Design method of primers and probes for detecting pathogens

Abstract

The invention provides a design method of primers and probes for detecting multiple subtypes of pathogens such as high-risk HPV. The designed primers and probes can be used for preliminary detection and type identification of multiple subtypes of the pathogens that is possible to exist in samples. Based on the affinity of multiple subtypes of the pathogens such as high-risk HPV in evolutionary trees, the primers and probes for multiple subtypes of the pathogens are designed, background fluorescence value can be significantly reduced, detection flux is obviously increased and the detection costis greatly reduced while detection accuracy, sensitivity and specificity are ensured.

Description

[0001] This application is an application number: 201610027771.9, application name: a divisional application for the method, oligonucleotide and kit for detecting high-risk HPV. technical field [0002] The invention belongs to the field of biological science and biotechnology, and in particular relates to a method for designing primers and probes for detecting multiple subtypes of pathogens. The designed primers and probes can be used to detect high-risk HPV that may exist in clinical samples, etc. Various subtypes of pathogens were initially detected and identified. Background technique [0003] 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 ear...

AI Technical Summary

Problems solved by technology

Among them, because SYBR Green I 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.

[0010] 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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