Research On Loop-mediated Isothermal Amplification Of Nucleic Acid Detection Technology Of HPV Genotypes

Human papillomavirus(HPV)was a kind of DNA virus,which could cause human skin and mucosa scale hyperplasia.The infections exhibited unusual warts and genital warts.The persistent infection of high-risk human papillomavirus was a major factor for the development of cervical cancer.It was trouble to distinguish the infected type from a variety of subtypes.Therefore,the classification played an important role in vaccine research,molecular epidemiology and control measures making,treatment and prognosis of HPV infection.There were many technologies available for HPV genotyping.The main method for detection of HPV infection was using the difference of gene sequences to judge the subtype at present,which based on the molecular level.The most widely used is polymerase chain reaction(PCR).Considering the expensive instruments and time-consuming,PCR was not the optimal.The relatively conserved L1 gene regions of different HPV genotypes was selected as the target fragments,which was found the all remarkable different sequences could be amplified successfully.To evaluate the analytical sensitivity and type specificity of the HPV assay,all recombinant plasmids were performed as positive standard controls of LAMP assay.In this study,two different molecular biology techniques,PCR reverse blot dot(RBD)and Real-time quantitative PCR(Taqman-qPCR),were used as the contrast to detect the HPV virus.In this paper,we selected the loop-mediated isothermal amplification to apply in high-risk type of HPV analysis,which had emerged to replace the conventional technologies.The reaction system was established by optimizing the reaction parameters.Calcein was a metal indicator,which could be used in LAMP reaction as a visual indicator to determine the results with manganese chloride through the experimental verification.We observed the change of color based on the visible dye directly.All procedures did not need the thermal cycling apparatus.The LAMP reaction was performed at 65? for 60 min.The analytical sensitivity of LAMP was tested using the HPV quantitative plasmid standard in an average range of 103 copies per reaction.LAMP generated positive results for target HPV plasmids as well as negative results for other subtypes.For the 450 viral specimens evaluated for HPV genotypes,results of the LAMP assay were 92.2%.As the central technology applying in molecular diagnostics market,reverse hybridization method was used as a control method in this study.The method could realize multiple reactions simultaneously.The sensitivity of type-specific LAMP reached at 103 copies in average.No cross-reactivity with other HPV genotype was observed.This method had a diagnostic specificity of 98% with the same 450 viral specimens.As the gold standard in diagnostic field,real-time quantitative PCR was used in the screening of the HPV infection.The detection limit of qPCR was 101 copies in most subtypes and the strong specificity was showed based on the outcomes.Compared with the qPCR method,the LAMP technology was less dependent on the instruments.It satisfied the limit requirements of the national standard.Compared with the hybridization method,the LAMP technology was simple and timesaving.The application of the technique not only saved manpower to bring a series of convenience,but also provided a new mentality of the rapid detection areas.In a word,the LAMP technology would be a kind of effective routine diagnostic tool according to its high efficiency,low-cost and accuracy.It had potential usefulness for the rapid screening of the HPV infection with the maturity.With the development of LAMP technology,the application of the technology in the field of molecular diagnostics had a bright future.