Method, oligonucleotide and kit for detecting high-risk HPV (human papilloma viruses)

Abstract

The invention provides a method, oligonucleotide and kit for detecting common high-risk HPV (human papilloma viruses). A fluorescence PCR (polymerase chain reaction) technology is adopted, HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 73 and 82 which possibly exist in a sample are subjected to initial detection and type identification. By the method, oligonucleotide and kit for detecting the common high-risk HPV, 18 high-risk types can be detected simultaneously, and the HPV 16 and 18 can be subjected to genetic typing simultaneously. On the basis of 18 types of high-risk HPV and affinity of other types on a phylogenetic tree, a high-risk HPV primer and a probe are designed, a background fluorescence value is reduced remarkably while detection accuracy, sensitivity and specificity are guaranteed, detection flux is increased remarkably, and detection cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of biological science and biotechnology, in particular to a method for detecting 18 common high-risk HPVs, oligonucleotides and kits, using fluorescent PCR technology to detect HPV16, 18, 31 that may exist in clinical samples , 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 73 and 82 for preliminary detection and type identification. Background technique [0002] Human papillomavirus (HPV) is a small DNA virus that can infect human epidermis and mucosal squamous epithelium, and can cause diseases such as genital warts and cervical cancer. Humans are the only host of HPV. The HPV genome is a double-stranded circular DNA, about 7900 base pairs (bp) long, containing 8 open reading frames (ORFs), consisting of 3 gene regions, including the early region (E region), the late region (L region) and non-coding regions or upstream regulatory regions. The E region can be divided into seven genes in sequence: E6, E7, E1, ...

AI Technical Summary

Problems solved by technology

Among them, because SYBRGreenI is an unsaturated dye, its specificity is not as good as that of the double-probe hybridization method and the Taqman probe method, and its specificity must be judged by observing the melting curve; and the cost of the double-probe hybridization method is relatively expensive.

[0009] At present, in the domestic market, many manufacturers have developed products based on the fluorescent PCR method to detect high-risk HPV, and most of them detect 13, 14 or 15 high-risk HPV at the same time. Only a few manufacturers use the fluorescent PCR method to develop simultaneous detection Products of 18 common high-risk HPV types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 26, 53, 66, 73 and 82), however, in Some of these products design a pair of type-specific primers and a type-specific probe for each high-risk HPV type, which will drastically increase the cost of detection, and the existence of so many probes will also significantly increase the background fluorescence signal; some It is still necessary to perform multi-tube detection, which will significantly increase the amount of samples and detection reagents, increase the cost of detection, and cannot type HPV16 and HPV18; what's more, primers and / or probes designed for different high-risk HPV The needles are limited to the same gene region, and the base sequence difference is too small, which will lead to crossover amplification reactions, false positives, and the specific types cannot be accurately distinguished

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