Potato virus detection primers and potato virus detection method

A potato virus and detection primer technology, applied in the field of biology, can solve problems such as multiple RT-PCR reaction systems, and achieve the effects of low cost, high detection efficiency and high sensitivity

Inactive Publication Date: 2014-12-03
CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no reports of multiplex RT-PCR reactions capable of detecting PVX, PVM, PVY, PVA, and PVS

Method used

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  • Potato virus detection primers and potato virus detection method
  • Potato virus detection primers and potato virus detection method
  • Potato virus detection primers and potato virus detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Potato virus detection method of the present invention, is according to the coat protein sequence of five kinds of viruses of potato virus X (PVX), potato virus M (PVM), potato virus Y (PVY), potato virus A (PVA) and potato virus S (PVS) Conserved regions were designed and synthesized above-mentioned primer pair of potato virus X, primer pair of potato M virus, primer pair of potato virus Y, primer pair of potato virus A and primer pair of potato virus S; then establish RT-PCR detection system, and use the potato virus Primer pair for Potato virus X in potato leaves, Primer pair for Potato virus M in potato leaves, Primer pair for Potato virus Y in potato leaves, Primer pair for Potato virus Y in potato leaves, Primer pair for Potato virus A in potato leaves Potato virus A and potato S virus primers were used to carry out RT-PCR reaction on the potato S virus in potato leaves, and finally the reaction products were analyzed by 1% agarose gel electrophoresis and cloned an...

Embodiment 2

[0042] The potato total RNA that chooses co-infection 5 kinds of viruses (PVX, PVM, PVY, PVA, PVS) is that embodiment 1 step (1) extracts the reverse transcription product of gained RNA as test template, according to 10 times gradient dilution method successively test Template dilution, the dilution factor is 10 0 、10 -1 、10 -2 、10 -3 、10 -4 、10 -5 and 10 -6 times, then carry out multiple RT-PCR amplification reaction (reaction system and reaction procedure are the same as embodiment 1), then carry out 1% agarose gel electrophoresis experiment, experimental result is as follows figure 2 shown by figure 2 It can be seen that 5 kinds of potato viruses (PVX, PVM, PVY, PVA, PVS) were diluted by 10 at the test template concentration. -4 Specific bands can still be amplified even when the concentration of the test template is diluted by 10 -4 The amplification products of the 5 kinds of potato viruses can still be detected at multiple times, which shows that the detection ...

Embodiment 3

[0044] The PCR amplified products obtained in the multiplex PCR reaction system in step (4) of Example 1 were recovered by tapping rubber, and then respectively connected with pMD18-T vector in vitro, then picked positive clones, and finally extracted plasmids for sequencing to check PVX, Amplification results of five viruses, PVM, PVY, PVA and PVS.

[0045]The sequencing results are: the amplification products of PVX, PVM, PVY, PVA and PVS are sequences composed of 138, 213, 369, 468 and 566 nucleotides respectively, wherein the PVX amplification product sequence is as SEQ ID NO: 11, the PVM amplification product sequence is shown in SEQ ID NO: 12, the PVY amplification product sequence is shown in SEQ ID NO: 13, and the PVA amplification product sequence is shown in SEQ ID NO: 14 The sequence of the PVS amplification product is shown in SEQ ID NO:15. The results of homology analysis by NCBI showed that the obtained amplified product sequence SEQ ID NO: 11 had a homology rat...

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Abstract

The invention respectively designs five pairs of primers for detecting potato viruses according to conserved regions of coat protein sequences of five types of viruses comprising a potato virus X, a potato virus M, a potato virus Y, a potato virus A and a potato virus S, and simultaneously discloses a method for detecting the potato viruses by utilizing the five pairs of primers. Specifically, the five types of viruses in potato leaves are detected in an amplification manner by virtue of the five pairs of specific primers via a multiplex RT-PCR method, and the verification is carried out by cloning, sequencing and comparing amplified products, so that the detection method capable of simultaneously and reliably detecting the five types of potato viruses via an one-step reaction is established. The method has the characteristics of high detection efficiency, high sensitivity and low cost.

Description

【Technical field】 [0001] The invention relates to a primer and a method for detecting a plant virus in the field of biology, in particular to a primer and a method for detecting a potato virus. 【Background technique】 [0002] Potato (Solanum tuberosum) is the main food crop in the world, and my country's planting area ranks first in the world (FAO2006). Potatoes are asexually propagated crops and are susceptible to virus infection. Once they are infected by viruses, their yield and quality will be severely reduced, generally by 30%-50%, and even more than 90% in severe cases. According to reports, there are more than 40 kinds of viruses and 2 virus-like viruses that can infect potatoes, of which 9 kinds of viruses and 1 virus-like virus have the most serious impact on potatoes. Potato virus diseases have occurred in various potato producing areas in my country, among which the common potato viruses are mainly potato virus X (Potato virus X, PVX), potato M virus (Potato viru...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/701C12Q1/686C12Q1/70C12Q2600/16C12Q2531/113C12Q2537/143
Inventor 李华伟汤浩罗文彬纪荣昌许泳清邱思鑫刘中华邱永祥
Owner CROP RES INST OF FUJIAN ACAD OF AGRI SCI
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