Development of Multiplex PCR Detection of Blood-borne Viruses by Nucleic Acid Hybridization

ABSTRACT

Polymerase chain reaction (PCR) has been used as a substitute for conventional serological methods in order to provide blood or blood products free from contaminating viruses and recently attempts have focused to detect 2 or 3 viruses by a single multiplex PCR (M-PCR) reaction. We were able to detect human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV) and human cytomegalovirus (HCMV) simultaneously by a single M-PCR. However detection by gel electrophoresis of the products from M-PCR suffers from drawbacks such as low sensitivity and product sizes. Here we report enhanced detection systems of M-PCR based on nucleic acid hybridization with arrays built on membrane. Membrane array was manufactured by spotting appropriate probe DNAs on nylon membrane. Single or multiplex PCR was performed and the PCR products were labeled with DIG and allowed to hybridize with the membrane array. Results indicate that nonspecific hybridization was not observed for membrane DNA array. Additionally, membrane array method could detect small amount of viruses that were not detectable by conventional gel electrophoresis. At least 25-fold, and in some cases more than 125-fold increases in sensitivity was obtained with DNA array method. Thus, the nucleic acid hybridization with membrane array could be applied for the detection of M-PCR of viruses in blood or blood products.