Primer probe group, kit and detection method for PCR detection of potato broom top virus

A technology of primer probes and broom top virus, which is applied in the field of primer probe sets for PCR detection of potato broom top virus, can solve the problems of inability to detect PMTV accurately and quickly, and achieves shortening detection time, simple preparation, and reduction of false positives and false positives. Effects of Negative Conditions

Active Publication Date: 2021-06-01
黑龙江省农业科学院马铃薯研究所
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] In order to solve the problem that the existing ELISA detection method and RT-PCR detection method cannot accurately and quickly

Method used

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  • Primer probe group, kit and detection method for PCR detection of potato broom top virus

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Embodiment 1

[0036]The present embodiment provides a kind of primer probe group that is used for PCR detection of potato broom top virus, comprises the RT-PCR primer probe group-PMTV2 of the RNA-CP RNA chain conservative region in the PMTV genome and for RNA-TGB RNA chain RT-PCR Primer Probe Set for Conserved Region - PMTV3.

[0037] RT-PCR primer probe set-PMTV2 comprises forward primer PMTV2-F, reverse primer PMTV2-R, probe PMTV2-P9 and probe PMTV2-P8; Wherein the nucleotide sequence of forward primer PMTV2-F is as SEQ Shown in ID No.1; The nucleotide sequence of reverse primer PMTV2-R is shown in SEQ ID No.2; The nucleotide sequence of probe PMTV2-P9 is shown in SEQ ID No.3; Probe PMTV2- The nucleotide sequence of P8 is shown in SEQ ID No.4.

[0038] Using the reverse-transcribed cDNA of PMTV RNA as a template, the forward primer PMTV2-F and the reverse primer PMTV2-R were used to perform PCR amplification to obtain a product of 196 bp, and its nucleotide sequence is shown in SEQ ID No...

Embodiment 2

[0044] This embodiment provides a detection kit containing the primer probe set of Example 1, specifically including forward primer PMTV2-F, reverse primer PMTV2-R, forward primer PMTV3-F and reverse primer PMTV3-R, Also includes the chip connected with probe PMTV2-P9, probe PMTV2-P8 and probe PMTV3-P5, also includes PCR Buffer, dNTP Mixture, Taq enzyme, ddH 2 O, hybridization solution, washing solution, chromogenic solution and blocking solution containing digoxin AP enzyme.

[0045] In the present embodiment, the chip connected with the probe is based on a 96-well plate, and is divided into a PMTV2 probe group and a PMTV3 probe group. The PMTV2 probe group is connected with a probe PMTV2-P9 and a probe PMTV2-P8, and the PMTV3 probe group The needle set is connected to the probe PMTV3-P5.

[0046] The specific preparation method of the probe connected to the chip is to use the gene chip sample solution to dilute the probe PMTV2-P9, probe PMTV2-P8 and probe PMTV3-P5 to a conc...

Embodiment 3

[0049] The present embodiment provides a kind of concrete detection method that utilizes embodiment 2 to carry out PMTV detection for potato broom top virus PCR detection kit, specifically comprises the steps:

[0050] Step 1, using the cDNA reverse-transcribed from the sample RNA to be tested as a template, using forward primer PMTV2-F, reverse primer PMTV2-R, forward primer PMTV3-F and reverse primer PMTV3-R to carry out double PCR reaction;

[0051] The amplification system of the double PCR reaction is: 1.0 μl of detection sample cDNA, 0.5 μl each of forward primer PMTV2-F and reverse primer PMTV2-R at a concentration of 10uM, and forward primer PMTV3-F and reverse primer at a concentration of 10uM. To each primer PMTV3-R 0.5μl, 10×PCR Buffer 2.5μl, concentration of 2.5mM dNTP Mixture 2.0μl, concentration of 5U / μl Taq enzyme 0.3μl and ddH 2 O 17.2 μl.

[0052] The reaction program of the double PCR reaction was: pre-denaturation at 94°C for 5 min, denaturation at 94°C for...

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Abstract

The invention relates to a primer probe group, a kit and a detection method for PCR detection of a potato broom top virus, and belongs to the technical field of molecular biology. In order to solve the problem that the PMTV cannot be accurately and rapidly detected by an existing detection method, the invention provides a primer probe group for PCR detection of the potato broom top virus. The primer probe group comprises a primer probe group-PMTV2 and a primer probe group-PMTV3; the invention also provides a detection kit comprising the primer probe group and a detection method. According to the invention, cDNA obtained through RNA reverse transcription of a sample to be detected is taken as a template, and primers in a primer probe group are adopted to carry out duplex PCR reaction; the obtained PCR reaction product is hybridized with a chip connected with the probes in the primer probe group, developing is conducted, and if the corresponding position of the chip is bluish violet, it is judged that the chip is positive. According to the invention, the defect that naked virus RNA cannot be detected by ELISA is avoided, and false positive and false negative conditions of an RT-PCR method are reduced.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer probe set, a kit and a detection method for PCR detection of potato broom top virus. Background technique [0002] Potato is the fourth largest food crop in my country, and it is susceptible to a variety of viruses during its growth. Among them, potato mop-top virus (PMTV) is highly pathogenic and harmful, and is listed as an imported plant in my country. List of quarantine prohibited items. Its genome consists of three linear forward-sense single-stranded (ss) RNA molecules each wrapped in a capsid protein. These RNAs were named RNA-Rep: 6043nt, RNA-CP: 3134nt and RNA-TGB: 2964nt based on their encoded products. RNA-Rep is associated with viral replication and encodes components of RNA-dependent RNA polymerase; RNA-CP encodes capsid and readthrough proteins, and RNA-TGB encodes motor proteins and a putative cysteine-rich miniature 8kDa protein Tr...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6837C12N15/11C12R1/94
CPCC12Q1/6837C12Q1/701C12Q2600/16C12Q2531/113C12Q2537/143C12Q2565/50
Inventor 白艳菊王腾
Owner 黑龙江省农业科学院马铃薯研究所
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