With the standardization of the sample-prep steps and the conventional qPCR reactions using SYBR® Green detection methodology, Dr. Crotty and his team developed a sensitive, robust assay for the detection of viral infection in vivo. The assay is more than 1,000-fold more sensitive than standard plaque assays for tracking LCMV infection in mice, he reports.
In the molecular diagnostics reference laboratory at Idexx Reference Laboratories, the requirement that real-time PCR provides analytical and diagnostic specificity and sensitivity is key to molecular diagnostics for infectious agents. On a daily basis, Christian Leutenegger, Ph.D., regional head of molecular diagnostics, and his laboratory are faced with two challenges: to maintain a portfolio of fully validated qPCR assays and to maintain the quality control for each assay.
As for the first challenge, PCR primer-sequence design is the solution. The specificity of the reaction is based on the balance between false-positive and false-negative signals. The former comes from cross-reactive signals from related but not etiological agents. False-negative signals come from sequence variation based on selection pressure and normal drift in the genetic isolates.
Regardless of the type of nucleic acid in the infectious agent including viral, parasites, fungi, or bacteria, all infectious agents demonstrate sequence variation. For optimal sequence design, it is important to query all the public databases for identification of both highly conserved and highly variable regions within infectious agents. This is a numbers game, so when the number of published sequence variants is high, the investigator can have confidence in the validity of the defined conserved and variable regions of the genomes.
Dr. Leutenegger and his colleagues routinely resequence positive samples to look for new sequence variants and then add the new sequence information to their working database. Being vigilant to do the bioinformatics workup for all qPCR assays is fundamental to proper assay design and validation. They apply this process in cycles, initially designing and validating the assay, then resequencing and redesigning, and finally revalidating the diagnostic assay.
PCR primers designed to bind within highly conserved regions risk high levels of cross reactivity to related strains that are not the etiological agent. PCR primers designed to bind within highly variable regions yield high specificity but risk high incidence of false-negative results. The answer is to design in a balance between these extremes.
Quality control in the laboratory is the other challenge, having the confidence in every negative result and every positive result. To maintain quality control in the laboratory process, controls from sample prep through amplification and finally resequencing are strictly enforced. The direction of the workflow is designed to minimize the chance for contamination.
“We accept the fact that no PCR laboratory is without contamination,” states Dr. Leutenegger, “so we employ a further level of control by incorporating the AmpErase system in all PCR assays.” This means that all amplification assays are subject to enzymatic digestion of any contaminating amplicons based on the incorporation of uracils into the product.