A universal avian endogenous real-time reverse transcriptase-polymerase chain reaction control and its application to avian influenza diagnosis and quantification.

ABSTRACT

Real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) is becoming an established first-line diagnostic assay as well as a precise quantification tool for avian influenza virus detection. However, there remain some limitations. First, we show that the sensitivity of RRT-PCR influenza detection can be 10- to 100-fold inhibited in oropharyngeal and cloacal swabs. Adding 0.5 U of heat-activated Taq DNA polymerase successfully reverses PCR inhibition. Second, an excellent strategy for detecting false negative samples is the coamplification of an internal control from each sample. We developed a universal avian endogenous internal control (bird beta-actin) and apply it to influenza A diagnosis. Moreover, this internal control proves useful as a normalizer control for virus quantification, because beta-actin gene expression does not change in infected vs. uninfected ducks. A combined panel of wild bird cloacal swabs, wild bird tissue samples, experimental duck swabs, and experimental duck and chicken tissue samples was used to validate the endogenous control. The application of an endogenous internal control proves an excellent strategy both for avoiding false negative diagnostic results and for standardizing virus quantification studies.