Competitor template RNA for detection and quantitation of hepatitis A virus by PCR.
PCR was used to introduce a 63-bp deletion into the putative RNA replicase coding sequence of hepatitis A virus. RNA was synthesized in vitro from the deletion mutant cloned into a transcription vector. Upon amplification by PCR, cDNA made from the competitor RNA generated an amplified fragment that could be easily distinguished from the product generated from wild-type hepatitis A virus genomic RNA by gel electrophoresis, when the same primers were used, without further manipulation. The competitor RNA was used as a positive control in PCR-based detection of very low copy numbers of hepatitis A virus genomic RNA in the presence 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 inhibited product formation from the underrepresented template. The competitor RNA should be useful as a control for reverse transcription and PCRs to determine hepatitis A virus genome RNA when accidental contamination of test samples by a wild-type positive control template would compromise the results.
Goswami
B B
BB
Division of Molecular Biological Research and Evaluation, Food and Drug Administration, Washington, DC 20204.
Koch
W H
WH
Cebula
T A
TA
D001483:Base Sequence; D006507:Hepatovirus / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*
D012367:RNA, Viral