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Duplex RT-PCR method for synchronous detection of WDV (wheat dwarf virus) and WYSV (wheat yellow striate virus)

A simultaneous detection and virus technology, applied in the field of agricultural biology, can solve problems such as high requirements for antibody specificity and inability to complete identification

Inactive Publication Date: 2018-08-03
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, as far as WDV and the new virus WYSV are concerned, since the two viruses are transmitted by the leafhopper and have similar symptoms, biological identification cannot complete the identification; serology has the advantages of fast, simple, high-throughput, etc. However, there are higher requirements for the specificity of antibody preparation

Method used

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  • Duplex RT-PCR method for synchronous detection of WDV (wheat dwarf virus) and WYSV (wheat yellow striate virus)
  • Duplex RT-PCR method for synchronous detection of WDV (wheat dwarf virus) and WYSV (wheat yellow striate virus)
  • Duplex RT-PCR method for synchronous detection of WDV (wheat dwarf virus) and WYSV (wheat yellow striate virus)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1. The preparation of the medium leafhopper and WDV and WYSV toxin source samples

[0048] 1.1 The source and preservation of the medium leafhopper

[0049] The different sand leafhoppers are collected from ordinary fields in Hancheng, Shaanxi, and are reared on healthy wheat after detoxification and purification. The feeding conditions are: 22±1°C, 20000lx light incubator.

[0050] 1.2 Source and preservation of wheat dwarf virus

[0051] The isolates of wheat dwarf virus (WDV) and the virus-transmitting agent (Leafhopper japonica) were collected from the diseased fields in Hancheng, Shaanxi. After being identified as a wheat dwarf virus (WDV) isolate by PCR detection and CP gene cloning and sequencing, the source of the virus was transmitted and propagated by the leafhopper heterosandra on a susceptible wheat variety ('Yangmai 12'). The poisonous source is propagated all the year round in the light incubator under the condition of 22±1℃ and 20 000 lx.

...

Embodiment 2

[0055] Example 2. Identification of Wheat Yellow Streak Virus (WYSV)

[0056] 2.1 Biological identification

[0057] In order to detect the specificity of virus transmission by mediators, three species of non-toxic brown planthopper (Nilaparvata lugens), white-backed planthopper (Laodelphax striatellus) and white-backed planthopper (Sogatella furcifera Horváth) and wheat long tube aphid (Sitobion avenae) were selected. ), wheat aphid (Schizaphis gramienum) and cereal aphid (Rhopalosiphum padi) were used as experimental insects for virus transmission experiments. In addition, barley (variety name: Longpimai No. 3) and oats (variety name: Anhei oat) were selected for preliminary host range identification. The feeding and transmission steps of the above experiment are the same as those in Example 1.3. All the tested plants were placed in the light incubator to grow, and the symptoms of the disease were observed after 2-3 weeks. The results of biological experiments show that WY...

Embodiment 3

[0070] Example 3. Extraction of Leafhopper total RNA

[0071] The total RNA of the leafhopper samples to be tested was extracted by TRIzol (Invitrogen, USA) method. The specific extraction process is as follows: put a single fresh or frozen sample into a 1.5mL centrifuge tube, grind for a few seconds with a pestle with a wireless motor (Beijing Tiangen Biochemical Technology Co., Ltd.), and then quickly transfer it to liquid nitrogen for freezing , repeat until the leafhopper is fully ground to powder, then add 200 μL TRIzol, shake vigorously to mix, and let stand on ice for 5 minutes; add 40 μL chloroform, shake vigorously for 15 seconds, and let stand on ice for 5 minutes. Centrifuge at 12,000rpm at 4°C for 10min; transfer the supernatant to a new centrifuge tube, add isopropanol equal to the volume of the supernatant, mix gently by inversion, and let stand on ice for 10min; centrifuge at 12,000rpm at 4°C for 15min, remove Supernatant; add 500 μL pre-cooled 75% ethanol (pre...

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Abstract

The invention relates to a duplex RT-PCR method for synchronous detection of WDV (wheat dwarf virus) and WYSV (wheat yellow striate virus). Specific primers shown as SEQ ID No .1\2 for the WDV and specific primers shown as SEQ ID No .3\4 for the WYSV are adopted, RNA of a sample containing the two viruses is taken as a template, SEQ ID No .2\4 are taken as primers for reverse transcription, viruscDNA is obtained, the four primers are added to one PCR reaction system, 773bp and 332bp products are obtained respectively, and various parameters in the reaction process are optimized simultaneously. The duplex RT-PCR method established on the basis of the primers and used for the WDV and the WYSV is high in specificity and sensitivity, the two viruses in mediators can be detected and identifiedin one system synchronously, rapidly and accurately, good monitoring and early warning can be realized, and diseases are controlled before pandemic of virus diseases finally.

Description

technical field [0001] The invention belongs to the technical field of agricultural biology, and in particular relates to a new virus, Wheat yellow striate virus (WYSV), and the ability to carry infecting wheat in the same PCR system synchronously on the medium Leafhopper leafhopper. Wheat dwarf virus (WDV) and wheat yellow striate virus (WYSV) were amplified to achieve rapid differentiation and efficient detection. Background technique [0002] The different sand leafhopper (Psammotettix alienus Linnaeus) is an agricultural insect belonging to the genus Psammotettix (Psammotettix) ). It is widely distributed in Europe, northern Africa, North America and north-central Asia. It is also the dominant species of wheat field leafhopper in the arid and semi-arid regions of Northwest and North China. The feeding hosts of the leafhopper are wheat, barley, oats, rye, highland barley, broomcorn millet, millet, naked oats, sorghum, corn, rice and other gramineous plants. Adults and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/94
CPCC12Q1/686C12Q1/70C12Q2537/143C12Q2521/107
Inventor 刘艳杜真真王锡锋
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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