Method for detecting sweet potato leaf curl viruses and special primer set thereof

A technology of sweet potato leafroll virus and primer set, which is applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., to achieve the effect of shortening the reaction time, overcoming the long detection cycle, and easy operation

Inactive Publication Date: 2015-12-09
BEIJING PLANT PROTECTION STATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no report on the detection of sweet potato leafroll virus using this technique

Method used

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  • Method for detecting sweet potato leaf curl viruses and special primer set thereof
  • Method for detecting sweet potato leaf curl viruses and special primer set thereof
  • Method for detecting sweet potato leaf curl viruses and special primer set thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1, design and preparation of primers

[0037] Based on a large number of sequence comparisons, primer design, primer screening and effect verification, a set of primers with the best effect for loop-mediated isothermal amplification was obtained. The sequences of each primer in the primer set are as follows:

[0038] SPLCV-F3 (outside upstream primer, sequence 1 of the sequence listing): 5'-GGCTGAACTTCGAGACAG-3';

[0039] SPLCV-B3 (outside downstream primer, sequence 2 of the sequence listing): 5'-AATCCGAGACACAGACAAAC-3';

[0040] SPLCV-FIP (inside upstream primer, sequence 3 of the sequence listing):

[0041]5'-CTCTTCATCCGGACGCCTCCTACACTGGGAATGCTGTC-3';

[0042] SPLCV-BIP (inside downstream primer, sequence 4 of the sequence listing):

[0043] 5'-GGTGACCGCATCCCGAAGTCTTGAACTCATAGTCCTGGA-3';

[0044] SPLCV-LF (loop upstream primer, sequence 5 of the sequence listing): 5'-TAGCTTCGGGCAGCAATT-3';

[0045] SPLCV-LB (loop downstream primer, sequence 6 of the s...

Embodiment 2

[0047] Embodiment 2, specific detection

[0048] Sweet potato leaf curl virus (SPLCV), sweet potato feathery mottle virus (SPFMV), sweet potato asymptomatic virus (SPSMV), sweet potato virus No. 2 (SPV2), sweet potato G virus (SPVG) and sweet potato C virus (SPVC) were selected as the samples to be tested. Virus. Among the above-mentioned viruses, the sweet potato leafroll virus is a DNA virus, and the other viruses are all RNA viruses.

[0049] 1. Extract the genomic DNA of the DNA virus to be tested, or extract the total RNA of the RNA virus to be tested and reverse transcribe it into cDNA.

[0050] 2. Using the genomic DNA or cDNA obtained in step 1 as template DNA, perform loop-mediated isothermal amplification, then observe and take pictures under normal light conditions. During the loop-mediated isothermal amplification process, a real-time fluorescent PCR instrument was used for real-time monitoring. Set up a blank control in which an equal volume of water was used i...

Embodiment 3

[0056] Embodiment 3, sensitivity detection

[0057] 1. Genomic DNA of sweet potato leafroll virus was extracted.

[0058] 2. Using the genomic DNA obtained in step 1 as template DNA, perform loop-mediated isothermal amplification, then observe and take pictures under normal light conditions. During the loop-mediated isothermal amplification process, a real-time fluorescent PCR instrument was used for real-time monitoring. Set up a blank control in which an equal volume of water was used instead of template DNA.

[0059] In the reaction system (25 μl) of loop-mediated isothermal amplification, the concentration of BstDNA polymerase is 0.32 U / μL, the concentration of SPLCV-F3 and SPLCV-B3 is 0.2 μmol / L, and the concentration of SPLCV-FIP and SPLCV-BIP The concentration is 1.6 μmol / L, the concentration of SPLCV-LF and SPLCV-LB is 0.8 μmol / L, the concentration of calcein fluorescent dye is 0.2 μmol / L, the concentration of betaine is 1mol / L, MgSO 4 The concentration of dNTP is 6...

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Abstract

The invention discloses a method for detecting sweet potato leaf curl viruses and a special primer set thereof. The primer set is composed of single-stranded DNA molecules shown by a first sequence, single-stranded DNA molecules shown by a second sequence, single-stranded DNA molecules shown by a third sequence, single-stranded DNA molecules shown by a fourth sequence, single-stranded DNA molecules shown by a fifth sequence and single-stranded DNA molecules shown by a sixth sequence. The primer set has the functions of authenticating or assisting to authenticate the sweet potato leaf curl viruses and detecting whether the sweet potato leaf curl viruses are contained in a to-be-detected biological sample or not. The primer set for detecting the sweet potato leaf curl viruses has the advantages that specificity is high, sensitivity is high, operation is easy and convenient, reaction time is obviously shortened, and the defect that an existing method needs to rely on expensive instruments is overcome. The method and the primer set have the high popularization value.

Description

technical field [0001] The invention relates to a method for detecting sweet potato leafroll virus and a special primer set thereof, more particularly to a method for detecting sweet potato leafroll virus based on loop-mediated isothermal amplification and a special primer set thereof. Background technique [0002] Sweetpotatoleafcurlvirus (Sweetpotatoleafcurlvirus, SPLCV), belongs to Geminiviridae (Germiniviridae) Begomovirus (Begomovirus), the main host is sweetpotato, split-leaf morning glory and round-leaf morning glory, the disease it causes can lead to sweet potato development Abnormal, severely impaired yield and quality and degraded variety. In the 1980s, the disease occurred on sweet potatoes in Taiwan and Japan. In recent years, with the increasing population and distribution of the transmission vector Bemisia tabaci, and the increasing frequency of human trade and migration, the rapid spread and widespread occurrence of sweet potato leafroll virus has attracted g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/94
CPCC12Q1/6844C12Q2531/119C12Q2563/107
Inventor 乔岩董杰杨建国李永强岳瑾王品舒张金良袁志强王伟青
Owner BEIJING PLANT PROTECTION STATION
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