Development of universal SYBR Green real-time RT-PCR for the rapid detection and quantitation of bovine and porcine toroviruses

Abstract

Toroviruses (ToVs) are a group of emerging viruses that cause gastroenteritis in domestic animals and humans. Currently, methods such as real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) have not yet been developed for the rapid detection and quantitation of bovine (BToV) and porcine (PToV) toroviruses. Using BToV and PToV RNA standards generated by in vitro transcription, the detection limit of the SYBR Green real-time RT-PCR assay was 2.54 x 10(2) BToV and 2.17 x 10(3) PToV copies/reaction (correlation coefficiency=0.99 and 0.97, respectively), whereas those of RT-PCR and nested PCR were 2.54 x 10(5) and 2.54 x 10(4) (BToV) and 2.17 x 10(7) and 2.17 x 10(5) (PToV) cRNA viral copies/reaction, respectively. Archived diarrhea specimens of calves (n=121) and piglets (n=86) were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR, 1 (0.8%) bovine and 7 (8.1%) porcine samples tested positive to BToV and PToV, respectively. With nested PCR, 13 (10.7%) bovine and 17 (19.8%) porcine samples tested positive. SYBR Green real-time RT-PCR assay detected BToV and PToV in 22 of 121 (18.2%) bovine and 31 of 86 (36.0%) porcine samples. These results indicate that SYBR Green real-time RT-PCR (P<0.05) is a more sensitive assay, which can be reproduced as a reliable, sensitive, and rapid tool for the detection and quantitation of toroviruses.

Fig. 1

Standards for the SYBR Green real-time RT-PCR assay for the quantitation of BToV and PToV cRNA. (A) Amplification of 10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ and 10⁻⁹ dilutions of the cRNA standard used in parallel with each SYBR Green-based real-time RT-PCR assay. (B) Standard curves of the real-time RT-PCR based on serial dilutions of BToV cRNA standards. In the standard curve of these dilutions, each dot represents the result of duplicate amplification of each dilution. The coefficient of determination (R²) and the slope(s) of the regression curve are indicated. (C) SYBR Green real-time RT-PCR products using serially diluted in vitro transcripts. M, molecular marker; lanes 1–10: 2.54 × 10¹¹, 2.54 × 10¹⁰, 2.54 × 10⁹, 2.54 × 10⁸, 2.54 × 10⁷, 2.54 × 10⁶, 2.54 × 10⁵, 2.54 × 10⁴, 2.54 × 10³ and 2.54 × 10² viral copies/reaction; N, negative control. (D) Amplification of 10⁰, 10⁻¹, 10⁻², 10⁻³, 10⁻⁴, 10⁻⁵, 10⁻⁶, 10⁻⁷, 10⁻⁸ and 10⁻⁹ dilutions of cRNA standard used in parallel with each SYBR Green-based real-time RT-PCR assay. (E) Standard curves of the real-time RT-PCR based on serial dilutions of PToV cRNA standards. In the standard curve of these dilutions each dot represents the result of duplicate amplification of each dilution. The coefficient of determination (R²) and the slope (s) of the regression curve are indicated. (F) SYBR Green real-time RT-PCR products using serially diluted in vitro transcripts. M, molecular marker; lanes 1–10: 2.17 × 10¹¹, 2.17 × 10¹⁰, 2.17 × 10⁹, 2.17 × 10⁸, 2.17 × 10⁷, 2.17 × 10⁶, 2.17 × 10⁵, 2.17 × 10⁴ and 2.17 × 10³ viral copies/reaction; N, negative control.