The use of mimics as internal standards to avoid false negatives in diagnostic PCR. 1996

A Ballagi-Pordány, and S Belák
Department of Virology, National Veterinary Institute, Uppsala, Sweden.

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.

UI MeSH Term Description Entries
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D011533 Proviruses Duplex DNA sequences in eukaryotic chromosomes, corresponding to the genome of a virus, that are transmitted from one cell generation to the next without causing lysis of the host. Proviruses are often associated with neoplastic cell transformation and are key features of retrovirus biology. Provirus
D012015 Reference Standards A basis of value established for the measure of quantity, weight, extent or quality, e.g. weight standards, standard solutions, methods, techniques, and procedures used in diagnosis and therapy. Standard Preparations,Standards, Reference,Preparations, Standard,Standardization,Standards,Preparation, Standard,Reference Standard,Standard Preparation,Standard, Reference
D001909 Leukemia Virus, Bovine The type species of DELTARETROVIRUS that causes a form of bovine lymphosarcoma (ENZOOTIC BOVINE LEUKOSIS) or persistent lymphocytosis. BLV,Bovine Leukemia Virus,Cattle Leukemia Virus,Leukemia Virus, Cattle,Virus, Bovine Leukemia,Virus, Cattle Leukemia
D004259 DNA-Directed DNA Polymerase DNA-dependent DNA polymerases found in bacteria, animal and plant cells. During the replication process, these enzymes catalyze the addition of deoxyribonucleotide residues to the end of a DNA strand in the presence of DNA as template-primer. They also possess exonuclease activity and therefore function in DNA repair. DNA Polymerase,DNA Polymerases,DNA-Dependent DNA Polymerases,DNA Polymerase N3,DNA Dependent DNA Polymerases,DNA Directed DNA Polymerase,DNA Polymerase, DNA-Directed,DNA Polymerases, DNA-Dependent,Polymerase N3, DNA,Polymerase, DNA,Polymerase, DNA-Directed DNA,Polymerases, DNA,Polymerases, DNA-Dependent DNA
D004587 Electrophoresis, Agar Gel Electrophoresis in which agar or agarose gel is used as the diffusion medium. Electrophoresis, Agarose Gel,Agar Gel Electrophoresis,Agarose Gel Electrophoresis,Gel Electrophoresis, Agar,Gel Electrophoresis, Agarose
D005188 False Negative Reactions Negative test results in subjects who possess the attribute for which the test is conducted. The labeling of diseased persons as healthy when screening in the detection of disease. (Last, A Dictionary of Epidemiology, 2d ed) False Negative Reaction,Reaction, False Negative,Reactions, False Negative
D005784 Gene Amplification A selective increase in the number of copies of a gene coding for a specific protein without a proportional increase in other genes. It occurs naturally via the excision of a copy of the repeating sequence from the chromosome and its extrachromosomal replication in a plasmid, or via the production of an RNA transcript of the entire repeating sequence of ribosomal RNA followed by the reverse transcription of the molecule to produce an additional copy of the original DNA sequence. Laboratory techniques have been introduced for inducing disproportional replication by unequal crossing over, uptake of DNA from lysed cells, or generation of extrachromosomal sequences from rolling circle replication. Amplification, Gene
D001483 Base Sequence The sequence of PURINES and PYRIMIDINES in nucleic acids and polynucleotides. It is also called nucleotide sequence. DNA Sequence,Nucleotide Sequence,RNA Sequence,DNA Sequences,Base Sequences,Nucleotide Sequences,RNA Sequences,Sequence, Base,Sequence, DNA,Sequence, Nucleotide,Sequence, RNA,Sequences, Base,Sequences, DNA,Sequences, Nucleotide,Sequences, RNA
D012367 RNA, Viral Ribonucleic acid that makes up the genetic material of viruses. Viral RNA

Related Publications

A Ballagi-Pordány, and S Belák
February 1993, BioTechniques,
A Ballagi-Pordány, and S Belák
January 2004, Indian journal of pediatrics,
A Ballagi-Pordány, and S Belák
November 2009, Regulatory peptides,
A Ballagi-Pordány, and S Belák
February 1994, PCR methods and applications,
A Ballagi-Pordány, and S Belák
September 1992, Journal of virological methods,
A Ballagi-Pordány, and S Belák
September 1995, BioTechniques,
A Ballagi-Pordány, and S Belák
January 1992, Applied and theoretical electrophoresis : the official journal of the International Electrophoresis Society,
A Ballagi-Pordány, and S Belák
January 1988, Urology,
Copied contents to your clipboard!