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Primer group and probe for detecting kashmir bee virus and detection method

A primer set and virus technology, which is applied in the field of animal health, can solve the problems of poor specificity and inaccurate detection results of honeybee Kashmir virus, and achieve the effect of short primer length requirements, improved sensitivity, and high sensitivity

Inactive Publication Date: 2021-03-23
INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Claims
  • Application Information

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Problems solved by technology

It solves the problems of poor specificity and inaccurate detection results in the detection of bee Kashmir virus in the prior art

Method used

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  • Primer group and probe for detecting kashmir bee virus and detection method
  • Primer group and probe for detecting kashmir bee virus and detection method
  • Primer group and probe for detecting kashmir bee virus and detection method

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Effect test

Embodiment 1

[0028] Example 1 Optimization of Real Time Fluorescence RT-PCR Annealing Temperature

[0029] The real-time fluorescent RT-PCR reaction procedure is: 50 ° C reverse transcription for 30 min; 95 ° C, 5 min; 95 ° C degeneration 15S, the annealing temperature is provided to 55 ~ 65 ° C (gradient temperature), annealing extension 30s, each gradient temperature setting 3 Repeat, a total of 40 cycles;

[0030] Real-time fluorescent RT-PCR reaction system: 10 × RT-PCR Buffer 2μL, 25mmol / L MGCL 2 2 μL, 2.5 mmol / l DNTPS 1.5 μL, 10 μmol / L of the upper and lower proders KbV-F / KBV-R Eared 0.5 μL, 5U / μL DNA polymerase 0.5 μL, 5U / μL Reverse transcriptase 0.5 μL, 40 U / μl RNase inhibitor 0.5 μL, bee Kehmir virus RNA 2 μl, then 9.0 μl of water, and the total volume of reaction was 20.0 μL.

[0031] The annealing temperature detected by the bee Kashmir virus real-time fluorescence RT-PCR is optimized by the above method. Such as figure 1 .

[0032] The results show that when the anne...

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Abstract

The invention relates to the technical field of animal hygiene, and provides a group of primers and probes capable of specifically detecting real-time fluorescent RT-PCR of kashmir bee virus, and a non-diagnostic detection method obtained by optimizing a reaction system and a reaction program of the real-time fluorescent RT-PCR by using the primer combination probes. The detection method has the following advantages: (1) good specificity and sensitivity: the detection of the kashmir bee virus has high specificity, no cross reaction with other bee viruses, and good repeatability; (2) high sensitivity: the sensitivity can reach about 8 copies / microliter; and (3) simple and fast operation, and the whole reaction can be completed within 60 minutes. The detection method can be used for the detection of the kashmir bee virus for non-diagnosis and the identification of the kashmir bee virus with other bee virus diseases, and has important significance for ensuring the healthy development ofthe apiculture industry and maintaining the natural ecology in China.

Description

Technical field [0001] The present invention relates to the field of animal hygiene, and more particularly to a primer group and probe for detecting a beekell malovirus and a detection method thereof. Background technique [0002] Kashmir Bee Virus (KBV) was originally isolated from the Western bee body offeeding the disease oriental bee Apis Cerana virus fluid, because these Eastern bees came from the Kashmir area, so the researchers used this. Name give this virus name. At present, in addition to India and Australia, KBV has been reported in Western honeycombs around the world, but people have limited knowledge of the spread of KBV in China. [0003] KBV diffuses through vertical propagation and horizontal transmission like other bee viruses. KBV first found in the bee body, the viral particles will cause the host to die in a few days after entering the lymph, so it is considered to be one of the most poisonous bee viruses. KBV will be infected with other viruses such as SBV, b...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/701C12Q1/686C12Q2561/113C12Q2561/101C12Q2563/107
Inventor 张体银邵碧英黄嫦娇王武军郑腾张志灯于师宇
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU