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Bee Israeli acute paralysis virus RT-PCR detection reagent kit and detection method thereof

A RT-PCR, detection kit technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of lack of diagnostic technology, lack of research, etc., and achieve good repeatability, high sensitivity, and easy operation. Effect

Inactive Publication Date: 2016-06-01
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

[0005] Since IAPV was only isolated and identified in 2007, as a new virus, there is little research on it, and there is currently a lack of perfect diagnostic techniques

Method used

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  • Bee Israeli acute paralysis virus RT-PCR detection reagent kit and detection method thereof
  • Bee Israeli acute paralysis virus RT-PCR detection reagent kit and detection method thereof
  • Bee Israeli acute paralysis virus RT-PCR detection reagent kit and detection method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0031] A RT-PCR kit for detecting honeybee Israel acute paralysis virus, including a forward primer and a reverse primer, wherein the sequence of the forward primer is 5'-AAGGCTCAGCTAGGATGACAC-3', and the sequence of the reverse primer is 5'-TCAGGATTTAAAGCCTCACG-3'; The kit includes the following reagents (30 reactions):

[0032] (1) Reverse transcription master mix: each reaction (20 μL system) includes 4 μL of 5× reverse transcription buffer; 1 μL of reverse primer with a concentration of 10 μmol / L; 2 μL of 5 mmol / L dNTP; 25 mmol / L of MgCl 2 2 μL; 40U / μL RNase inhibitor 0.5 μL, 9.5 μL in total, 285 μL in total for 30 reaction systems, and store at -20°C for later use.

[0033](2) PCR master mix: each reaction (25 μL system) includes 12.5 μL of 2×PCRBuffer; 0.5 μL of forward primer and reverse primer with a concentration of 10 μmol / L; 1 μL of 5 mmol / L dNTP; 25 mmol / L of MgCl 2 2 μL, 16.5 μL in total, 495 μL in total for 30 reaction systems, and store at -20°C for future use....

Embodiment 2

[0040] The detection method of honeybee Israel acute paralysis virus RT-PCR detection kit comprises the following steps:

[0041] (1) Reverse transcription reaction: Add 3 μL of total RNA of the sample to be tested in the PCR tube, RNase-freedH 2 O7 μL, incubate at 70°C for 10 min, briefly centrifuge and ice-bath for 5 min, then add 9.5 μL of reverse transcription reaction solution and 0.5 μL of reverse transcriptase at a concentration of 200 U / μL. Incubate at 42°C for 60min, inactivate reverse transcriptase at 95°C for 5min, and ice-bath for 5min to synthesize cDNA.

[0042] (2) PCR reaction: Add 16.5 μL of PCR reaction solution to the PCR tube, 3 μL of cDNA synthesized in step (1), 0.5 μL of 5 U / μL TaqDNA polymerase, RNase-freedH 2 O5 μL for a total volume of 25 μL. PCR reaction program: pre-denaturation at 95°C for 5 minutes; then denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 30 s, a total of 35 cycles; extension at 72°C for 10 min, and ...

Embodiment 3

[0044] Example 3: Specificity determination of honey bee Israel acute paralysis virus RT-PCR detection kit

[0045] (1) Extraction of total RNA from honeybee samples: Israeli acute paralysis virus (IAPV), acute paralysis virus (ABPV), Kashmir honey bee virus (KBV), cystic larvae virus (SBV) and black queen bee virus (BQCV) ) honeybee samples as materials, put 0.1g each in a sterilized mortar, add 1mL PBS buffer solution to grind, centrifuge at 10000g for 5min at 4°C, transfer the supernatant to a sterilized 1.5mL centrifuge tube, add 1mL Trizol reagent , shake vigorously for 15s, let stand at room temperature for 5min; centrifuge at 12000g for 10min at 4°C, transfer the supernatant to another sterilized 1.5mL centrifuge tube; add 300μL of chloroform, shake vigorously for 15s, let stand at room temperature for 5min; 4°C, 12000g Centrifuge for 15 minutes, transfer the upper aqueous phase into another sterilized 1.5mL centrifuge tube; add an equal volume of isopropanol, invert an...

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Abstract

The invention relates to a bee Israeli acute paralysis virus RT-PCR detection reagent kit and a detection method thereof. The reagent kit and the detection method are specially used for detection of bee Israeli acute paralysis viruses. The reagent kit comprises an inverse transcription premixed solution, a PCR premixed solution, reverse transcriptase enzyme, Taq DNA polymerase, positive control, negative control and RNase-free ddH2O. The reagent kit has the advantages that stability and specificity are good, high specificity is achieved in bee IAPV detection, cross reaction with other bee viruses does not exist, and repeatability is good; sensitivity is high and sensitivity can reach 103 copies; operation is easy and convenient, the reaction premixed solution is adopted, operation steps are reduced, and pollution risks are reduced. The method can be used for IAPV infection detection of bees, and plays an important role in early prevention and in-time prevention of the bee Israeli acute paralysis viruses.

Description

technical field [0001] The invention relates to a RT-PCR detection kit for bee Israel acute paralysis virus and a detection method thereof, which belongs to the technical field of animal quarantine and is suitable for rapid detection and monitoring of Israel acute paralysis virus of entering and exiting bees and bee breeding enterprises. Background technique [0002] From the winter of 2006 to the spring of 2007, colony collapse disorder (CCD) swept across 22 states in the United States, and then became popular in countries such as France, Sweden, Germany and Australia, causing the loss of local beekeepers' bee colonies to reach 50 %~90%. The survey found that the probability of Israeli acute paralysis virus (IAPV) in bee colonies with CCD symptoms was 83.3%, while the probability of existence in bee colonies without CCD symptoms was only 1%, showing a strong correlation with CCD Therefore, the main pathogen of CCD is currently identified as IAPV. [0003] IAPV is a positi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/701C12Q1/686
Inventor 张体银郑腾蔡一龙白泉阳王武军张志灯于师宇
Owner INSPECTION & QUARANTINE TECH CENT OF FUJIAN ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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