BACKGROUND: Cervical cancer screening is conducted by a cytological Papanicolaou (Pap) test. For screening, it is becoming increasingly important to introduce a more objective result, based on human papillomavirus (HPV) DNA test. We describe here a practical method allowing the mass detection of HPV-DNA by PCR followed by fluorogenic DNA intercalation. METHODS: Samples used were cervical scrapes or biopsy specimens obtained from women who had undergone cytological testing for cervical cancer. Crude DNAs were extracted by a simplified proteinase K-boil method. Common and type-specific primers were newly designed for major types of high-risk HPVs. A fluorogenic DNA intercalator, SYBR Green I was directly added to the specific PCR products. The resultant fluorescence was measured by a conventional fluorometric microplate reader. RESULTS: The proposed PCR/microfluorometry (MFL) allowed a simple, rapid and economical detection of HPV-DNA without any use of labeling primers or probes. HPV-DNAs were found in 48.2% (123/255) of the cervical scrapes. The detection rate of HPV in cervical cancer biopsy specimen was 92.4% (61/66). CONCLUSIONS: PCR/MFL detection of HPV-DNA, followed by combined type-specific PCR, is expected to be an extremely useful tool in cervical cancer screening.
BACKGROUND:Cervical cancer screening is conducted by a cytological Papanicolaou (Pap) test. For screening, it is becoming increasingly important to introduce a more objective result, based on human papillomavirus (HPV) DNA test. We describe here a practical method allowing the mass detection of HPV-DNA by PCR followed by fluorogenic DNA intercalation. METHODS: Samples used were cervical scrapes or biopsy specimens obtained from women who had undergone cytological testing for cervical cancer. Crude DNAs were extracted by a simplified proteinase K-boil method. Common and type-specific primers were newly designed for major types of high-risk HPVs. A fluorogenic DNA intercalator, SYBR Green I was directly added to the specific PCR products. The resultant fluorescence was measured by a conventional fluorometric microplate reader. RESULTS: The proposed PCR/microfluorometry (MFL) allowed a simple, rapid and economical detection of HPV-DNA without any use of labeling primers or probes. HPV-DNAs were found in 48.2% (123/255) of the cervical scrapes. The detection rate of HPV in cervical cancer biopsy specimen was 92.4% (61/66). CONCLUSIONS: PCR/MFL detection of HPV-DNA, followed by combined type-specific PCR, is expected to be an extremely useful tool in cervical cancer screening.
Authors: S R Gadelha; D M V Soares-Barreto; G B Costa; V C N Leal; L G S Gomes; U R Santos; G C S Ferreira; L D Carvalho; S M V Soraes-Almeida; M A G Mello; A P M Mariano; S M B Sousa; A R Vago; L J Marin Journal: Epidemiol Infect Date: 2017-11-23 Impact factor: 4.434