COMPETITOR TEMPLATE RNA FOR DETECTION AND QUANTITATION OF HEPATITIS-AVIRUS BY PCR

PCR was used to introduce a 63-bp deletion into the putative RNA repli case coding sequence of hepatitis A virus. RNA was synthesized in vitr o from the deletion mutant cloned into a transcription vector. Upon am plication by PCR, cDNA made from the competitor RNA generated an ampli fied fragment that could be easily distinguished from the product gene rated front wild-type hepatitis A virus genomic RNA by gel electrophor esis, when the same primers were used, without further. manipulation. The competitor RNA was used as a positive control in PCR-based detecti on of very low copy numbers of hepatitis A virus genomic RNA in rite p resence of unrelated hard-shell clam RNA. When the competitor RNA was used for competitive PCR to quantitate wild-type RNA, the presence of one template at a 10-fold to 100-fold higher level almost completely i nhibited product formation from the underrepresented template. The com petitor RNA should be useful as a control for reverse transcription an d PCRs to determine hepatitis A virus genome RNA when accidental conta mination of test samples by a wild-type positive control template woul d compromise the results.