Universal shell type fluorescent reverse transcription-polymerase chain reaction (RT-PCR) detection method of bird flu virus and detection kit

A technology of RT-PCR and avian influenza virus, which is applied in the field of universal nested fluorescent RT-PCR detection and detection kits for avian influenza virus, can solve the problems of long diagnostic cycle, low sensitivity and high sensitivity, and achieve error propagation Low probability of amplification, increase sensitivity and specificity, and avoid the effect of non-specific amplification

CN102071264AInactive Publication Date: 2011-05-25中华人民共和国珠海出入境检验检疫局
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Publication Date
2011-05-25
Estimated Expiration
Not applicable · inactive patent

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Abstract

The invention discloses a high-efficiency high-sensitivity universal shell type fluorescent reverse transcription-polymerase chain reaction (RT-PCR) detection method of bird flu virus and a detection kit. The detection method is a method in which two sets of polymerase chain reaction (PCR) primers (one pair of outer primers and one pair of inner primers) are utilized to carry out two rounds of PCR amplification, namely, the products of the outer primers subjected to the PCR amplification are used as templates of the inner primers to be subjected to the PCR amplification, the bonding sites of the inner primers and the template DNAs are positioned at the inner sides of the DNA fragments amplified by the outer primers, and shell type PCR is extremely effective for reducing or eliminating nonspecific amplification and improving sensitivity. Compared with the fact that the temperate with an extremely low concentration (one or multiple copies) is difficult to detect by using the common detection method, the efficiency and fidelity of amplification can be greatly improved by using the detection method in the invention; and the detection method disclosed by the invention is significantly effective for the amplification of extremely trace target genes in environmental samples, is extraordinarily beneficial to the amplification of the trace temperate of the bird flu virus in a fish farming water body, and can fully meeting the requirements of the sensitivity and specificity of the bird flu virus detection in fishpond farming water.
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Description

technical field

[0001] The invention relates to a universal nested fluorescent RT-PCR detection method and a detection kit for avian influenza virus. Background technique

[0002] The isolation and identification of avian influenza virus needs to collect throat swabs or cloacal swabs from birds, or collect internal organs from sick and dead birds, use chicken embryos for virus isolation and culture, and then identify hemagglutinin (HA) and neuraminic acid Enzyme (NA), and finally the determination of pathogenicity through artificial infection of chickens. This classic detection method of avian influenza virus is recommended by OIE as an international reference method due to its high sensitivity. It is carried out in a third-level laboratory, so it cannot be used for rapid detection, and the existing avian influenza virus detection methods, such as conventional RT-PCR technology, colloidal gold immunoassay and ELISA, cannot be directly used in poultry and poultry due to their...

Examples

Embodiment Construction

[0042] The specific construction steps of the present invention are further elaborated below, mainly including the following steps:

[0043] 1. Design and synthesis of nested fluorescent RT-PCR primers and probes:

[0044] Use Lasergene gene analysis software to compare the nucleotide sequences of H5N1, H5N2, H5N9, H2, H6, H9 AIV, NDV, and EDSV downloaded from NCBI, find highly conserved regions, and select appropriate AIV NP gene-related sequences , import the probe design software Primer Express 2.0 to design AIV universal nested fluorescent RT-PCR primers and Tagman probes, dissolve them in DEPC-treated water, and store them in the dark at -80°C. The outer primers P1, P2, inner primers P3, P4 and probes are shown in the table below:

[0045]

[0046] 2. Establishment and optimization of nested fluorescent RT-PCR reaction system:

[0047] (1) Optimization of the concentration of internal primers and probes: the concentration of primers and probes in the fluorescent PCR ...