Pan-serotypic detection of foot-and-mouth disease virus using a minor groove binder probe reverse transcription polymerase chain reaction assay

A novel assay for the pan-serotypic detection of foot-and-mouth disease virus (FMDV) was designed using a 5' conjugated minor groove binder (MGB) probe real-time RT-PCR system. This assay targets the 3D region of the FMDV genome and is capable of detecting 20 copies of a transcribed RNA standard. The linear range of the test was eight logs from 2 × 101 to 2 × 10? copies and amplification time was approximately 2 h. Using a panel of 83 RNA samples from representative FMDV isolates, the diagnostic sensitivity of this test was shown to be equivalent to a TaqMan real-time RT-PCR that targets the 5' untranslated region of FMDV. Furthermore, the assay does not detect viruses causing similar clinical diseases in pigs such as swine vesicular disease virus and vesicular stomatitis virus, nor does it detect marine caliciviruses causing vesicular exanthema. The development of this assay provides a useful tool for the differential diagnosis of FMD, potentially for use in statutory or emergency testing programmes, or for detection of FMDV RNA in research applications.     

Serotyping of foot-and-mouth disease virus by antigen capture reverse transcriptase/polymerase chain reaction.

The technique of capturing of foot-and-mouth disease virus (FMDV) from clinical material in microcentrifuge tubes coated with type-specific antibodies and amplifying the viral sequences by RT/PCR in the same tube, promoted the detection and serotyping of FMDV with high sensitivity and specificity. The efficiency of antigen capturing and shelf life of the coated tubes was improved by glutaraldehyde fixation of antibodies to the tubes. Virus in infected tissues, even after storage for 25-30 years at 70 degrees C, could be successfully typed by this method. Conserved sequences flanking the variable region of immunoreactive VP1 gene of FMDV were used as primers in the assay and hence the nucleotide sequence analysis of the product could reveal the strain variation. The test has been found to be at least 125-fold more sensitive than type specific ELISA and of comparable sensitivity as other protocols for detection of FMDV by RT/PCR.     

Detection of differential expression of porcine IFN-alpha subtypes by reverse transcription polymerase chain reaction.

The porcine interferon-alpha (IFN-alpha) multigene family is a new IFN-alpha system in recent research. Characterization of the PoIFN-alpha multigene family has been described in our previous work, and 14 functional PoIFN-alpha genes were detected in the porcine genome. In this study, we designed subtype-specific primers and consensus primers for PoIFN-alpha. The expression of PoIFN-alpha was detected using the two PCR strategies in three systems, namely, poly(I).poly(C)-DEAE-dextran-induced PK15 cells, pseudorabies virus-infected PK15 cells, and infected PK15 cells with an attenuated strain of swine fever virus, respectively. In poly(I).poly(C)-DEAE-dextran-induced PK15 cells, the expression of IFN-alpha2, -alpha3, -alpha4, -alpha8, and -alpha9 after 6-h/24-h inducement in PK15 cells were observed. In pseudorabies virus-infected PK15 cells, the expression of PoIFN-alpha2, -alpha3, -alpha8, -alpha9, -alpha10, and -alpha13 was observed after 6-h/24-h infection, and in the attenuated strain of swine fever virus-infected PK15 cells, upregulation of PoIFN-alpha2, -alpha3, -alpha4, -alpha8, -alpha9, and -alpha10 was detected. The results of realtime quantitative PCR analysis suggested that the expression was time-dependent in pseudorabies virus/poly(I).poly(C)-DEAE-dextran-induced PK15 cells, but in the attenuated swine fever virus-infected PK15 system, the expression level of IFN-alpha subtypes was not obviously time dependent.     

Evaluation of real-time reverse transcription polymerase chain reaction assays for the detection of swine vesicular disease virus

Differential detection of swine vesicular disease virus (SVDV) from the other vesicular disease viruses of foot-and-mouth disease (FMD), vesicular stomatitis (VS) and vesivirus is important as the vesicular lesions produced by these viruses are indistinguishable in pigs. Two independent sets of primers and probe, designed from nucleotide sequences within the 5' untranslated region (UTR) of the SVDV genome, were evaluated in a real-time (5' nuclease probe-based or fluorogenic) PCR format. Although both primers/probe sets failed to detect one isolate, the assays successfully amplified RNA extracted from epithelial suspensions (ES) and cell culture grown virus preparations from clinical samples representing all currently designated phylogenetic groups of SVDV. Furthermore, no cross-reactivity was demonstrated when these primer/probe sets were tested with RNA prepared from all seven serotypes of FMD virus (FMDV) and from selected isolates of VS virus (VSV), vesivirus and teschoviruses. These assays provide sensitive and rapid alternatives to supplement the routine procedures of ELISA and virus isolation for SVDV diagnosis. The two independent sets of primers/probe can be used routinely while only one of the primers/probe sets would typically be used in SVDV diagnosis during an outbreak.