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.

Design and Evaluation of New Light-Upon-Extension (Lux) Real-Time PCR Primer System for Detection ofPolyomavirus Hominis 2(JC Virus) in Clinical Samples

“…We believe that this technology is suitable for routine laboratory diagnostics. in addition lUX real-time PcR can lower the performance cost of real-time PcR based research (1). the developed new approach for PcR-based detection of microorganisms utilizes genomic sequences to find speciesspecific regions for design of PCR primers targeted to these sequences.…”

Comparison of In-house and Commercial Real-time PCR Systems for the Detection of Enterobacteriaceae and their Evaluation Within an Interlaboratory Study Using Infant Formula Samples

“…Such fluorogenic PCR has the potential to be routinely used in food industries because of rapidity, simplicity, and lower cost compared with real-time PCR systems using probes for instance. LUX™ primers have been designed for use in a number of studies mainly in virology (Aitichou et al 2005;Antal et al 2007;Chen et al 2004;Nordgren et al 2008;Slavov et al 2008). However, some applications in bacteriology have been reported (Balcazar et al 2007;Kunchev et al 2007;McCrea et al 2007;Mitchell et al 2009;Xu et al 2008).…”

Rapid differentiation of vaccine strain and Chinese field strains of transmissible gastroenteritis virus by restriction fragment length polymorphism of the N gene

“…Standard serological tests that are used to detect TGEV also require considerable time to complete, and have not been able to distinguish between different strains. The results can be obtained using RT-PCR more rapidly than with the VI method or serological tests, and the RT-PCR technique has been used increasingly as a supplementary method in TGEV diagnosis [19][20][21][22][23][24][25][26][27]. Paton et al [25] detected TGEV in clinical fecal specimens using single-round PCR and was able to discriminate TGEV from PRCV.…”

“…They detected TGEV in ten intestinal and nine fecal samples, and among the nine positive fecal samples, three samples were culture negative. Chen et al [19] established a novel realtime RT-PCR assay with the LUX primer for the detection of TGEV from different TGEV strains and clinical specimens. They found that this method is more rapid, reliable and sensitive than a gel-based RT-PCR method.…”