Development of a real-time RT-PCR method for detection of porcine rubulavirus (PoRV-LPMV)

Abstract

In order to provide a rapid and sensitive method for detection of the Porcine rubulavirus La Piedad-Michoacan-Mexico Virus (PoRV-LPMV), we have developed a specific real-time reverse transcriptase polymerase chain reaction assay. The detection of PoRV-LPMV, represents a diagnostic challenge due to the viral RNA being present in very small amounts in tissue samples. In this study, a TaqMan(®) real-time PCR assay was designed based on the phosphoprotein gene of PoRV-LPMV, to allow specific amplification and detection of viral RNA in clinical samples. Assay conditions for the primers and probe were optimized using infected PK15 cells and ten-fold serial dilutions of a plasmid containing the whole P-gene. The sensitivity of the developed TaqMan(®) assay was approximately 10 plasmid copies per reaction, and was shown to be 1000 fold better than a conventional nested RT-PCR. The performance of this real-time RT-PCR method enables studies of various aspects of PoRV-LPMV infection. Finally, the assay detects all current known variants of the virus.

Immunogenicity of a recombinant hemagglutinin neuraminidase-<i>Porcine rubulavirus</i> produced by <i>Escherichia coli</i> of <i>Porcine rubulavirus</i> gives protective immunity of litter after challenge

“…Viral infection was confirmed in five euthanized-suckling piglets by qPCR of each group; no statistical difference in virus load was shown in midbrain samples collected at necropsy. The average of Ct value found in their brains was 24.73 ( r HN-PorPV-ISCOMs) and 24.356 ( r HN-PorPV-protein alone) and 24.75 in the control group; corresponding approximately to 9.50 × 10 4 and 6.32 × 10 3 copy numbers, respectively, according to the standard curve by qRT-PCR assay [ 10 ] ( Table 3…”

“…The qPCR assay was performed on the midbrain from suckling piglets to estimate the PRV viral load, specific primers to the P-gene were designed as described by Cuevas-Romero et al ., 2013 [ 10 ]. Briefly, 50 ng of total RNA in a volume of 2 μL was used as templates for the SuperScript II Reverse Transcriptase, followed by qPCR amplification (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.…”

Salud porcina: historia, retos y perspectivas

“…La prueba de inhibición de la hemaglutinación es la más empleada, aunque frecuentemente puede dar falsos positivos, si no es estandarizada correctamente (84) . Se ha reportado la detección y cuantificación del RVP mediante RT-PCR en tiempo real (85,86) ; estas pruebas pueden resultar costosas si son aplicadas en grandes poblaciones. Por lo tanto, existen campos de oportunidad para el desarrollo de pruebas rápidas aplicables en campo.…”

Molecular and epidemiological studies ofPorcine rubulavirusinfection – an overview

“…Other serological techniques frequently used are indirect fluorescent antibody, serum-virus neutralisation ( 40 , 41 , 46 , 57 ) and a blocking ELISA ( 46 , 54 , 57 ). Classical PCR technologies have been used for different research purposes 16 , 41 , 46 ), and a real-time PCR (qRT-PCR) method specific to PorPV has been developed for PorPV to study epidemiology aspects of the disease ( 58 , 59 ). This qRT-PCR is based on the detection of viral RNA in clinical samples.…”