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Nasba amplification primers, kits and detection methods for detecting hop dwarf viroid

A technology for dwarfing viroids and hops, which is applied in the field of plant pathogen detection, can solve the problems of low viral RNA content, limited application and low repeatability, and achieves the effects of high sensitivity, low mismatch rate and low repeatability

Inactive Publication Date: 2016-08-24
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 amplification primers, kits and detection methods for detecting hop dwarf viroid
  • Nasba amplification primers, kits and detection methods for detecting hop dwarf viroid
  • Nasba amplification primers, kits and detection methods for detecting hop dwarf viroid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] 1 material

[0050] 1.1 Viruses

[0051] Hop dwarf viroids include imported French grape rootstock seedling isolates, imported Italian grape seedling isolates, Xinjiang grape seedling and hop isolates, apple scar skid viroid (ASSVd), pear blister canker Pear Blister Canker Viroid (PBCVd), Peach latentmosaic viroid (PLMVd), Apple stem pitting virus (ASPV), Grapevine yellow speckle viroid-2 (Grapevine yellow speckle viroid, GYSVd-2) are kept by the applicant's laboratory.

[0052] 1.2 Reagents

[0053] 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;

[0054] 1.3 Primers

[0055] According to the full-length sequence of the hop dwarf viroid gene in the NCBI nucleic acid sequence database (accession number HM357802.1), the design NASBA reaction primers (NA-P1, NA-P2) with T7 prom...

Embodiment 2

[0067] Embodiment 2: specificity experiment

[0068] 1. Extract the RNA of hop dwarf virus, apple rust virus, pear blister canker virus, peach latent mosaic virus, apple stem pox virus, and grape macular virus-2, and use NASBA method for detection.

[0069] 2. Extraction of viral RNA

[0070] 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 m...

Embodiment 3

[0079] Embodiment 3 Sensitivity experiment

[0080] 1. Use DEPC water to make a 10-fold gradient dilution of the hop dwarf viroid virus RNA template solution downwards, in order of 1×10 0 , 1×10 -1 , 1×10 -2 , 1×10-3 , 1×10 -4 , 1×10 -5 , 1×10 -6 , 1×10 -7 , 1×10 -8 μg / μL, each 2 μL was used as a template for NASBA amplification reaction.

[0081] 2. Extraction of viral RNA

[0082] 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 pl...

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Abstract

The invention discloses an NASBA (nucleic acid sequence based amplification) primer, a kit and a detection method for detecting hop stunt viroids. The sequences of the upstream primer and the downstream primer of the NASBA primer are SEQ ID NO: 1-2 respectively, and the kit comprises NASBA reaction liquid A and NASBA reaction liquid B. The detection method includes the steps: 1) extracting RNA (ribonucleic acid) of samples; 2) performing NASBA for the hop stunt viroids; 3) detecting electrophoreses. The NASBA primer is applicable to rapidly detecting and confirming the hop stunt viroids, can be widely applied to disease surveillance and control in agricultural production and environments and monitoring and detecting of the viroids in import and export trade and is simple and convenient to operate, the number of the needed samples is small, and the quality requirement for template RNA is low.

Description

Technical field: [0001] The invention relates to a NASBA amplification primer for detecting hop dwarf viroid, a kit and a detection method, and belongs to the technical field of detection of plant pathogens. Background technique: [0002] Viroids are the smallest pathogenic factors known to cause plant diseases so far. They are circular RNA molecules with a full-length genome consisting of 246-399 nucleotides, which can cause severe diseases in many commercial crops. According to their sequence and structure homology and replication characteristics, they are divided into two groups, namely potato spindle tuber viroid group (PSTVd) and avocado sun spot viroid group (ASBVd). Hop stunt vroid (Hop stunt vroid) belongs to the genus Hop stunt vroid of Potato spindle tuber viroid family, usually contains about 307 nucleotides. In 1970, Yamamoto et al. in Japan first discovered hop dwarf viroids on dwarf hops. In 1985, Sano of Japan discovered HSVd on grapes for the first time. Af...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6865C12Q1/701C12Q2531/143
Inventor 吴兴海张成标魏晓棠甘琴华张京宣邵秀玲历艳尼秀媚
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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