Reverse transcription loop-mediated isothermal amplification for the detection of highly pathogenic porcine reproductive and respiratory syndrome virus

Abstract

A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay targeting the open reading frames 1a of highly pathogenic porcine reproductive and respiratory syndrome virus genome was developed. The 10 reference strains, 1 clinical isolation strain and 122 positive samples were tested. Positive reactions were confirmed for all strains and specimens by reverse transcription loop-mediated isothermal amplification and nested reverse transcription polymerase chain reaction (RT-PCR). The results showed this detection technique is more reliable and convenient for rapid and sensitive diagnosis of highly pathogenic porcine reproductive and respiratory syndrome virus infection.

Fig. 1

(A) Agarose gel (2.5%) electrophoresis RT-LAMP products. Lines M, markers DL2000 (2000, 1000, 750, 500, 250 and 100 bp); lane 1, negative control; lane 2, highly pathogenic PRRSV (JXA1); lanes 3–5, highly pathogenic PRRSV blood, semen and lung samples (GD). (B) Sensitivity of nested RT-PCR determined by agarose gel electrophoresis of nested RT-PCR products (236 bp) from spiked with 5-fold dilution of the highly pathogenic PRRSV RNA (HPBEDV). Lines M, markers DL2000; lane 1, negative control; lanes 2–7, different highly pathogenic PRRSV RNA copy numbers of nested RT-PCR (1, 5, 25, 125, 625 and 3125 copies/tube, respectively). (C) Sensitivity of RT-LAMP determined by agarose gel electrophoresis of RT-LAMP products from spiked with 5-fold serial dilution of the highly pathogenic PRRSV RNA (HPBEDV). Lines M, markers DL2000; lanes 1–6, different highly pathogenic PRRSV RNA copy numbers of RT-LAMP (1, 5, 25, 125, 625 and 3125 copies/tube, respectively); lane 7, negative control. Nested RT-PCR products showed a specific amplification for the HPBEDV ORF1a with a detection limit of 25 copies, whereas detection limit of RT-LAMP is 5 copies/tube.