Detection of swine transmissible gastroenteritis coronavirus using loop-mediated isothermal amplification

Abstract

A conserved nucleic acid fragment of the nucleocapsid gene of Swine Transmissible Gastroenteritis Coronavirus (TGEV) was chosen as the target, six special primers were designed successfully. Loop-mediated isothermal amplification (LAMP) was developed to detect the TGEV by incubation at 60°C for 1 h and the product specificity was confirmed by HphI digestion. Standard curves with high accuracy for TGEV quantization was constructed by adding 1 × SYBR greenI in the LAMP reaction. The assay established in this study was found to detect only the TGEV and no cross-reaction with other viruses, demonstrating its high specificity. By using serial sample dilutions as templates, the detection limit of LAMP was about 10 pg RNA, 10 times more sensitive than that of PCR and could be comparable to the nest-PCR.

Figure 1

The conserved fragment of the nucleocapsid gene from TGEV.

Figure 2

Analysis of TGEV by LAMP with agarose gel electrophoresis. 1, LAMP products of TGEV; 2, LAMP products digested with Hph I; 3, Negative control.

Figure 3

Dynamic curves of the different TGEV standards.

Figure 4

Standard curves of real-time fluorescence TGEV-LAMP.

Figure 5

Specificity of LAMP for TGEV detection. 1-6, TGEV, PRRSV, PRV, CSFV, PPV and FMDV respectively; 7, Negative Control.

Figure 6

Sensitivity of LAMP for TGEV detection. A, LAMP; B, PCR; C, Nest-PCR; 1-7, TGEV RNA sample at 10^-1, 10^-2...10^-7 dilutions respectively; 8. Negative Control.