Recovery of DNA for the detection and genotyping of human papillomavirus from clinical cervical specimens stored for up to 2 years in a universal collection medium with denaturing reagent

• Published in: Journal of virological methods 2008-02, Vol.147 (2), p.333-337
• Main Authors: Campos, Elisabete A., Simões, José Antonio, Rabelo-Santos, Silvia H., Sarian, Luis Otávio, Pitta, Denise Rocha, Levi, José Eduardo, Derchain, Sophie
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; Cervical cancer; Cervical Intraepithelial Neoplasia - virology; Cervix Uteri - virology; Denaturing reagent; Detection; DNA, Viral - isolation & purification; Female; Fundamental and applied biological sciences. Psychology; Genotype; HPV DNA; Human papillomavirus; Humans; Microbiology; Papillomaviridae - classification; Papillomaviridae - genetics; Papillomaviridae - isolation & purification; Papillomavirus Infections - virology; Polymerase Chain Reaction - methods; Sensitivity and Specificity; Specimen Handling; Storage; Techniques used in virology; Time Factors; Universal collection medium; Vaginal Smears; Virology
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2007.09.014

The recovery and stability of DNA for the detection and genotyping of HPV in UCM-containing specimens, after exposure to denaturing reagents and stored for up to 2 years were evaluated. Samples were collected from 60 women who had cervical cytology specimens harboring cervical intraepithelial neoplasia (CIN) 2 or 3. All samples were stored in UCM and had been frozen at −20°C following the addition of the denaturing reagent (sodium hydroxide) and the removal of the aliquot required for Hybrid Capture 2 testing for the identification of HPV DNA. The samples had been stored for 6, 12 and 24 months (20 samples for each storage time). HPV DNA extraction was performed according to a protocol designed specifically and the presence and quality of DNA was confirmed by human β-globin detection using the consensus primers G73 and G74. HPV DNA was amplified using the consensus primers PGMY09 and PGMY11, and reverse line-blot hybridization was used to detect type-specific amplicons for 37 HPV types. The DNA extracted from the denatured specimen was recovered in 57/60 (95%) of the samples. HPV DNA was detected in 56/57 (98%) of the recovered samples. Twenty-six of the 56 samples recovered (48%) were genotyped successfully.