Detection of Serum HCV RNA by Rapid Cycle PCR Using Hot Air Thermocycler with Capillary Tubes

Youn, Mi Yae; Lee, Hye Soo; Kim, Dal Sik; Choi, Sam Im; Wittwer, Carl T

Abstract

BACKGROUND: Reverse transcription-polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of Hepatitis C virus (HCV) RNA from serum. The PCR by conventional heat block thermocycler using small plastic tube is time and reagent consuming procedure, but rapid cycle PCR (RPCR) by hot air thermocycler using glass capillary tube is very rapid and economic. Therefore, RPCR have been recognized as a very convenient method for routine diagnostic test in clinical laboratories, but there are few reports about its usage for the detection of HCV RNA.
METHODS
We selected two sets of primer pair from 5'noncoding region of HCV RNA genome, and optimized RPCR condition using hot air rapid thermocycler with master mix in capillary tubes. And RT-RPCR for detection of HCV RNA were performed on the serum of 58 patients, which were tested anti -HCV antibody by EIA.
RESULTS
The optimized RPCR conditions were: denaturation; 94 degrees C for "0" sec, annealing; 55 degrees C (first) and 50 degrees C (nested) for "0" sec, elongation; 72 degrees C for "0" sec, and amplification cycles were 30 cycles. The consuming times per cycle were 30 sec (first) and 40 sec (nested), respecti vely, so the total involving times for nested RPCR were 35 min. Of the 42 EIA positive samples, 26 (62%) were RT-RPCR positive.
CONCLUSIONS
RT-RPCR using hot air thermocycler with glass capillary tubes for detection of HCV RNA in serum is very rapid and economic than conventional PCR using heat block thermocycler. Therefore HCV RNA detection by RT-RPCR appears to be very useful for routine clinical laboratory diagnostic method.