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Detection of human papillomavirus DNA by PCR/microfluorometry for screening of cervical cancer
Akihiro Yamaguchi1, Naoko Hashimoto, Wataru Tsutae
1Sapporo Immunodiagnostic Laboratory, Shinkawa 2-2-12-20, Kita-ku, Sapporo 001-0922, Japan.
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.
