Detection of hepatitis A virus by the nucleic acid sequence-based amplification technique and comparison with reverse transcription-PCR

A nucleic acid sequence-based amplification (NASBA) technique for the detec tion of hepatitis A virus (HAV) in foods was developed and compared to the traditional reverse transcription (RT)-PCR technique. Oligonucleotide prime rs targeting the VP1 and VP2 genes encoding the major HAV capsid proteins w ere used for the amplification of viral RNA in an isothermal process result ing in the accumulation of RNA amplicons. Amplicons were detected by hybrid ization with a digoxigenin-labeled oligonucleotide probe in a dot blot assa y format. Using the NASBA, as little as 0.4 ng of target RNA/ml was detecte d per comparison to 4 ng/ml for RT-PCR. When crude HAV viral lysate was use d, a detection limit of 2 PFU (4 X 10(2) PFU/ml) was obtained with NASBA, c ompared to 50 PFU (1 X 10(4) PFU/ml) obtained with RT-PCR. No interference was encountered in the amplification of HAV RNA in the presence of excess n ontarget RNA or DNA. The NASBA system successfully detected HAV recovered f rom experimentally inoculated samples of waste water, lettuce, and blueberr ies. Compared to RT-PCR and other amplification techniques, the NASBA syste m offers several advantages in terms of sensitivity, rapidity, and simplici ty. This technique should be readily adaptable for detection of other RNA v iruses in both foods and clinical samples.