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RPA primer for detecting chicken infectious laryngotracheitis virus and its detecting method

A technology for tracheitis and larynx, which is applied in the field of detection of RPA primers for chicken infectious laryngotracheitis virus and its detection field, can solve the problems of false negative results, low sensitivity, difficult application, etc., and achieve strong specificity and high sensitivity , The effect of accurate test results

Active Publication Date: 2017-10-24
HENAN AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the virus isolation method is the gold standard for detecting pathogens, but this method takes a long time and has low sensitivity; serological detection methods are widely used, simple to operate and high in sensitivity, but for cases of acute infection, this method has shown its advantages. Limitation, easy to produce false negative results; PCR method, including fluorescent quantitative PCR, is a fast, sensitive and accurate detection method, but this method requires expensive equipment, and its application is difficult for the field and the laboratory with poor conditions

Method used

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  • RPA primer for detecting chicken infectious laryngotracheitis virus and its detecting method
  • RPA primer for detecting chicken infectious laryngotracheitis virus and its detecting method
  • RPA primer for detecting chicken infectious laryngotracheitis virus and its detecting method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1: Synthesis of primers for detecting chicken infectious laryngotracheitis virus

[0041] With reference to the chicken infectious laryngotracheitis virus K317 strain genome sequence (accessionnumber: JX458824) published in GenBank, select the sequence of the 648bp-685bp position in the genome nucleotide sequence as the upstream primer of the RPA amplification reaction, and in its 5 'End labeling with carboxyfluorescein (FAM): 5'-FAM-CTGCCTAAGCGAGGCTCCGCACCGGAGACTCAGGAATG-3'. Use the reverse complementary sequence of the sequence of the 762bp-799bp position in the genome nucleotide sequence of chicken infectious laryngotracheitis virus K317 strain as the downstream primer of the RPA amplification reaction, and label biotin (Biotin) at its 5' end: 5'-Biotin-CAATTCAGCCGAGGATTTGGGCCGCTCACCGCCAGAGC-3'.

[0042] Primers were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.

Embodiment 2

[0043] Embodiment 2: the preparation of colloidal gold lateral flow immunochromatography test strip

[0044] (1) Preparation of streptavidin-coated gold nanoparticles: Take 1 mL of gold nanoparticles solution (0.15 pmol / mL), add 1-2 μL of 200 mM borax solution, and adjust its pH value to 9.5. Meanwhile, 2 μL of streptavidin (2 mg / mL) was mixed with 398 μL of borax solution (2 mM) in another tube to obtain diluted streptavidin. The diluted streptavidin was added to the aforementioned gold nanoparticle solution in an amount of 50 μL, and stirred while adding, to obtain a mixture. Place the mixture at room temperature for 45 minutes, add 155.6 μL of 2 mM borax solution containing 10% BSA, continue to stand at room temperature for 10 minutes, then centrifuge at 4500 g for 15 minutes, discard the liquid, and wash with 1 mL of washing solution (containing 10 g / L BSA) 2mM borax solution) to resuspend the pellet, centrifuge at 4500g for 15 minutes, and discard the liquid to obtain a ...

Embodiment 3

[0046] Embodiment 3: DNA template extraction

[0047] The extraction of chicken infectious laryngotracheitis virus DNA is carried out as follows:

[0048] a. Take 350 μl chicken infectious laryngotracheitis virus solution in a 1.5mL EP tube, add 350 μL tissue lysate, and then add proteinase K at a final concentration of 0.2mg / mL, mix well and place in a water bath at 56°C for 1 hour;

[0049] b. Add 700 μL of Tirs balanced phenol to each tube, invert and mix 10 times, let stand at room temperature for 5 minutes, and then centrifuge at 1000 g for 5 minutes;

[0050] c. Take the supernatant into a new 1.5mL EP tube, add 350μL Tirs balance phenol and 350μL chloroform, invert and mix 10 times, then centrifuge at 10000×g for 5min;

[0051] d. Take 400 μL of the supernatant, add 1 mL of dehydrated ethanol pre-cooled at -20°C, and add 40 μL of NaAC (3M, pH=5.2) at the same time, mix well and let stand at -20°C for 2 hours;

[0052] e. Discard the supernatant after centrifugation at...

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Abstract

The invention discloses a RPA primer for detecting chicken infectious laryngotracheitis virus. The nucleotide sequence of the RPA primer is shown as SEQ ID NO.1 and SEQ ID NO.2; the RPA primer is strong in specificity, high in sensitivity, and accurate in detection result. The invention further provides a RPA method for detecting the chicken infectious laryngotracheitis virus. The detecting method is easy to operate, low in equipment requirement, strong in specificity, high in sensitivity, and accurate and reliable in detection result; the detecting method provides a low-cost, fast and specific field diagnosis method for effectively detecting and diagnosing the chicken infectious laryngotracheitis virus.

Description

technical field [0001] The invention belongs to the field of molecular biology detection, and in particular relates to an RPA primer for detecting chicken infectious laryngotracheitis virus and a detection method thereof. Background technique [0002] Chicken infectious laryngotracheitis is an acute, contact upper respiratory infectious disease caused by infectious laryngotracheitis virus (ILTV). The disease spreads quickly and has a high mortality rate. It occurs and prevails in many areas of our country and endangers the development of the chicken industry. Minimize losses and further spread of disease. [0003] Conventional polymerase chain reaction (Polymerase Chain Reaction, PCR) technology has been used as the gold standard method for diagnosing various diseases because of its ability to detect trace amounts of pathogenic DNA. However, PCR requires special thermal cycle equipment, low-temperature storage reagents and technical operation requirements to avoid cross-co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/701C12Q2531/119C12Q2565/625C12Q2521/507C12Q2537/1376C12Q2522/101
Inventor 赵军王川庆高冬生杨霞王新卫李永涛常洪涛陈陆刘红英姚惠霞
Owner HENAN AGRICULTURAL UNIVERSITY
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