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Linked gene detection kit for potato yellow dwarf virus and detecting method

A yellow dwarf virus and potato technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the time-consuming, unseen real-time fluorescent quantitative RT-PCR detection kit and detection method , PYDV detection and other issues have not been seen yet

Active Publication Date: 2018-10-16
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantage of the ordinary RT-PCR method is that the result judgment needs to be detected by agarose gel electrophoresis and gene sequencing, which is easy to pollute and takes a long time
But so far, there has been no real-time fluorescent quantitative RT-PCR method report specifically for PYDV detection, let alone a real-time fluorescent quantitative RT-PCR detection kit and detection method based on combined gene detection

Method used

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  • Linked gene detection kit for potato yellow dwarf virus and detecting method
  • Linked gene detection kit for potato yellow dwarf virus and detecting method
  • Linked gene detection kit for potato yellow dwarf virus and detecting method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] Embodiment 1, potato yellow dwarf virus combined gene detection kit and detection method

[0089] 1. A complete set of specific primers and fluorescent probes for the detection of Potato Yellow Dwarf Virus

[0090] The invention designs and screens two sets of specific primers and fluorescent probes according to the conserved region of the RdRp gene of the potato yellow dwarf virus and the conserved region of the NP gene of the potato yellow dwarf virus.

[0091] The primers and fluorescent probes designed in the present invention for detecting the RdRp gene of potato yellow dwarf virus are composed of primer PYDV-f1, primer PYDV-r1 and fluorescent probe PYDV-Probe1. The sequences of primers and fluorescent probes are as follows:

[0092] PYDV-f1: 5'-GGTCAAATCGGAAATGAG-3' (SEQ ID NO: 1);

[0093] PYDV-r1: 5'-CTGGTTACAGTGATCAGA-3' (SEQ ID NO: 2);

[0094] PYDV-Probe1: 5'-FAM-TAAGCGGCAACCAACTGTCG-TAMRA-3' (SEQ ID NO: 3);

[0095] The primers and fluorescent probes desig...

Embodiment 2

[0117] Embodiment 2, the detection method of potato yellow dwarf virus combined gene detection kit

[0118] 1. Establishment of real-time fluorescent quantitative RT-PCR reaction system and optimization of reaction conditions for potato yellow dwarf virus combined gene

[0119] The sample infected with Potato Yellow Dwarf Virus was used as the sample to be tested, and the kit in Example 1 was used to detect the Potato Yellow Dwarf Virus in the sample to be tested. Specific steps are as follows:

[0120] 1) Reverse transcription: Add 3 μL of total RNA of the sample to be tested, RNase-free ddH to the PCR tube 2 7 μL of O and 1 μL of random primers with a concentration of 100 μmol / L, mix well, bathe in water at 70°C for 10 minutes, then ice-bath for 5 minutes, add 5 μL of 5×RT Buffer, 1 μL of reverse transcriptase at a concentration of 200 U / μL, and a concentration of 10 mmol / L 2 μL of dNTPs in L and 1 μL of RNase inhibitor at a concentration of 40 U / μL, bathed in water at 42°...

Embodiment 3

[0134] Embodiment 3, detect the detection method of potato yellow dwarf virus for single gene

[0135] One, be used for detecting the method for the primer of potato yellow dwarf virus RdRp gene, fluorescent probe detection potato yellow dwarf virus

[0136] A sample infected with Potato Yellow Dwarf Virus was used as a test sample, and the Potato Yellow Dwarf Virus in the test sample was detected by real-time fluorescent quantitative RT-PCR for the Potato Yellow Dwarf Virus RdRp gene. Specific steps are as follows:

[0137] 1) Reverse transcription: Add 3 μL of total RNA of the sample to be tested, RNase-free ddH to the PCR tube 2 7 μL of O and 1 μL of random primers with a concentration of 100 μmol / L, mix well, bathe in water at 70°C for 10 minutes, then ice-bath for 5 minutes, add 5 μL of 5×RT Buffer, 1 μL of reverse transcriptase at a concentration of 200 U / μL, and a concentration of 10 mmol / L 2 μL of dNTPs in L and 1 μL of RNase inhibitor at a concentration of 40 U / μL, ...

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Abstract

The invention discloses a linked gene detection kit for a potato yellow dwarf virus and a detecting method. The kit comprises one set of specific primers and fluorescence probes for a potato yellow dwarf virus RdRp gene, one set of specific primers and fluorescence probes for a potato yellow dwarf virus NP gene, RT Buffer, a random primer, a RNA enzyme inhibiting factor, dNTPs, reverse transcriptase, TaqMan PCR mix, a positive control sample and a negative control sample. The invention further discloses a reaction system, a reaction condition and result determination for performing real-time fluorescent quantitative RT-PCR detection on the potato yellow dwarf virus through a linked gene. According to the experiment, the method can effectively detect potato yellow dwarf viruses of differentplant systems, has the advantages of short detection time, high specificity, high sensitivity, simple and convenient operation, safe and high flux, and is very suitable for quick detection on entry and exit port quarantine and the potato yellow dwarf virus on agricultural production.

Description

technical field [0001] The invention relates to a combined gene detection kit and detection method of potato yellow dwarf virus, belongs to the technical field of plant quarantine, and is suitable for rapid detection and identification of potato yellow dwarf virus in entry and exit port quarantine and agricultural production. Background technique [0002] At present, there are more than 30 types of potato viruses that have been reported, among which Potato yellow dwarf virus (PYDV) is listed as a quarantine pest that is prohibited from entering my country. PYDV is a member of the genus Nucleorhabdovirus, which can cause a devastating potato virus disease - yellow dwarf disease. It can be spread over a large area by vector insects. PYDV is divided into two strains, SYDV and CYDV. Among them, SYDV is transmitted through Aceratagallia sanguinolenta, but Agalliaconstricra cannot be transmitted, and CYDV is transmitted through Agallia constricta, but Aceratagallia sanguinolenta ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2600/166C12Q2531/113C12Q2563/107C12Q2545/113
Inventor 沈建国高芳銮张永江陈细红廖富荣
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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