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

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.

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...

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Problems solved by technology

[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, and use chicken embryos for virus Isolate and cultivate, then identify hemagglutinin (HA) and neuraminidase (NA), and finally measure the pathogenicity through artificial infection of chickens. This classic detection method for avian influenza virus is recommended by OIE as the most sensitive method because of its high sensitivity. International reference method, but due to the long diagnostic cycle and the need to carry out in a biosafety level 3 laboratory, it cannot be used for rapid detection, while the existing detection methods for avian influenza virus, such as conventional RT-PCR technology, colloidal gold immunoassay and ELISA, etc., cannot be directly used for the detection of AIV in poultry and poultry products due to their low sensitivity

[0003]The content of avian influenza viruses (AIV) in fish farming water is very low, and its content is far lower than that of ordinary poultry tissues. The detection of poultry is much more difficult than that of ordinary poultry tissues. The existing more sensitive chicken embryo virus isolation, as well as the conventional RT-PCR (reverse transcription-polymerase chain reaction) and fluorescent RT-PCR (real-time RT-PCR) detection methods are all Unable to meet the needs of rapid detection and high sensitivity of avian influenza virus in fish farming water

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