[Detection of the genome of bovine viral diarrhea virus (BVDV) using the polymerase chain reaction after reverse transcription (RT-PCR): comparison of methods for the isolation of ribonucleic acid (RNA) from clinical samples].
The RT-PCR is an in vitro technique that is increasingly being used for diagnosis of viral animal pathogens. Due to its high sensitivity it is considered as an alternative to current standard methods for detecting BVDV especially in pooled samples, e.g. from bulk tank milk. A prerequisite for the performance of RT-PCR is an efficient and simple method for sample preparation. The aim of this work was to compare the efficiency of three commercially available kits for RNA extraction, and their suitability for sample preparation for the detection of the BVDV genome by RT-PCR in blood, milk and tissue 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 was performed by acidic guanidinium-isothiocyanate-phenol-chloroform extraction with the Trizol (Gibco) reagent. Using a kit based on the binding of RNA to silica membrane in a spin column, positive results in RT-PCR were obtained from all samples, but with lower sensitivity. The advantage of the column-based kits is that they are less time-consuming, easier to handle and suitable for automatisation of sample preparation. A kit using salt precipitation of the desoxribose nucleic acid (DNA) and proteins was unsuitable for the isolation of viral RNA from the samples.
Scheibner
H
H
Institut für Virologie der Tierärztlichen Hochschule Hannover.
Frey
H R
HR
Eicken
K
K
Meyer
H
H
Greiser-Wilke
I
I
D000818:Animals; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000175:diagnosis*; D002417:Cattle; D001908:Diarrhea Viruses, Bovine Viral / Q000235:genetics / Q000302:isolation & purification*; D005260:Female; D016679:Genome, Viral; D008892:Milk / Q000821:virology*; D012367:RNA, Viral / Q000302:isolation & purification*; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic