Evaluation of a portable, 'real-time' PCR machine for FMD diagnosis.

SIR, - Claims have been made during the UK foot-and-mouth disease (FMD) epidemic that the use of portable 'real-time' PCR instruments would have enabled on-farm rapid diagnosis, and thereby accelerated control procedures and reduced the amount of culling.

The performance of a portable 'real-time' PCR instrument has been compared with a laboratory-based 'real-time reverse-transcriptase PCR system at the Institute for Animal Health, Pirbright. Before the trial, the portable PCR machine was calibrated by a representative from the supply company so that it performed optimally. The reagents used in the assay were recommended by the manufacturer of the instrument.

In tests which included identical DNA/cDNA aliquots and a range of clinical specimens from experimentally infected animals, the portable machine functioned faster than the laboratory-based system, but with some samples it was up to 100 times less sensitive and it failed to detect a series of weak positive samples. The use of such an instrument for diagnosis during the FMD epidemic could have been disastrous.

In attempts to improve the performance of the portable instrument, the reagents normally employed in our laboratory-based system were used in the instrument instead of the reagents recommended by the manufacturer. This was found to raise the diagnostic sensitivity of the portable machine to very close to that of the laboratory-based system. While these results are encouraging, further tests are required to validate the instrument for specific diagnostic purposes. When the investigations are completed the data will be submitted for publication.

It is important to point out that the nucleic acid in FMD virus is RNA and that, before a PCR can be performed, the nucleic acids must be extracted from the test sample so that the RNA can be converted into cDNA. Currently this is a time-consuming exercise and not well suited to the conditions likely to be encountered in the field. Furthermore, multiple steps in the assay complicated the process, increase the risk of contamination and limit the number of samples that can be tested within a short period. These requirements appear to have been overlooked by those who have promoted the use of portable 'real-time' PCR instruments.

Alex I. Donaldson, Anna Hearps, Soren Alexandersen, Institute for Animal Health, Pirbright laboratory, Woking, Surrey GU24 0NF