摘要
Objective To develop a simple,cheap,quick,accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA. Methods Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR,then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6,11,16,18,31,33,35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence. Results Compared with the results of DNA sequencing,the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection,but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them,there were 14 HPV double infections [HPV6B and 11 (9 cases),HPV11 and 16 (4),HPV11 and 18 (1)],5 HPV triple infections [HPV6B,11 and 16 (4),HPV11,16 and 18 (1)],and one HPV quadruple infection (HPV6B,11,16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2),HPV11 and 16 (1),HPV6B and 16 (1),HPV16 and 18 (1),HPV18 and 58 (1)], 3 HPV triple infections [HPV6B,11 and 16 (2),HPV11,16 and 18 (1)] and one HPV quadruple infection (HPV6B,11,16 and 18) were detected in cervical cancer scrapes.Conclusions The proposed method allowed a high throughout,special,simple,rapid,automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.
Objective To develop a simple,cheap,quick,accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA. Methods Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR,then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6,11,16,18,31,33,35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence. Results Compared with the results of DNA sequencing,the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection,but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them,there were 14 HPV double infections [HPV6B and 11 (9 cases),HPV11 and 16 (4),HPV11 and 18 (1)],5 HPV triple infections [HPV6B,11 and 16 (4),HPV11,16 and 18 (1)],and one HPV quadruple infection (HPV6B,11,16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2),HPV11 and 16 (1),HPV6B and 16 (1),HPV16 and 18 (1),HPV18 and 58 (1)], 3 HPV triple infections [HPV6B,11 and 16 (2),HPV11,16 and 18 (1)] and one HPV quadruple infection (HPV6B,11,16 and 18) were detected in cervical cancer scrapes.Conclusions The proposed method allowed a high throughout,special,simple,rapid,automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.
