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RT-PCR (reverse transcription-polymerase chain reaction) primer group for detecting passion fruit woodiness virus diseases and kit and application of RT-PCR primer group

An RT-PCR and lignification technology, applied in the field of RT-PCR primer sets for detecting early passion fruit lignification virus disease, can solve the problems of expensive instruments and equipment, long time-consuming biological detection, limited environmental conditions, etc., and meet sample requirements The effect of small amount, early diagnosis, and perfect detection technology

Inactive Publication Date: 2020-01-07
GUANGXI ZHUANG AUTONOMOUS REGION ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Biological detection takes a long time and is limited by environmental conditions
The detection results of electron microscopy are intuitive and accurate. Due to the expensive equipment, complex production and operation techniques are difficult to master, and require high technical level of operators, they have not been widely used.
Serological methods are fast and intuitive, but the accuracy of the results often depends on the quality of the antiserum, and cannot detect unstable viruses

Method used

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  • RT-PCR (reverse transcription-polymerase chain reaction) primer group for detecting passion fruit woodiness virus diseases and kit and application of RT-PCR primer group
  • RT-PCR (reverse transcription-polymerase chain reaction) primer group for detecting passion fruit woodiness virus diseases and kit and application of RT-PCR primer group
  • RT-PCR (reverse transcription-polymerase chain reaction) primer group for detecting passion fruit woodiness virus diseases and kit and application of RT-PCR primer group

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Extraction of total RNA from passion fruit infected by lignification virus:

[0022] (1) Take 100 mg of passion fruit lignification virus-infected leaves, grind it fully with liquid nitrogen, transfer it to a 2 mL centrifuge tube, add 1 mL of TRIzol reagent from Invitrogen Company, mix well and incubate at room temperature for 5 minutes to facilitate the full lysis of the sample. get lysate;

[0023] (2) Add 0.2mL chloroform per 1mL TRIzol reagent, add chloroform to the lysate obtained in step (1), cover the sample tube cap carefully, shake vigorously up and down for 15 seconds, then incubate at room temperature for 2-3 minutes, at 4°C Centrifuge at 12,000×g for 15 minutes at low temperature;

[0024] (3) After centrifugation, the liquid in the tube is divided into 3 layers. Carefully absorb the upper colorless water phase and transfer it to a new RNase-free 1.5mL centrifuge tube, then add 0.5mL isopropanol, incubate at room temperature for 10 minutes, and cool at 4°C ...

Embodiment 2

[0028] Synthesis of cDNA first strand:

[0029] (1) Add 4 μL of total RNA to an RNase-free PCR tube at a concentration of 10 μmol L -1 The sequence is CKTGCAGTRTGCCTTTCAGT lignification virus disease detection specific primer 1 μL, and with DEPC-treated sterilized water to supplement the volume to 12 μL, to obtain a mixture;

[0030] (2) Place the mixture obtained in step (1) in a PCR instrument and incubate at 65°C for 5 minutes, then immediately ice-bath for 2 minutes, then add 4 μL of 5×Reaction Buffer and 1 μL of Ribonuclease Inhibitor (50 U / μL) to the mixture , dNTPmix (10mM) 2μL, M-MuLV Reverse Transcriptase (200U / μL) 1μL, centrifuge and mix well, place in a PCR instrument and incubate at 42°C for 60 minutes; then incubate at 85°C for 5 minutes to end the reaction and obtain the first strand of cDNA .

Embodiment 3

[0032] Passion fruit lignification virus RT-PCR detection:

[0033] (1) Design of primers

[0034] According to the conserved region of the coat protein gene sequence of passion fruit lignification virus, Primer Premier 5.0 software was used to design specific primers. The size of the primer amplification product was 365bp, and the name and sequence were as follows:

[0035] Upstream primer PWV-F: 5'-GTGTGGGTRATGATGGATGG-3'

[0036] Downstream primer PWV-R: 5'-CKTGCAGTRTGCCTTTCAGT-3'

[0037] (2) Perform PCR reaction in the following PCR reaction system: 2×PCR buffer for KOD FX 12.5 μL, 2mMdNTPs 5.0 μL, PWV-F (10 μmol L -1 )0.75μL, PWV-R (10μmol L -1 )0.75μL, KOD FX (1.0U·μL -1 ) 0.5 μL, cDNA first strand 3.0 μL, ddH 2 O 2.5 μL.

[0038] (3) Set the following reaction program in the PCR instrument: pre-denaturation at 94°C for 2 minutes to fully denature the template cDNA; then enter the amplification cycle of denaturation at 98°C for 10 s, annealing at 53°C for 30 s, an...

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Abstract

The invention discloses an RT-PCR (reverse transcription-polymerase chain reaction) primer group for detecting passion fruit woodiness virus diseases. The primer group comprises a specific primer PWV-F (passion fruit woodiness virus-F) and a specific primer PWV-R, wherein the specific primer PWV-F has a nucleotide sequence of SEQ ID No.1 shown in the description; and the specific primer PWV-R hasa nucleotide sequence of SEQ ID No.2 shown in the description. The method disclosed by the invention is small in sample demand amount, small in technique difficulty, high in accuracy and applicable tolarge-scale sample analysis, not only are samples with suspected passion fruit woodiness virus infection symptoms diagnosed, but also samples with viruses at a latency stage without conspicuous symptoms can be identified, and early-stage diagnosis on affected plants can be achieved.

Description

technical field [0001] The invention belongs to the technical field of detection of fruit quarantine diseases, in particular to an RT-PCR primer set, a kit and an application thereof for detecting early passion fruit lignification virus disease. Background technique [0002] With the good market situation of passion fruit in Guangxi and the bumper harvest of planting years, passion fruit has become popular in many places and has become a "short, flat and fast" targeted poverty alleviation project. In recent years, the planting area of ​​passion fruit in Guangxi has continued to expand, reaching 178,000 mu in 2016, 300,000 mu in 2017, and 410,000 mu in 2018. Due to extensive planting management, lack of attention to seedling selection at the source, and lack of timely prevention and control of plant diseases and insect pests by fruit farmers, most orchard blight, stem rot, and viral diseases are common, resulting in the production and quality of passion fruit in Guangxi. Low...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/701C12Q1/686
Inventor 蒋胜理魏源文曹辉庆黄诚梅罗海斌陆珍邓智年徐林吴凯朝
Owner GUANGXI ZHUANG AUTONOMOUS REGION ACAD OF AGRI SCI
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