Detection of hepatitis A virus in environmental samples by antigen-capture PCR

Abstract

The efficacy of the antigen-capture PCR (AC-PCR) method for the detection of hepatitis A virus (HAV) in environmental samples was demonstrated. HAV was captured from a seeded liquid waste or a shellfish sample with homologous antibody and then heat denatured and subjected to reverse transcription and the PCR, all in the same tube. Subsequently, the AC-PCR products were analyzed by oligonucleotide probe hybridization in solution, agarose gel electrophoresis, and autoradiography. The AC-PCR detected as little as 0.053 PFU of cell culture-adapted HAV strain HM175/18f. The results of cDNA-RNA hybridization indicated that the particle/PFU ratio of this virus strain is approximately 79:1. Therefore, the detection limit of the AC-PCR was estimated to be four virus particles. No amplified products were observed when poliovirus 1, coxsackievirus A9, coxsackievirus B3, echovirus 6, reovirus 1, adenovirus type 40, human rotavirus type 1, and bovine enterovirus type 2 were tested, confirming the specificity of the assay. There were no differences between the nucleotide sequences of AC-PCR products of HAV strain HM175/18f and the sequences of wild-type HAV strain HM175 derived from molecularly cloned cDNA. Of 121 waste and shellfish samples tested by both plaque assays (PA) in cell cultures and the AC-PCR, 81 (67%) were positive and 31 (26%) were negative as determined by both methods, whereas 9 (7%) were positive as determined by the AC-PCR and negative as determined by the PA, and none were positive as determined by the PA and negative as determined by the AC-PCR.