Bird flu and newcastle disease double-droplet digital PCR absolute quantitative detection kit

Abstract

The invention discloses a bird flu and newcastle disease double-droplet digital PCR absolute quantitative detection kit. A primer probe combination is provided and consists of a primer probe set AIV-F1R1P1 and a primer probe set NDV-F1R1P1; the primer probe set AIV-F1R1P1 consists of a primer AIV-F1, a primer AIV-R1 and a probe AIV-P1; the primer AIV-F1 is as shown in the sequence 1, and the primer AIV-R1 is as show in the sequence 2; the nucleotide sequence of the probe AIV-P1 is the sequence 3; the primer probe set NDV-F1R1P1 consists of a primer NDV-F1, a primer NDV-R1 and a probe NDV-P1; and the primer NDV-F1 is as shown in the sequence 4, the primer NDV-R1 is as shown in the sequence 5, and the nucleotide sequence of the probe NDV-P1 is the sequence 6. The bird flu and newcastle disease double-droplet digital PCR absolute quantitative detection kit has a significant application value on prevention and control of bird flu virus and / or newcastle disease virus, and contributes to controlling epidemic situations from the source and effectively preventing large-scale outbreak of bird epidemic diseases.

Description

technical field [0001] The invention belongs to the field of virus detection, and in particular relates to a double droplet digital PCR absolute quantitative detection kit for bird flu and Newcastle disease. Background technique [0002] Avian influenza virus (AIV) is a type A influenza virus. Influenza viruses belong to the Orthomyxoviridae family of RNA viruses and are divided into three types: A, B, and C. Among them, influenza A virus is mainly found in poultry, and it is an infectious disease caused by influenza A virus in poultry with various symptoms ranging from respiratory system to severe systemic sepsis. The International Veterinary Bureau defines it as a Class A infectious disease. In 1994, 1997, 1999, and 2003, bird flu broke out in Australia, Italy, Hong Kong, and the Netherlands, respectively. In 2005, it broke out mainly in Southeast Asia and Europe. It is mainly transmitted through the respiratory tract, through close contact with infected poultry and thei...

AI Technical Summary

Problems solved by technology

Traditional detection methods, such as virus neutralization test (VNT), immunofluorescence, immunoelectron microscopy, enzyme-linked immunosorbent assay, etc., take a long time to exist, rely on experienced testing personnel, complex test operations, and involve live virus operations , high requirements for the detection environment, and many shortcomings such as low sensitivity and poor repeatability

Although nucleic acid detection methods such as RT-PCR have the advantages of stronger specificity, higher sensitivity, higher degree of automation, and better repeatability of results than traditional methods, they can only achieve qualitative and semi-quantitative detection of avian influenza and Newcastle disease. detection, it is impossible to perform accurate absolute quantitative detection of avian influenza and Newcastle disease nucleic acid, so the detection results cannot reflect the real situation of virus infection in the sample

At the same time, the RT-PCR method still has certain limitations in sensitivity and specificity. It is more sensitive to certain chemical substances in the extraction process of viral nucleic acid, which in turn inhibits the PCR reaction, resulting in "false negative" results.

[0005] The concept of digital PCR (Digital PCR, dPCR) was adopted and published by Bert Vogelstein as early as 1999, and its original intention was to obtain a large number of normal somatic cells from clinical samples (such as urine, lymph, plasma, stool, etc.) A small amount of mutant cells were detected in the sample, but because the consumables that could be used to dilute samples at that time were only 384-well plates, it could not reflect the core concept of digital PCR - "terminal dilution" (terminal dilution) very well.