Detection of bovine virus diarrhoea virus (BVDV) using the polymerase chain reaction after reverse transcription (RT-PCR): Comparison of methods for the isolation of RNA from clinical samples

The RT-PCR is an in vitro technique that is increasingly being used for dia gnosis of viral animal pathogens. Due to its high sensitivity it is conside red as an alternative to current standard methods for detecting BVDV especi ally in pooled samples, e.g. from bulk tank milk. A prerequisite for the pe rformance of RT-PCR is an efficient and simple method for sample preparatio n. The aim of this work was to compare the efficiency of three commercially available kits for RNA extraction, and their suitability for sample prepar ation for the detection of the BVDV genome by RT-PCR in blood, milk and tis sue samples. The kits were based on different methods for extraction of RNA and differed in costs, labour and time consumption. The most sensitive RT- PCRs (exception: heparinised blood) were obtained when sample preparation w as performed by acidic guanidinium-isothiocyanate-phenol-chloro-form extrac tion with the Trizol (Gibco) reagent. Using a kit based on the binding of R NA to silica membrane in a spin column, positive results in RT-PCR were obt ained from all samples, but with lower sensitivity. The advantage of the co lumn-based kits is that they are less time-consuming, easier to handle and suitable for automatisation of sample preparation. A kit using salt precipi tation of the desoxribose nucleic acid (DNA) and proteins was unsuitable fo r the isolation of viral RNA from the samples.