Diagnosis of FMD by RT - PCR : prospects for mobile and portable assays

Abstract

Rapid and accurate diagnosis is needed for effective control of foot-and-mouth disease (FMD). Laboratory-based methods such as virus isolation, antigen-ELISA and real-time RT-PCR (rRT-PCR) can provide an objective result within a few hours of sample receipt. However, the time taken to transport suspect material to a centralized laboratory can be unacceptably long, often precluding laboratory confirmation in the event of an outbreak. There are opportunities to deploy mobile rRT-PCR assays inside a vehicle, or in local laboratories, for rapid diagnosis of suspect cases. These assays typically require pre-processing steps of samples (such as RNA extraction) which demands trained personnel. Portable PCR technology has also recently been developed with the potential for pen-side or “point of care” diagnosis. These field-based assays could be operated by untrained personnel without prior knowledge of molecular biology. In contrast to laboratory-based assays, where high sample throughput is a major prerogative, portable assays focus on speed in order to generate a diagnostic result within one hour of sample collection. To determine whether mobile and portable platforms might be used to carry out molecular diagnosis closer to suspect cases of FMD, two instruments were evaluated: (i) a “mobile” nucleic acid extraction robot (BioRobot EZ1, Qiagen) was compared with centralized robotic extraction equipment currently used to prepare RNA for FMD virus (FMDV) detection and (ii) the portable Bioseeq platform (Smiths Detection) was assessed as to whether this instrument could be used for pen-side diagnosis of FMD by rRT-PCR. Both instruments provide realistic options for performing molecular assays for FMDV away from centralised laboratories. Introduction: Rapid and accurate diagnosis is needed for effective control and eradication of FMD. Laboratory-based methods such as virus isolation (VI), antigen detection ELISA (Ferris and Dawson, 1988) and real-time RT-PCR (rRT-PCR) (Reid et al., 2003; Shaw et al., 2004) can provide an objective result within a few hours of sample receipt. However, the time taken to transport suspect material to a centralized laboratory can be unacceptably long, often precluding laboratory confirmation in the event of an outbreak. Using existing available equipment, there are opportunities to deploy mobile rRT-PCR assays inside a vehicle, or in local laboratories, for rapid diagnosis of suspect cases (Callahan et al., 2002; Hearps et al., 2002). These assays typically require pre-processing of samples (such as RNA extraction) which demands trained personnel. Portable PCR technology has also recently been developed with the potential for pen-side or “point of care” diagnosis. Field-based assays like these could be operated by untrained personnel without prior knowledge of molecular biology. In contrast to laboratory-based assays, where high sample throughput is a major prerogative, portable assays focus on speed in order to generate a diagnostic result within one hour of sample collection. To determine whether mobile and portable platforms might be used to carry out molecular diagnosis closer to suspect cases of FMD, two instruments were evaluated. Firstly, the performance of a “mobile” nucleic acid extraction robot (BioRobot EZ1, Qiagen) was compared with centralized robotic extraction equipment currently used to prepare RNA for FMDV detection. Secondly, the portable Bioseeq platform (Smiths Detection, Watford, UK) was assessed as to whether this instrument could be used for pen-side diagnosis of FMD by rRTPCR. Materials and methods: Comparison of the BioRobot EZ1 RNA extraction instrument with other extraction methods for diagnosis of FMD An initial evaluation of the Qiagen BioRobot EZ1 was performed by comparing RNA extracted by this potential mobile device with other robotic instruments, namely the Qiagen BioRobot 9604 and the MagNA Pure LC (Roche) (Fig. 1). The QIAamp Viral RNA Mini Kit (Qiagen) was also included to compare the performance of a manual extraction method. Decimal dilutions of an ~10% epithelial suspension of the reference FMDV strain O1 Manisa (TUR 8/69) were prepared in suspensions of uninfected negative bovine epithelium and 200μl of each dilution was lysed in 300μl of the accompanying lysis buffer as appropriate (Table 1). For the manual RNA extractions, 140μl of the dilutions were lysed in 560μl of AVL

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Cite this paper

@inproceedings{Reid2007DiagnosisOF, title={Diagnosis of FMD by RT - PCR : prospects for mobile and portable assays}, author={Scott M. Reid and Juliet P. Dukes and Katja Ebert and Nigel P. Ferris and Donald P. King}, year={2007} }