Development of a ssRNA internal control template reagent for a multiplex RT-PCR to detect turkey astroviruses

Abstract

Turkey astrovirus (TAstV), a positive sense single-stranded RNA (ssRNA) virus, is an important causative agent of enteritis of poults. The detection and diagnosis of astroviruses have been mainly dependent on electron microscopy (EM). However, EM is not very sensitive. Reverse transcription-polymerase chain reaction (RT-PCR) has high specificity and sensitivity. Thus, monoplex RT-PCR and multiplex RT-PCR for detection of TAstVs were developed in our laboratory. RT-PCR could be adversely affected by many factors, which would result in lowering the sensitivity of the reaction. To minimize this pitfall of RT-PCR, a ssRNA internal control (IC) template reagent containing two sets of primer sequences (SRV and AFCP) specific to the capsid region of TAstV genomes was developed, and applied to the multiplex RT-PCR. Sixty-four fecal specimens from 2-week-old poults with enteritis were tested using the multiplex RT-PCR with the IC serving as a co-amplification template. An overall test inhibition rate of 12.5% was found for the RT-PCR, which can be used to decrease the false-negative rate. The approach to the generation of the IC developed in this study is simple, convenient and productive, and can be used as a universal protocol to generate a ssRNA IC template reagent for RT-PCR.