Development of one-step TaqMan® real-time reverse transcription-PCR and conventional reverse transcription PCR assays for the detection of equine rhinitis A and B viruses

Abstract

been used to detect specific antibodies against the infections, but these assays are also restricted due to their antibody-detected limitation and/or potential cross-reactivity to other pathogens. For example, reading of IFAT results is very difficult to read results and not objective at borderline between positive and negative titers. Results of IFAT may also change not only with different origin of antigens, but also with different lots of FITC-conjugated anti-horse IgG antibody. Recently, new diagnostics such as immunochromatographic test (ICT) and loop-mediated isothermal amplification (LAMP) assay have been developed for the detection of B. caballi and/or T. equi infections. ICT has similar sensitivity to ELISA and is a rapid and simple method to detect antibodies [1]. LAMP assay can rapidly amplify DNA samples within one hour under an isothermal condition (at 60 to 65 C). LAMP has higher specificity and sensitivity than those of PCR [2]. Therefore, both ICT and LAMP are practical pen-side diagnostic methods for equine piroplasmosis. The objectives of this presentation are to review diagnostic methods and problems of the current diagnostic methods, to introduce newly developed diagnostics, and to discuss how these methods can contribute the strengthening of quarantine for equine piroplasmosis. References[1] Huang X, et al. Immunochromatographic test for simultaneous serodiagnosis of Babesia caballi and B. equi infections in horses. Clin Vaccine Immunol 2006;13:553-5. [2] Alhassan A, et al. Comparative evaluation of the sensitivity of LAMP, PCR and in vitro culture methods for the diagnosis of equine piroplasmosis.Equine rhinitis virus A and B (ERAV and ERBV) are common equine respiratory viruses belonging to the family Picornaviridae. There is evidence that these two viral infections are prevalent in countries in which sero-surveillance studies have been undertaken. Currently there is a lack of rapid and reliable diagnostic methods for virus detection and antibody determination. The sensitivity of virus isolation varies between laboratories and is confounded by difficulties in isolating virus from the urine of carrier horses. The objective of this study was to develop molecular diagnostic assays (real-time RT-PCR [rRT-PCR] and conventional RT-PCR [cRT-PCR] assays) capable of detecting and distinguishing ERAV from ERBV without the inherent problems associated with the current laboratory diagnosis of these infections. Three rRT-PCR assays targeting the 5'-UTR of ERAV and ERBV were developed. One assay was specific for ERAV, whereas the remaining two assays were specific for ERBV. In addition, six cRT-PCR assays targeting the 5'-UTR and 3D polymerase regions of ERAV and ERBV were also developed. RNA extracted from prototype strains of ERAV and ERBV as well as 21 archived tissue culture fluid samples which were previously determined to be positive by virus isolation for ERAV or ERBV with mono-specific rabbit antisera were used to evaluate the sensitivity and specificity of these assays. Using serial decimal dilu...

Field-Deployable Reverse Transcription-Insulated Isothermal PCR (RT-iiPCR) Assay for Rapid and Sensitive Detection of Foot-and-Mouth Disease Virus

“…Nucleic acid from the remaining viruses was extracted using QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA) as per the manufacturer's instructions. Before use in the FMDV RT‐iiPCR assay, the presence of viral RNA in each extraction was confirmed using published (Forsyth and Barrett, ; Tsunemitsu et al., ; Paton et al., ; Pasick et al., ; King et al., ; Deregt et al., ; Toussaint et al., ; Hindson et al., ; Lung et al., , ; Lu et al., ; Klima et al., ) or unpublished but laboratory‐validated (for PCV‐1) pathogen‐specific real‐time or conventional RT/PCR assays (data not shown).…”