Amplification and sequencing method for identification of various HPV types and genomic integration analysis

Abstract

The invention relates to an amplification and sequencing method for identification of various HPV types and genomic integration analysis. The method comprises the steps: firstly, DNA is interrupted, then the interrupted DNA is screened and purified by magnetic beads, and fragments having the main peak of about 350 bp are retained; then, normal terminal repairing is carried out and a joint is connected, and purification is carried out once by the magnetic beads; and finally, PCR amplification and enrichment are carried out, a library building product of the previous step is used as a template,a mixed solution of 29 primers and P7 terminal primers are amplified, a sequencing library is obtained after purification is carried out once by the magnetic beads, and sequencing is carried out withcooperation of an illumina sequencing platform. The method can simultaneously obtain two results of the HPV types and the human genomic integration sites by one-time sequencing; the number of reads generated by sequencing can be used as a basis for quantitative analysis to further judge the development of disease; and the result information of the HPV types obtained by sequencing is more accurateand comprehensive than that by qPCR.

Description

technical field [0001] The invention belongs to the technical field of gene diagnosis, and in particular relates to an amplification sequencing method for typing identification of various HPVs and genome integration analysis. Background technique [0002] Cervical cancer refers to a malignant tumor that occurs in the cervix and cervix. It is a common gynecological tumor, and its incidence rate is second only to breast cancer. In the past 30 years, the incidence of cervical cancer has increased by 0.6% every year, and 200,000 people in the world die from cervical cancer every year, and nearly 50,000 people die from cervical cancer every year in China. According to the pathological descriptive diagnostic method, cervical precancerous lesions are divided into high-grade squamous intraepithelial lesions (HSIL) and cervical precancerous lesions are divided into low-grade squamous intraepithelial lesions (LSIL), that is, invasive cervical cancer (ICC). ), cervical intraepithelial...

AI Technical Summary

Problems solved by technology

[0004] The existing HPV typing technology is mainly based on the qPCR platform taqman probe method detection kit. There are many brands and types, but due to methodological limitations, only 20 types of HPV typing can be detected at most.

However, the qPCR probe method is powerless for other types of HPV typing detection

The method of next-generation sequencing has also been developed and used to type HPV, but because the sensitivity is lower than that of qPCR probe method, it often misses detection and cannot be used as the gold standard for typing

Moreover, this integration will affect the gene expression of the host cell and cause changes in the cell cancer.

The conventional integration site detection method is to amplify by designing primers in the hotspot region, but the problem with this method is that firstly, the hotspot integration region is large, and a single or several amplifications cannot completely cover the entire region, and secondly, although it is defined as a hotspot In the integration area, the frequency probability is not as high as imagined. Another problem is that there are many integrations outside the hotspot integration area, and the location is very random. It cannot be detected by simple amplification and next-generation sequencing.

At present, another method for detecting integration sites is to design capture probes for known integration sites through the capture method of next-generation sequencing. The main advantage of this method is that the probes can cover many known integration sites, and the throughput The sum of information is much larger than that of simple amplification, but the disadvantage is that it is limited to the capture of HPV integration into the genome position information, and the typing results of infected free HPV cannot be obtained at the same time

Images