The development of a rapid SYBR one step real-time RT-PCR for detection of Porcine Reproductive and Respiratory Syndrome Virus

Abstract

Background: Prompt detection of PRRSV in the field samples is important for effective PRRS control, thereby reducing the potentially serious economic damage which can result from an outbreak. In this study, a rapid SYBR-based, one step real-time RT-PCR quantitative reverse transcription PCR (qRT-PCR) has been developed for the detection of porcine reproductive and respiratory syndrome virus (PRRSV). Primers were designed based on the sequence of highly conservative region of PRRSV N gene.

Results: The sensitivity of the real-time qRT-PCR assay was achieved through PRRSV ch-1a RNA for the generation of a standard curve. The detection limit of the assay was found to be 9.6 RNA copies per reaction mixture. This assay had excellent intra- and inter-assay reproducibility as in total 65 field samples were screened for the presence of PRRSV by conventional RT-PCR in parallel with qRT-PCR, and the detection rate increased from 60.0% to 76.9%. Moreover, the specificity result indicated that this assay could reliably differentiate PRRSV from the other swine viral diseases, such as classical swine fever virus (CSFV), swine vesicular disease virus (SVDV) and vesicular exanthema of swine virus (VESV).

Conclusion: The real-time qRT-PCR assay described in this report allows the rapid, specific and sensitive laboratory detection of PRRSV in field samples.

Figure 1

Standard ch-1a curve of the PRRSV real-time qRT-PCR assay. A 10-fold serial dilutions ranging from 9.6 to 9.6 × 10⁵ copies of PRRSV RNA were tested in the real-time qRT-PCR. The standard formula is y = - 3.528x + 15.60 and the correlation co-efficient is 0.999.

Figure 2

The specificity and sensitivity of one step SYBR green real-time RT-PCR. Plot of the amplification of a 10-fold serial dilution of ch-1a RNA to calculate the detection limit and sensitivity of real-time RT-PCR by analyzing the fluorescence curve of the 228 bp DNA amplification product. NTC is the negative control; ND is the non-diluted sample (9.6 × 10⁵); CSFV is classical swine fever virus; SVDV is swine vesicular disease virus; VESV is vesicular exanthema of swine virus; ch-1a dilutions are 10^-2 to 10^-7 with copies from 9.6 × 10⁵ down to 9.6 per reaction mixture.

Figure 3

Dissociation plot of the amplification products of ch-1a RNA. Negative control including NTC, CSFV, SVDV AND VESV, melting peaks of ch-1a ten-fold serial dilutions and negative control, the positive samples showed an identical melting curve profile.

Figure 4

The sensitivity of real-time qRT-PCR for detection of the PRRSV. A 10-fold dilution series of total RNA extracted from a field sample ranging from 10^-1 to 10^-7 dilutions were tested in parallel in the qRT-PCR assay and in the conventional RT-PCR. The detection limit for the real-time qRT-PCR assay was a 10^-5 dilution of sample RNA.

Figure 5

The sensitivity of conventional RT-PCR for detection of the PRRSV. The detection limit for the conventional RT-PCR assay was a 10^-4 dilution of sample RNA. Lanes 1--7: 10-fold dilution series of sample total RNA ranging from 10^-1 to 10^-7 dilutions; lane N: negative control having no template; lane M: DL2000 DNA ladder Maker (TAKARA).