M Daskou1, D Tsakogiannis2, T G Dimitriou1, G D Amoutzias3, D Mossialos1, C Kottaridi4, C Gartzonika5, P Markoulatos1. 1. Microbiology-Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Biopolis, 41500, Larissa, Greece. 2. Microbiology-Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Biopolis, 41500, Larissa, Greece. Electronic address: ditsakog@uth.gr. 3. Bioinformatics Laboratory, University of Thessaly, School of Health Sciences, Department of Biochemistry & Biotechnology, BIOPOLIS, Larissa, Greece. 4. Department of Cytopathology, National and Kapodistrian University of Athens, Medical School, "ATTIKON" University Hospital, 1 Rimini, Haidari, 12462, Athens, Greece. 5. Department of Microbiology, University of Ioannina, Medical School, Ioannina, Greece.
Abstract
BACKGROUND AND OBJECTIVES: Persistent infection with High-Risk HPV genotypes is the principal cause for the development of cervical cancer with HPV16 and HPV18 to be the most frequently identified HPV genotypes observed in approximately 70% of cervical cancer cases worldwide. The present study focused on the development of a simple molecular methodology based on WarmStart colorimetric LAMP for the specific identification of HPV16 and HPV18. METHODS: The method was developed by designing LAMP type-specific primer sets that target the E6 gene. The assay was applied using HPV-positive clinical samples along with control cases in order to evaluate the specificity of the newly designed isothermal protocol. In addition, an experimental cutoff value was estimated through reconstitution experiments with HPV-DNA plasmids. LAMP amplicons were visualized by color changes, thus eliminating the requirement for post-amplification processing steps. RESULTS: The WarmStart colorimetric LAMP facilitates the isothermal amplification of 10 copies per reaction of both HPV16 and HPV18 DNA, while it exhibits 100% specificity for the detection of the corresponding genotypes in LSIL and HSIL cases. Moreover, the assay demonstrates 100% PPV and 100% NPV. Finally, the sensitivity of conventional PCR with the type-specific LAMP primer sets (B3/F3) for the HPV16, HPV18 DNA detection was 100 copies/reaction and 10 copies/reaction, respectively. CONCLUSIONS: The newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes.
BACKGROUND AND OBJECTIVES: Persistent infection with High-Risk HPV genotypes is the principal cause for the development of cervical cancer with HPV16 and HPV18 to be the most frequently identified HPV genotypes observed in approximately 70% of cervical cancer cases worldwide. The present study focused on the development of a simple molecular methodology based on WarmStart colorimetric LAMP for the specific identification of HPV16 and HPV18. METHODS: The method was developed by designing LAMP type-specific primer sets that target the E6 gene. The assay was applied using HPV-positive clinical samples along with control cases in order to evaluate the specificity of the newly designed isothermal protocol. In addition, an experimental cutoff value was estimated through reconstitution experiments with HPV-DNA plasmids. LAMP amplicons were visualized by color changes, thus eliminating the requirement for post-amplification processing steps. RESULTS: The WarmStart colorimetric LAMP facilitates the isothermal amplification of 10 copies per reaction of both HPV16 and HPV18 DNA, while it exhibits 100% specificity for the detection of the corresponding genotypes in LSIL and HSIL cases. Moreover, the assay demonstrates 100% PPV and 100% NPV. Finally, the sensitivity of conventional PCR with the type-specific LAMP primer sets (B3/F3) for the HPV16, HPV18 DNA detection was 100 copies/reaction and 10 copies/reaction, respectively. CONCLUSIONS: The newly established WarmStart colorimetric LAMP can be considered as a powerful molecular tool that it can be easily implemented in small clinical and research laboratories for a rapid and efficient identification of the most tumorigenic HPV genotypes.
Authors: Jiasi Wang; Jeannette P Staheli; Andrew Wu; Jason E Kreutz; Qiongzheng Hu; Jingang Wang; Thomas Schneider; Bryant S Fujimoto; Yuling Qin; Gloria S Yen; Bob Weng; Kara Shibley; Halia Haynes; Rachel L Winer; Qinghua Feng; Daniel T Chiu Journal: Anal Chem Date: 2021-02-03 Impact factor: 6.986