Detection of viral sequences by internally calibrated gene amplification.
Inherent pitfalls of the polymerase chain reaction (PCR) can become serious difficulties when transferring research applications to high-volume routine procedures such as biofermentation process control and clinical diagnostics. Difficulties include 1) the danger of accidental sample contamination with positive control templates; 2) variable amplification due to positional effects in thermocycler blocks and unequal primer efficiency for sense/anti-sense strands; and 3) the need for reliable controls, which provide confidence for reporting negative reactions. Using the PCR detection system for Epstein-Barr virus as a model, we have developed a quick process to generate mutant internal co-amplification templates. These can be used for titration of amplification sensitivity. More importantly, single tube co-amplification without titrations allows determination of the minimum sensitivity achieved in each individual reaction; critical information when reporting negative diagnostic results. Mutant and native fragments are easy to distinguish by size, and sample cross contamination can be readily identified. The system should be easily adaptable to gene amplification procedures, which aim to routinely detect the presence of a given gene fragment in a controlled fashion.
Cheung
R K
RK
Department of Pediatrics, University of Toronto, Hospital For Sick Children, Research Institute, Ontario, Canada.
Hui
M F
MF
Dosch
H M
HM
Ewart
T E
TE
D000956:Antigens, Viral / Q000235:genetics; D001483:Base Sequence; D001709:Biotechnology; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D019309:Epstein-Barr Virus Nuclear Antigens; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016296:Mutagenesis; D021141:Nucleic Acid Amplification Techniques
D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens