aqueous phase without disturbing the middle and lower phases. Add 500 μL of isopropanol to mix the supernatant, and place it at 15°C~30°C for 10min. 2°C~8°C, 12000g, centrifuge for 10min. Remove the supernatant, add 1 mL of 75% ethanol to the precipit...

Embodiment 3

[0084] Embodiment 3 Sensitivity experiment

[0085]1. Use DEPC water to make a 10-fold gradient dilution of the peach latent mosaic virus RNA template solution downwards, in order of 1×10 -1 , 1×10 -2 , 1×10 -3 , 1×10 -4 , 1×10 -5 , 1×10 -6 , 1×10 -7 ng / μL, each 2 μL was used as a template for NASBA amplification reaction. .

[0086] 2. Extraction of viral RNA

[0087] Take 0.1 g sample, add liquid nitrogen and grind it into powder, transfer the ground material into a 1.5 mL centrifuge tube quickly, add 1 mL Trizol Reagent, mix by inversion, 2°C~8°C, 12000 g, centrifuge for 10 min. Take the supernatant, keep it at 15°C~30°C for 5min; add 0.2 mL of chloroform, shake vigorously by hand (do not vortex) for 15s. 15°C~30°C, stand for 2min~3min; 2°C~8°C, 12000 g, centrifuge for 15min. Carefully pipette 600 μL of the upper aqueous phase without disturbing the middle and lower phases. Add 500 μL of isopropanol to mix the supernatant, and place it at 15°C~30°C for 10min. ...

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Abstract

The invention discloses an NASBA primer, a kit and a method for detecting peach latent mosaic viroid. The sequences of an upstream primer and a downstream primer are respectively SEQ ID No. 1-2. An NASBA amplification kit comprises the following components: (1) NASBA amplification reaction liquid A; (2) NASBA amplification reaction liquid B. The detection method is as follows: 1) extracting sample RNA; 2) carrying out PLMVd NASBA amplification; and 3) carrying out electrophoresis detection. The NASBA primer, kit and method disclosed by the invention are suitable for quick detection and verification of peach latent mosaic viroid and can be widely used for monitoring epidemic situations in agricultural production and environment and monitoring and detecting the kind of viruses in import and export trades, the operation is very simple and convenient, the necessary sample amount is small and the requirements on the quality of template RNA are quite low.

Description

Technical field: [0001] The invention relates to a NASBA primer, a kit and a method for detecting peach latent mosaic virus, belonging to the technical field of plant pathogen detection. Background technique: [0002] Peach latent mosaic virus ( Peach latent mosaic viroid , PLMVd) belongs to the Avsunviroidae family, usually containing about 338 nucleotides, without a central conserved region. It occurs in peach cultivation areas all over the world, and mainly infects stone fruit trees such as peaches, pears, plums, and apricots: PLMVd is a latent infection on many peach tree (Prunus persica) varieties, and the incubation period can reach 5 ~7 years, usually no obvious leaf symptoms, but the growth of poisonous peach trees is slow, the tree vigor declines, and the resistance decreases. After the infected variety is infected, white or yellow creamy mosaic leaves (Peach calico) or printed lesions, chlorotic spots (Peach blotch) are formed on the leaves, or mosaic symptoms...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6865C12Q1/701C12Q2531/143
Inventor 吴兴海张成标魏晓棠张云霞宋涛赵丽青王简尼秀媚
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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