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Nasba primers, kits and methods for detecting apple rust viroids

The technology of a rust-like virus and a kit is applied in the field of plant pathogen detection, which can solve the problems of low viral RNA content, low repeatability, poor stability, etc., and achieve the effects of high sensitivity, low repeatability and fewer cycles.

Inactive Publication Date: 2016-08-17
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 primers, kits and methods for detecting apple rust viroids
  • Nasba primers, kits and methods for detecting apple rust viroids
  • Nasba primers, kits and methods for detecting apple rust viroids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] 1 material

[0051] 1.1 Viruses

[0052] Apple rust virus was provided by the Chinese Academy of Inspection and Quarantine, hop stunt vroid (HSVd), pear blister canker viroid (PBCVd), peach latent mosaic virus (Peach latent mosaic viroid, PLMVd), apple stem pitting virus (Apple stem pitting virus, ASPV) are preserved by our laboratory.

[0053] 1.2 Reagents

[0054] Reverse transcription polymerase AMV, RNaseH, RNase inhibitor, T7 RNA polymerase, dNTP, ssRNAmarker, rNTP were all purchased from NEW ENGLAND BIOLAB; PCR amplification reagents, DNA markers, etc. were purchased from Beijing Tiangen Company;

[0055] 1.3 Primers

[0056] According to the full-length sequence of the apple rust viroid gene (accession number EU825613.1) in the NCBI nucleic acid sequence database, the highly conserved region of the apple rust viroid was compared and analyzed under the premise of ensuring the amalgamation and versatility of the amplification, and the design NASBA reaction prim...

Embodiment 2

[0068] Embodiment 2 specificity experiment

[0069] 1. Extract the RNA of apple rust virus-like virus, hop dwarf virus-like virus, pear blister ulcer virus-like virus, peach latent mosaic virus and apple stempox virus, and use NASBA method to detect.

[0070] 2. Extraction of viral RNA

[0071] Take 0.1 g sample, grind it into powder with liquid nitrogen, 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 about 15s. 15°C~30°C, stand for 2min~3min; 2°C~8°C, 12000 g, centrifuge for 15min. Carefully pipette approximately 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...

Embodiment 3

[0080] Embodiment 3 Sensitivity experiment

[0081] 1. Use DEPC water to make a 10-fold gradient dilution of the apple rust virus-like 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 ng / μL, each 2 μL was used as a template for NASBA amplification reaction. .

[0082] 2. Extraction of viral RNA

[0083]Take 0.1 g sample, grind it into powder with liquid nitrogen, 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 about 15s. 15°C~30°C, stand for 2min~3min; 2°C~8°C, 12000 g, centrifuge for 15min. Carefully pipette approximately 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 a NASBA primer, a reagent kit, and a method for detecting apple scar skin viroid; the sequences of the upstream primer and the downstream primer are respectively SEQ ID NO: 1-2; the NASBA augmentation reagent kit comprises the constituents as follows: (1) NASBA augmentation reaction liquid A; (2) NASBA augmentation reaction liquid B; the detection method is as follows: 1) extracting sample RNA; 2) augmenting NASBA of ASSVd; 3) detecting with electrophoresis. The NASBA primer, the reagent kit, and the method for detecting apple scar skin viroid are applicable to quickly detecting and confirming apple scar skin viroid, can be widely applied for epidemic surveillance in agricultural production and environment as well as monitoring and detecting the viroid in import and export trade; the operation is very simple; the amount of the required sample is small; and the quality requirements to template RNA is lower.

Description

Technical field: [0001] The invention relates to a NASBA primer, a kit and a method for detecting apple rust viroids, and belongs to the technical field of plant pathogen detection. Background technique: [0002] Apple scar skid viroid (ASSVd) belongs to the genus Apscaviroid, and its granule consists of 330 nucleotides. The infection hosts of apple rust fruit viroids are apples and pears. The susceptible apples showed five dominant diseases due to the changes of varieties and environmental conditions: rusty fruit type, flower face type, rust fruit-flower face type, ring spot type and green spot type, and most of the susceptible pears showed recessive symptoms. , a few are deformed fruits. Due to the replication and accumulation of viroids in the nucleus of host plants, viroids can be detected in the leaves, stems, skins, rootstocks of susceptible hosts, as well as in the epidermis, pulp and seeds of fruits. Therefore, fruit trees infected with viroids carry the virus thr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6865C12Q1/701C12Q2531/143
Inventor 吴兴海张成标魏晓棠宋涛姜英辉邵秀玲历艳尼秀媚
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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