Rapid pre-clinical detection of classical swine fever by reverse transcription loop-mediated isothermal amplification

Abstract

The usefulness of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid pre-clinical detection of classical swine fever virus (CSFV) infection was evaluated. The RT-LAMP reaction could be finished in 60 min under isothermal condition at 65 degrees C by employing a set of four primers targeting the 5' untranslated region of CSFV. The RT-LAMP assay of CSFV showed higher sensitivities than that of RT-PCR, with a detection limit of 5 copies per reaction. No cross-reactivity was observed from the samples of other related viruses including porcine circovirus type 2, porcine parvovirus, porcine pseudorabies virus, Japanese encephalitis virus, and porcine reproductive and respiratory syndrome virus. The detection rates of CSFV RT-LAMP, RT-PCR and virus isolation for samples including blood, tonsil, nasal and rectal swabs from uninoculated pigs without any clear clinical symptom were 89%, 78% and 71%, respectively. Furthermore, all of the assays showed higher sensitivity for blood and tonsil swabs samples than nasal and rectal swabs. These results indicate that the CSFV RT-LAMP assay is a valuable tool for its rapid, cost-effective detection and has potential usefulness for rapid pre-clinical detection and surveillance of classical swine fever in developing countries.

Fig. 1

Comparative sensitivities of RT-LAMP and RT-PCR for the detection of CSFV by agarose gel electrophoresis analysis. From left to right: Lane M, DNA Marker DL-2000 (Takara); Lanes 1–6, different CSFV RNAs copy numbers of RT-PCR (1, 5¹, 5², 5³, 5⁴ and 5⁵ copies/tube, respectively); Lanes 7–12, different CSFV RNAs copy numbers of the RT-LAMP assay (1, 5¹, 5², 5³, 5⁴ and 5⁵ copies/tube, respectively). RT-PCR products showed a specific amplification for CSFV with a detection limit of 5³ copies per tube, whereas detection limit of RT-LAMP is 5 copies per tube.

Fig. 2

Electrophoresis analysis of cross-reaction of CSFV RT-LAMP with PCV2, PPV, PRV, JEV and PRRSV. Lane M, DNA Marker DL-2000. Lane 1, negative control. Lanes 2, RNA of CSFV-LT. Lane 3, RNA of CSFV-GS. Lane 4, DNA of PCV2. Lane 5, DNA of PPV. Lane 6, DNA of PRV. Lane 7, RNA of PRRSV. Lane 8, RNA of JEV.