Pyrosequencing Applied In Hypotyping The High-risk HPV

Human paplomaviruses (HPV) belong to the Papovaviridae family. HPV were found by electron microscopy in 1949, carry circular double-stranded DNA, approximately 8 kb in length. More than 100 different HPV types, more than 30 of which infect the cervical mucosa, about 20 of which were related to tumor, have been identified on the basis of DNA sequence homology. HPV were divided into low -risk and high-risk HPV based on their relation to carcinogenic. It is important to type HPV in clinical diagnostic, for differences in the pathogenesis mechanism and the risk. HPV typing were carried out by fluorescence real-time PCR, which can't meet the clinical needs for it's low throughput. At present, HPV typing by the nucleic acid hybrid is prevail in clinical, but its sensitivity and the specificity needed to be improved. In the molecular diagnostic field, the most directly and the explicit technology is sequencing. The traditional sequencing method is often carried through the Sanger's enzymic method, by which only one sequence can be read at once .But there is more and more multi-type infection, it is very difficult to obtain the ideal result. The pyrosequencing technology is a novel sequence analysis method, the pyrophosphoric acid released after mixing the nucleotides and the template triggers the cascade reaction, urges the fluorescein to shine. This method is high-throughput, high efficiency and 5 hypotypes can be typed simultaneously, it will be popularized in detecting of increasing mixed-type HPV infection.Object: To improve the sensitivity and the accuracy of diagnostic for mixed infection, the method of typing the high-risk hypotype HPV by pyrosequencing was developed.Method: The special HPV gene hypotype sequencing primers were designed and grouped into 3 primers pools by bioinformatics; obtained the PCR products amplified by high-fidelity Taq polymerase, pyrosequenced the PCR products.Results: 18 HPV high-risk hypotype sequencing primers were designed, and were grouped into 3 primers pools. The pyrosequencing is simple, high efficiency, high flux, high-throughput high sensitivity and high accuracy method of typing HPV.