Specific primer and probe combination and reagent kit for fluorescent quantitative PCR (polymerase chain reaction) detection on 15 types of HPV (human papillomavirus)

Abstract

The invention provides a combination of specific primers and probes and a kit for fluorescent quantitative PCR detection of type 15 HPV. 3 upstream and 2 downstream primer sets and 15 specific Taqman probe sets designed for the L1 region of the HPV gene in the present invention, the corresponding kits can be quickly and easily detected in the same tube under specific PCR conditions, including16,18 , 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and other 15 high-risk HPV types and 16, 18 HPV types, reduce the cost, and do not need to open The lid operation prevents contamination, and the human actin gene is used as the internal standard gene to reduce the occurrence of false negatives.

Description

Technical field [0001] The invention relates to a qualitative kit for detecting one or more high-risk human papillomaviruses in a sample by using the fluorescent quantitative PCR (polymerase chain reaction) technology. Background technique [0002] Human papillomavirus (HPV) is a common pathogen that seriously harms human health and causes many human diseases. Its existence is closely related to the occurrence and development of a variety of skin and mucosal hyperplasia, especially cervical cancer. HPV is an epithelial double-stranded circular DNA virus. Its particles are spherical, icosahedral and symmetrical, with a diameter of 45-55 nm, and a full-length genome of about 8000 bp. At present, more than 110 HPV subtypes have been discovered in the world based on the differences in specific segments of the genome. Different genotypes of HPV have different pathogenic risks. Low-risk HPV mainly causes anal skin and external genital warts, while persistent infection of high-risk HPV...

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Problems solved by technology

[0012] The present invention is based on multiplex PCR (MultiplexPCR) technology, adding more than two pairs of primers in the same PCR reaction to simultaneously amplify the corresponding target fragments, and at the same time, in order to avoid the decrease in amplification efficiency caused by the mutual interference of too many specific primers, the present invention is based on On the basis of retaining the universal primers of different subtypes and taking into account the differences in base sequences between different types, 3 upstream primers and 2 downstream primers were used to amplify the L1 region of 15 HPV DNAs, which overcomes the traditional multiplexing When PCR detects multiple types of HPV, the dimer caused by too many primers seriously leads to the problem of decreased sensitivity and the problem of low amplification efficiency of individual types when a pair of universal primers amplifies multiple types. Design specific probes to bind to corresponding HPV subtype target sequences

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