The use of mimics as internal standards to avoid false negatives in diagnostic PCR

Mol Cell Probes. 1996 Jun;10(3):159-64. doi: 10.1006/mcpr.1996.0022.


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.

Publication types

  • Comparative Study

MeSH terms

  • Base Sequence
  • DNA Primers / genetics
  • DNA-Directed DNA Polymerase / genetics
  • Electrophoresis, Agar Gel
  • False Negative Reactions
  • Gene Amplification
  • Leukemia Virus, Bovine / genetics
  • Molecular Sequence Data
  • Polymerase Chain Reaction / methods*
  • Proviruses / metabolism
  • RNA, Viral
  • Reference Standards


  • DNA Primers
  • RNA, Viral
  • DNA-Directed DNA Polymerase