A NONRADIOISOTOPIC REVERSE-PHASE DIPSTICK HYBRIDIZATION METHOD FOR DETECTION OF POLYMERASE CHAIN-REACTION AMPLIFIED PRODUCT

A rapid and simple nonradibisotopic method has been developed for dete ction of polymerase chain reaction (PCR) amplified product. Digoxigeni n-11-dUTP (DIG-11-dUTP) was incorporated in the amplified product by i ncluding it in the PCR reaction mixture. The PCR product was detected colorimetrically either directly or by reverse phase hybridization met hod where an unlabelled oligonucleotide probe was immobilized on a nit rocellulose dipstick and the digoxigenin labelled PCR product was in t he liquid phase. With this system the PCR product could be detected ev en after 10 cycles of amplification by both direct and hybridization m ethods, The method was applied on the amplified product of DNA from pe ripheral blood mononuclear cells from 10 HIV-1 ELISA positive and 8 EL ISA negative individuals, PCR was positive in all ELISA positive, West ern blot positive individuals from whom HIV-1 was also isolated, PCR w as negative in all ELISA negative individuals.