title: The use of mimics as internal standards to avoid false negatives in diagnostic PCR.
abstract: The PCR laboratories may face not only the frequently documented false positive results, but also unexpected false negatives. The latter are mostly due to inhibitory effects of some ingredients and/ or to pipetting errors. In order to reveal the errors, it is advisable to apply standard molecules as indicators of the efficacy of the reactions. In the present paper a rapid and simple method is presented to create internal standards for two test PCR assays. One of the assays detects proviral DNA of bovine leukemia virus (BLV-PCR), the other assay amplifies cDNA of feline infectious peritonitis virus (FIPV-RT-PCR). The internal standard molecules, termed 'mimics', were constructed to have the same primer-binding nucleotide sequences as the viral nucleic acids, but to flank a heterologous DNA fragment of different size. As heterologous DNA, a part of human beta-acin gene was used for the mimic construction. The identical primer-binding nucleotide sequences allowed co-amplification of the viral nucleic acid and the mimic in the same tube, and simultaneously, the size differences allowed easy discrimination between their PCR products. By running a rapid agarose gel electrophoresis after co-amplification, the presence or absence of the mimic PCR products provided proper information on the efficacy of the PCR in each reaction tube. We came to the conclusion that 5 to 20 mimic molecules, co-amplified with the samples, significantly increased the reliability of the diagnostic PCR assays.
authors.affiliation: Department of Virology, National Veterinary Institute, Uppsala, Sweden.
authors.forename: A
authors.initials: A
authors.lastname: Ballagi-Pordány
authors.forename: S
authors.initials: S
authors.lastname: Belák
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D005188:False Negative Reactions; D005784:Gene Amplification; D001909:Leukemia Virus, Bovine / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000378:metabolism; D012367:RNA, Viral; D012015:Reference Standards
chemical_list: D017931:DNA Primers; D012367:RNA, Viral; D004259:DNA-Directed DNA Polymerase