Use of reverse transcription and PCR to discriminate between infectious and non-infectious hepatitis A virus.
Hepatitis A virus (HAV) is a major cause of infectious hepatitis worldwide. Detection of HAV in contaminated food or water is a priority research area in laboratories worldwide. Our laboratory has reported previously the development of reverse transcription-polymerase chain reaction (RT-PCR) based detection and typing methods for HAV in contaminated shellfish and produce. It is commonly held that RT-PCR can detect viral genome, but cannot distinguish between infectious and inactivated virus. Therefore, signals obtained after PCR should be considered as false positives unless it can be shown that the sample contains virus capable of infecting a suitable host cell line in culture. We present data to show that this general assumption is not valid. Evidence is provided that demonstrate that signals generated after RT-PCR amplification of viral genome correlated well with the presence of infectious virus in the sample. Viral samples inactivated by heat or UV treatment produced significantly lower signal strength that paralleled infectivity of the sample in cultured cells. The loss of signal strength is most likely the result of damage to the viral RNA that renders it unsuitable for RT-PCR. The correlation between PCR signal and infectivity was better following UV inactivation than heat treatment. The procedure may be adapted to other viruses and inactivating agents.
Bhattacharya
Siddhartha S
SS
Division of Molecular Biology, HFS-025, Office of Applied Research and Safety Assessment, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD 20708, USA.
Kulka
Michael
M
Lampel
Keith A
KA
Cebula
Thomas A
TA
Goswami
Biswendu B
BB
D001483:Base Sequence; D017931:DNA Primers; D016679:Genome, Viral; D030041:Hepatitis A virus / Q000302:isolation & purification* / Q000472:pathogenicity* / Q000528:radiation effects; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014466:Ultraviolet Rays; D014779:Virus Replication; D014871:Water Microbiology
D017931:DNA Primers; D012367:RNA, Viral