Diagnosis of pestivirus infections of pigs by simplified RT-nested PCR

Pestiviruses cause diseases in ruminants, pigs and wild boars. Pestivirus, known as Classical Swine Fever virus (CSFV), causes one of the most virulen t diseases of swine and wild boars found in Europe. The aim of the study wa s to apply the simplified procedure of reverse transcription and polymerase chain reaction (RT-nested PCR) for CSF diagnosis. Total RNA was extracted from 250 mu l of virus-containing cell culture supernatant by using TRIZOL (Life Technologies). Five microliters of RNA were reversely transcribed and amplified by two methods: standard, three steps and modified and closed on e-tube. Two sets of PCR primers were used for each method. The first, based on 5'UTR of pestivirus genome, was specific for all pestiviruses. The seco nd, designed on E2 protein coding region was used for specifically detectin g CSFV strains. The standard method consisting of 3 steps was performed in 3 separate reaction tubes: RT, PCR and nested-PCR. In the single-tube metho d all three steps were performed in a single, closed tube. In this method r e-agents for RT-PCR step were deposited in tube bottom, while re-agents for nested PCR were immobilised in a tube cap using carbohydrate trehalose. Af ter the RT-PCR step was completed the tube was vortexed, centrifuged and th e nested PCR was performed. It was concluded that the closed, one tube RT-n ested PCR method was very sensitive and less prone to giving false positive results compared to standard RT-nested PCR, carried out iu separate reacti on tubes.