Revista medico-chirurgicala a Societatii de Medici si Naturalisti din Iasi · 2009
[Enhanced molecular techniques for the diagnosis of human papillomavirus infections].
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Ursu Ramona Gabriela, Onofriescu M, Nemescu D, Iancu Luminiţa Smaranda
Аннотация
OBJECTIVES: optimisation of Real Time PCR technique for quantifying oncogenic types 16 and 18 of Human Papilloma Viruses, genotyped through classic PCR, followed by hybridisation.
MATERIAL AND METHOD: DNA/ HPV was purified with High Pure PCR Template Preparation kit (ROCHE DIAGNOSTICS), genotyping was performed with Linear Array HPV Genotyping (ROCHE DIAGNOSTICS) and PCR reaction was realized with ABI 9700 Gold Plate System. Absolute quantification of HPV 16 and 18 was performed with Path-HPV16/18 Real-time PCR detection kit for Human Papillomavirus, 2 x Precision Mastermix kits (PrimerDesign), and the instrument used was MX3000P STRATAGENE.
RESULTS: I. HPV genotyping was optimised through testing of 12 cervical samples, collected from patients who have signed the informed consent approved by the local Bioethical Committee. Among the tested samples, 5 were negative for any HPV type, 3 patients had unique infections with oncogenic HPV type, and 2 patients had multiple infections, with oncogenic and non-oncogenic HPV types. Negative and positive controls were validated, identical as the internal control - beta globin gene. II. Absolute quantification for HPV 16 and 18 were performed on two samples tested by the previous method. The number of viral copies was determined using the standard curves procedure, whose parameters values were between the accepted limits.
CONCLUSIONS: We fulfilled the quality criteria for both techniques: genotyping assay and viral load quantification by Real Time PCR. This allows us to start the study for monitoring persistent infections with HPV 16 and HPV 18.
MATERIAL AND METHOD: DNA/ HPV was purified with High Pure PCR Template Preparation kit (ROCHE DIAGNOSTICS), genotyping was performed with Linear Array HPV Genotyping (ROCHE DIAGNOSTICS) and PCR reaction was realized with ABI 9700 Gold Plate System. Absolute quantification of HPV 16 and 18 was performed with Path-HPV16/18 Real-time PCR detection kit for Human Papillomavirus, 2 x Precision Mastermix kits (PrimerDesign), and the instrument used was MX3000P STRATAGENE.
RESULTS: I. HPV genotyping was optimised through testing of 12 cervical samples, collected from patients who have signed the informed consent approved by the local Bioethical Committee. Among the tested samples, 5 were negative for any HPV type, 3 patients had unique infections with oncogenic HPV type, and 2 patients had multiple infections, with oncogenic and non-oncogenic HPV types. Negative and positive controls were validated, identical as the internal control - beta globin gene. II. Absolute quantification for HPV 16 and 18 were performed on two samples tested by the previous method. The number of viral copies was determined using the standard curves procedure, whose parameters values were between the accepted limits.
CONCLUSIONS: We fulfilled the quality criteria for both techniques: genotyping assay and viral load quantification by Real Time PCR. This allows us to start the study for monitoring persistent infections with HPV 16 and HPV 18.