Designing and optimization of real-time RT-PCR technique for the detection of hepatitis C virus (HCV) genome in blood serum as internal laboratory quality control

• Published in: Przegląd epidemiologiczny Vol. 65; no. 2; p. 325
• Main Authors: Machnik, Grzegorz, Pelc, Ewa, Zapała, Małgorzata, Gasecka-Czapla, Monika, Kaczmarczyk, Grzegorz, Kozłowska, Danuta, Okopień, Bogusław
• Format: Journal Article
• Language: Polish
• Published: Poland 2011
• Subjects: Hepacivirus - genetics; Hepacivirus - isolation & purification; Hepatitis C, Chronic - blood; Hepatitis C, Chronic - virology; Humans; Poland; Quality Control; Reverse Transcriptase Polymerase Chain Reaction - methods; RNA, Viral - blood; Sensitivity and Specificity; Viral Load; Poland
• ISSN: 0033-2100

The need of hepatitis C virus (HCV) monitoring in serum samples of infected persons is of particular importance, because of chronic and non-symptomatic disease course of hepatitis C infection. We developed a novel "in-house" method variant for the detection of HCV genetic material in human blood serum. Detection technique is based on reverse transcription-real time polymerase chain reaction (RT-rPCR). We designed and analyzed several HCV 5' UTR-complementary PCR starter and probe sequence sets and we chose one set of highest HCV detection potency. Optimal concentration of starters and probe has been found. The 226-base pair long fragment of constitutively expressed glyceraldehyde 3-phosphate dehydrogenase gene served as internal endogenous control and should be added to each analysis in order to ensure that no PCR inhibitors are present. All parameters were optimized for Mx3005 QPCR System (Agilent Technology).