Invasive Species Compendium

Detailed coverage of invasive species threatening livelihoods and the environment worldwide

Abstract

Establishment and application of a real-time RT-PCR based on SYBR Green II for detection of porcine sapelovirus.

Abstract

A TaqMan real-time RT-PCR assay was established for the detection of porcine sapelovirus. A pair of special primers was designed according to the highly conserved fragment of the 5′-UTR of gene in GenBank and a real-time reverse transcription polymerase chain reaction (RRT-PCR) based on SYBR Green II was developed for quantization of porcine sapelovirus. The results indicated that no amplification was detected from other DNA samples by this method, such as CSFV, PRRSV, PKoV, PEDV, TGEV and ProV A, respectively. The detection limit of the method was 6.37×102 copies/µL of initial templates, which was 100-fold more sensitive than that of the conventional PCR. And the melting curve analysis using SYBR Green II dye showed one specific peak with no primer-dimer peak. Excellent reproducibility was obtained for detecting constructed positive plasmid DNA with intra-assay of 0.44% to 1.14% and inter-assay of 0.70% to 1.32%. A total of 58 clinical specimens collected from Sichuan Province were detected by the established RT-PCR and the positive rate is 56.9% (33/58). The results of the quantitative RT-PCR were the same as that of the conventional RT-PCR. The results proved that the RRT-PCR was specific and sensitive to detect porcine sapelovirus, which can support the clinic detection, quantitative analysis and molecular epidemiological investigation.