Development of SYBR Green real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus

Abstract

Unclassified bovine enteric calicivirus (BECV) is a newly recognized bovine enteric calicivirus that differs from bovine norovirus, and which causes diarrhea in the small intestines of calves. To date, methods such as real-time reverse transcription-polymerase chain reaction (RT-PCR) have not been developed for the rapid detection, quantitation and diagnosis of BECV. Presently, a BECV-specific SYBR Green real-time RT-PCR assay was evaluated and optimized. Diarrheic specimens (n=118) collected from 2004 to 2005 were subjected to RT-PCR, nested PCR and SYBR Green real-time RT-PCR. By conventional RT-PCR and nested PCR, 9 (7.6%) and 59 (50%) samples tested positive, respectively, whereas the SYBR Green assay detected BECV in 91 (77.1%) samples. Using BECV RNA standards generated by in vitro transcription, the SYBR Green real-time RT-PCR assay sensitively detected BECV RNA to 1.1 x 10(0)copies/microl (correlation coefficiency=0.98). The detection limits of the RT-PCR and nested PCR were 1.1 x 10(5) and 1.1 x 10(2)copies/microl, respectively. These results indicate that the SYBR Green real-time RT-PCR assay is more sensitive than conventional RT-PCR and nested PCR assays, and has potential as a reliable, reproducible, specific, sensitive and rapid tool for the detection, quantitation and diagnosis of unclassified BECV.

Fig. 1

SYBR green real-time RT-PCR assay for the quantitation of BECV cRNA standard. (A) Amplification of 1 × 10⁰, 1 × 10¹, 1 × 10², 1 × 10³, 1 × 10⁴, 1 × 10⁵, 1 × 10⁶, 1 × 10⁷ and 1 × 10⁸ copies of cRNA standard used in parallel with each SYBR Green-based real-time RT-PCR assay. (B) SYBR Green real-time RT-PCR products using serially diluted in vitro transcripts. M: molecular marker; lanes 1–9: 1.1 × 10⁸, 1.1 × 10⁷, 1.1 × 10⁶, 1.1 × 10⁵, 1.1 × 10⁴, 1.1 × 10³, 1.1 × 10², 1.1 × 10¹ and 1.1 × 10⁰ viral copies/μl; N: negative control. (C) Standard curves of the real-time RT-PCR based on serial dilutions of BECV 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.

Fig. 2

Sensitivity of conventional RT-PCR and nested PCR with in vitro transcripts. (A) One-step conventional RT-PCR was performed in the same tube with serially diluted in vitro transcripts. (B) Nested PCR products with one-step conventional RT-PCR products. M: molecular marker; lanes 1–9: 1.1 × 10⁸, 1.1 × 10⁷, 1.1 × 10⁶, 1.1 × 10⁵, 1.1 × 10⁴, 1.1 × 10³, 1.1 × 10², 1.1 × 10¹ and 1.1 × 10⁰ viral copies/μl, respectively; N: negative control.