The detection of foot-and-mouth disease virus in oesophagealpharyngeal samples by a polymerase chain reaction technique

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Abstract

A polymerase chain reaction (PCR) technique was used to detect the presence of foot-and-mouth disease virus (FMDV) in oesophageal-pharyn-geal(OP) samples from experimentally infected steers. Ten-fold dilutions of OP samples were also diluted, inoculated onto lamb kidney cell cultures, and incubated overnight. The cultures that did not show overt cytopathogenic effects (CPE) of FMDV infection were frozen and thawed; both the fluid and the cell pellet were tested by the PCR technique. The PCR detected FMDV in the fluids of 57% of the cell cultures inoculated with 2–20 tissue culture infective doses-50% (TCID50) and of 33% of cell cultures inoculated with the 0.4–2 TCID50. The PCR detected FMDV in cell pellets of all cell cultures inoculated with 20 TCID50, of 71% of cell cultures inoculated with 2–20 TCID50 and of 50% of cell cultures inoculated with 0.2-2 TCID50. A diagnostic scheme using PCR and cell culture that would provide rapid and sensitive detection of FMDV in OP samples is proposed.

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    Many factors, such as specific primers, PCR-related reagents and conditions, quality of samples, virus strains used, can influence the sensitivity of the RT-PCR (Reid et al., 1998). The detection limits of PCR for FMDV detection have been described in several previous reports and it was shown that the sensitivity of the PCR, depending on different primer pairs used, varied between 0.01 TCID50 to 104 TCID50 (Amaral-Doel et al., 1993; Clavijo et al., 2003; Giridharan et al., 2005; House and Meyer, 1993; Meyer et al., 1991; Rodriguez et al., 1994). In this report, the limits of FMDV detection of one-step multiplex RT-PCR using the new primers varied between 101.5 and 103.5 TCID50/ml, it was most sensitive for serotypes A (A22/Iraq), followed by serotype O (O1/Manisa), and Asia 1 (As1/Shamir/89).

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