Development of a SYBR Green I real-time PCR for the detection of the orf virus

In this study, we developed a sensitive and specific SYBR Green I real-time PCR assay to quantitatively detect ORFV. Viral detection can utilize several different methods. Viral isolation is considered to be the gold standard for detection of pathogen infection. Endpoint dilution assays and plaque assays have also been used to quantify viruses. Compared with these methods, the SYBR Green I real-time PCR assay developed in this study offers several advantages, including more rapid results, reduced labor, and high-throughput capability. Moreover, viral isolation, endpoint dilution assays, and plaque assays are only able to identify infectious viral particles. In the case of field isolates that have not been adapted for growth in vitro, viral quantification via such methods is difficult.

A conventional PCR assay based on amplification of the ORFV B2L gene was developed to detect many recognized Parapoxvirus species (Gallina et al. 2006; Sullivan et al. 1994). When combined with DNA sequencing, this method can be used to distinguish among the different Parapoxvirus species (Inoshima et al. 2000). However, conventional PCR methods are not quantitative and can sometimes include non-specific products of the same size. To avoid these issues, a real-time PCR assay based on SYBR Green I was developed for the detection and quantification of ORFV. Many studies have demonstrated that real-time PCR is accurate and effective in quantifying viral DNA (Espy et al. 2000; Liu et al. 2013; Lo and Chao 2004; Mohamed et al. 2013; Niesters 2001). Even in the case of moderate DNA quality, such as DNA extracted from paraffin- or formalin-fixed tissues, real-time PCR can be effectively used for viral detection (Lu et al. 2016; Norlelawati et al. 2016). Compared with conventional PCR, a real-time PCR-based assay eliminates the requirement for gel electrophoresis of the reaction product. Reaction results can instead be observed in real time and clearly interpreted. Another advantage to the real-time PCR method is that the entire process requires only approximately 1.5 h, making it particularly suitable for high-throughput detection of clinical and laboratory pathogens.

Generally, conventional PCR assays often involve the amplification of long DNA fragments, reducing their sensitivity. To improve the sensitivity, we designed our assay to produce a short amplicon of 514 bp, enabling sensitive and reliable detection of ORFV DNA. Because the detection limit of our assay was significantly lower than that of conventional PCR, real-time PCR should be especially preferred when detecting pathogens at low copy numbers. Although the variation in the B2L gene sequences of different viral species reaches 15.6% (Gallina et al. 2006; Liu et al. 2013), specificity can be further improved through careful selection of primers. In our study, neither PCR amplification products nor fluorescence signals were observed in the control condition, indicating high specificity of the established real-time PCR assay. Traditional PCR typically shows good performance for specific detection. However, false negatives will occur when the virus content is low. When the virus content is relatively low, the performance of real-time PCR is even better than that of traditional PCR.

SYBR Green I real-time PCR relies on detection of a fluorescent signal using the fluorescent chimeric dye SYBR Green I, eliminating the need for a specific probe. Compared to the TaqMan probe method, it therefore does not require the design of a separate probe, which can be complex and expensive. Moreover, the sensitivity of the SYBR Green I assay compares quite well with the published sensitivity of 50 copies for the TaqMan probe-based real-time PCR assay (Gallina et al. 2006). Indeed, the sensitivity of the SYBR Green I-based assay is even higher than that of the TaqMan probe-based assay, with a detection limit of 20 copies. In addition, the SYBR Green I method provides information regarding the amplification of the PCR reaction in the form of the melting curve. Using the melting curve and Tm value, we could intuitively assess whether the product of the reaction is the intended target. Lastly, the SYBR Green I method also eliminates problems related to probe contamination, in which substandard quality or weak fluorescence signals cause false positive results, or mismatch of the probe with the template, where the lack of a fluorescent signal or low detection rate leads to false negative results.

Thus, our SYBR Green I real-time PCR assay exhibits high specificity for detecting ORFV; among common DNA pathogens, only ORFV returned a positive result. This novel method can detect as few as 20 copies of viral DNA, and the sensitivity is 1000 times higher than that of conventional PCR, effectively protecting against false negative results. In the reproducibility analysis, the inter- and intra-assay CV values ranged from 0.11 to 0.39%, demonstrating that this method possesses high stability and repeatability. Clinical sample testing also showed that real-time PCR exhibits higher sensitivity that conventional PCR in ORFV detection.

In conclusion, our results demonstrate that the established SYBR Green I real-time PCR assay is suitable for diagnosis of ORFV infection in high-throughput clinical samples, epidemiological surveillance, and laboratory research.