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Primer and probe combination for RAA-LFD detection of chicken infectious laryngotracheitis virus and application of primer and probe combination

A tracheitis and virus genome technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of complex operation, low accuracy, and long cycle, and achieve simple operation, short cycle, The effect of simple detection methods

Pending Publication Date: 2019-09-20
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problems of high cost, complicated operation, low accuracy, long cycle and easy non-specific reaction in the existing ILTV detection method, the present invention provides a primer and probe for RAA-LFD detection of chicken infectious laryngotracheitis virus Composition and its application

Method used

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  • Primer and probe combination for RAA-LFD detection of chicken infectious laryngotracheitis virus and application of primer and probe combination
  • Primer and probe combination for RAA-LFD detection of chicken infectious laryngotracheitis virus and application of primer and probe combination
  • Primer and probe combination for RAA-LFD detection of chicken infectious laryngotracheitis virus and application of primer and probe combination

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Experimental program
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Effect test

Embodiment 1

[0035] Design of primers and probes for detection of chicken infectious laryngotracheitis virus by RAA-LFD

[0036] Comparing the TK gene sequence of ILTV, combined with the RT-RAA nucleic acid amplification kit requires the design of 4 pairs of primers (as shown in Table 1). Genomic DNA of ILTV Wanggang strain was extracted using a virus genomic DNA / RNA extraction kit and used as a template. Use RT-RAA Nucleic Acid Amplification Kit for amplification. Reaction system: 25 μL of fluorescent basic buffer (included with the kit), 2.1 μL of upstream and downstream primers (10 μM), 1 μL of template (220ug / μL), 17.3 μL of purified water and magnesium acetate 2.5 μL. React at 39°C for 20min, and after the reaction, detect the RAA reaction product by 1% agarose gel electrophoresis (such as figure 1 As shown, M is DL2000Marker, from left to right is the negative control, the amplification products of primers ILTV-1, ILTV-2, ILTV-3 and ILTV4), the results show that all 4 pairs of prim...

Embodiment 2

[0044] Utilize the method that above-mentioned primer and probe combination detect chicken infectious laryngotracheitis virus, comprise the steps:

[0045] (1) Genomic DNA of the ILTV standard virulent Wanggang strain (AV195) was extracted using a viral genome DNA / RNA extraction kit;

[0046] (2) Using the DNA obtained above as a template, use the RT-RAA nucleic acid amplification kit and the upstream primer shown in SEQ ID No.1, the downstream primer shown in SEQ ID No.2 and the probe shown in SEQ ID No.11 RAA amplification, RAA amplification reaction system is: 25 μL of fluorescent basic buffer, 2.1 μL of upstream primer and downstream primer, 0.6 μL of probe, 1 μL of DNA template, 16.7 μL of purified water and 2.5 μL of magnesium acetate, and the concentration of DNA template is 220ug / μL, the concentration of primers and probes is 1250nmol / L, react at 37°C for 20min, and obtain the RAA amplification product;

[0047] (3) LFD was used to detect the RAA amplification produc...

Embodiment 3

[0049] Utilize the method that above-mentioned primer and probe combination detect chicken infectious laryngotracheitis virus, comprise the steps:

[0050] (1) Genomic DNA of the ILTV standard virulent Wanggang strain (AV195) was extracted using a viral genome DNA / RNA extraction kit;

[0051] (2) Using the DNA obtained above as a template, use the RT-RAA nucleic acid amplification kit and the upstream primer shown in SEQ ID No.1, the downstream primer shown in SEQ ID No.2 and the probe shown in SEQ ID No.11 RAA amplification, RAA amplification reaction system is: 25 μL of fluorescent basic buffer, 2.1 μL of upstream primer and downstream primer, 0.6 μL of probe, 1 μL of DNA template, 16.7 μL of purified water and 2.5 μL of magnesium acetate, the concentration of DNA template is 200ug / μL, the concentration of primers and probes is 1200nmol / L, react at 35°C for 18min, and obtain the RAA amplification product;

[0052] (3) LFD was used to detect the RAA amplification product, a...

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Abstract

The invention relates to the technical field of biological detection, and particularly discloses a primer and probe combination for RAA-LFD detection of chicken infectious laryngotracheitis virus and application of the primer and probe combination. The primer comprises an upstream primer and a downstream primer, wherein the sequence of the upstream primer is shown as SEQ ID No. 1, and the sequence of the downstream primer is shown as SEQ ID No. 2; and the probe sequence is shown in SEQ ID No. 11. The primer and probe combination provided by the invention is used for detecting chicken infectious laryngotracheitis virus, and the corresponding RAA-LFD method is simple, rapid, convenient, reliable, strong in specificity, high in sensitivity and accuracy, and is more suitable for clinical field detection.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a RAA-LFD (recombinase-mediated isothermal nucleic acid amplification technology-lateral flow chromatography test strip) primer and probe combination for detecting chicken infectious laryngotracheitis virus and its application. Background technique [0002] Infectious laryngotracheitis (ILT) is a highly contagious respiratory disease caused by the Infectious laryngotracheitis virus (ILTV) of the α-herpesviridae subfamily of the Herpesviridae family. The viral nucleic acid is double-stranded DNA, mainly present in chicken tracheal tissue and its exudate. ILT mainly infects adult chickens, while chicks have a certain resistance to the disease and will not have obvious clinical symptoms after infection. ILT has the characteristics of acute onset and rapid spread. Adult chickens infected with ILTV will have shortness of breath, runny nose, cough and other symptoms. Wi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/705C12Q1/6844C12Q2521/507C12Q2522/101C12Q2565/625
Inventor 张铁王文静王春光李希明孟凡国张鹏姚姗姗刘静茹梁存军
Owner HEBEI AGRICULTURAL UNIV.
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