Development of a novel real-time RT-PCR assay with LUX primer for the detection of swine transmissible gastroenteritis virus

Abstract

Real-time RT-PCR assay, based on light upon extension (LUX) fluorogenic primer and LightCycle technology, was developed for rapid detection of transmissible gastroenteritis virus (TGEV). Viral RNA from different TGEV isolates and clinical specimens was detected. To evaluate the sensitivity of the assay, a gel-based RT-PCR method targeted at the same 101 bp sequence was also developed. Serial 10-fold dilutions of TGEV RNA were detected by the two methods. Although the real time method used only 2 microl RNA for each reaction, a 10-fold increase of sensitivity over that of the gel-based method, which used 10 microl RNA was demonstrated. The study indicates that the LUX assay reported below is rapid, reliable and sensitive and it has the potential for use as an alternative molecular method for TGEV diagnosis.

Fig. 1

The results obtained by the LUX and RT-PCR assays on the dilution series of TGEV-Purduce115 RNA. LightCycle real-time fluorescence signal curve and the C_T value for each dilution is shown. The inset depicts agarose gel electrophoresis of the RT-PCR products (101 bp). M, 100 bp DNA ladder; N, negative control.