Hybrid capture assay

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A New PCR-Based Assay Amplifies the E6-E7 Genes of Most Mucosal Human Papillomaviruses (HPV)

Journal Title, Volume, Page: 
Virus Research Volume 67, Issue 2, Pages 127-139
Year of Publication: 
2000
Authors: 
Walid Basha
School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan
Current Affiliation: 
Faculty of Medicine & Health Sciences, Department of Biomedical Sciences, An-Najah National University, Nablus, Palestine
Toshiyuki Sasagawa
School of Health Sciences, Faculty of Medicine, Kanazawa University, 5-11-80 Kodatsuno, Kanazawa, Ishikawa 920-0942, Japan
Yuzuru Minemoto
Graduate School of Natural Science and Technology, Kanazawa University, 13-1, Kanazawa, Takara-machi 920-0934, Japan
Hiroshi Yamazaki
Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 13-1 Takaramachif Kanazawa, Ishikawa 920-8641, Japan
Mitsuo Nakamura
Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 13-1 Takaramachif Kanazawa, Ishikawa 920-8641, Japan
Hideo Yoshimoto
Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 13-1 Takaramachif Kanazawa, Ishikawa 920-8641, Japan
Jun Sakaike
Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 13-1 Takaramachif Kanazawa, Ishikawa 920-8641, Japan
Masaki Inoue
Department of Obstetrics and Gynecology, School of Medicine, Kanazawa University, 13-1 Takaramachif Kanazawa, Ishikawa 920-8641, Japan
Preferred Abstract (Original): 

We established a new assay to detect the E6–E7 DNA of mucosal human papillomaviruses (HPV) by a PCR-based method using four pairs of degenerate LCR and E7 primers (LCR-E7 PCR). This assay amplifies the full length of E6 and the N-terminal part of E7. HPV typing was performed using restriction-fragment-length polymorphism (RFLP), and by analyzing the sequences of cloned PCR products. We compared this assay with the first generation hybrid captured assay (HCA-I) and the MY09/11-PCR method. LCR-E7 PCR was able to detect more than 34 mucosal HPV types and theoretically should detect two additional types. LCR-157 PCR and HCA-I detected HPV DNA in 70% (69/99) and 55% (54/99) of low-grade cervical intraepithelial lesions (LSIL), 89% (105/118) and 76% (90/118) of high-grade cervical intraepithelial lesions (HSIL), and 90% (56/62) and 79% (49/62) of invasive squamous cell carcinomas (SCC), respectively. LCR-E7 PCR was more sensitive than the HCA-1 test. Discordant results between the LCR-E7 and MY 11/09-PCR tests were observed in one of 185 (0.5%) normal samples, seven of 85 (8.2%) LSIL samples, seven of 82 (8.5%) HSIL samples, and four of 72 (5.6%) SCC samples. The discordant results were mostly observed in samples with a low-copy number of the HPV genome or with multiple HPV infection. The sensitivity of LCR-E7 PCR was equivalent to that of MY 11/09 ECR, and false positives were less frequent in LCR-E7 PCR. LCR-E7 PCR may be useful for determining the biological activity of detected HPV types, since this method amplifies the entire E6 gene.

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