Author/Editor		Barlič-Maganja, Darja; Grom, J
Title		Hitra metoda za določanje virusa goveje virusne diareje (BVDV) v serumu trajno okuženega goveda s polimerazno verižno reakcijo
Translated title		Rapid detection of bovine viral diarrhea virus (BVDV) in the serum of persistently infected cattle by a nested polymerase chain reaction
Type		članek
Source		Zb Vet Fak Univ Ljublj
Vol. and No.		Letnik 34, št. 2
Publication year		1997
Volume		str. 171-6
Language		slo
Abstract		The polymerase chain reaction is very specific, rapid and highly sensitive method, which represents an improvement over existing technology for the identification of BVD virus. Serum samples were collected from animals belonged to the herd highly infected with BVDV. For RT-PCR assay negative serum samples on BVD antibodies were chosen. The reverse transcription reaction (RT) was done without previously isolating RNA from serum samples. After RT reaction the first PCR reaction with outer primers were performed in the same tube. The secound PCR reaction was prepared in a new tube using 5 micro l of the first PCR reaction and internal primers. Both sets of primers were chosen from highly conversed 5'-untranslated region of the virus genome. After amplification with internal primers PCR product was analysed by agarose gel electrophoresis. We obtained a 288 bp fragment clearly visible onUV transiluminator. The described RT-PCR method for detection of BVD virus in serum samples is fast, simple and very sensitive. Its cotst currently makes it unsuitable for large-scale testing. However, its has been very useful as a confirmatory test in cases where the viral titre was very low or where the ELISA results were in the suspicious range.
Summary		Metoda polimerazne verižne reakcije je zlo specifična, hitra in zelo občutljiva. Predstavlja izboljšavo obstoječih metod za dokazovanje virusa goveje virusne diareje (BVD). Zbrali smo serumske vzorce živali iz črede, ki je bila zelo prekužena z virusom BVD. Serumske vzorce, ki niso imeli protiteles proti virusu BVD, smo testirali z metodo RT-PCR. Reakcijo reverzne transkripcije smo naredili brez poprejšnje izolacije RNK iz serumskega vzorca. Po reverzni transkripciji smo pripravili prvo reakcijo PCR z zunanjimi oligonukleotidi kar v isti epruveti. V novi epruveti smo pripravili drugo reakcijo PCR, za katero smo uporabili 5 micro reakcijske zmesi iz prve reakcije PCR in notranje oligonukleotide. Oba para oligonukleotidov smo izbrali na zelo ohranjenem 5'-nekodirajočim delu virusnega genoma. Po pomnoževanju z notranjimi oligonukleotidi smo produkt analizirali z agarozno gelsko elektroforezo. Fragment v velikosti 288 bp je bil jasno viden na UV transiluminatorju. Opisana metoda RT-PCR za odkrivanje virusa BVD v serumskih vzorcih je hitra, enostavna inzelo občutljiva. Na splošno zaradi visoke cene ni primerna za testiranje velikega števila vzorcev. Kljub temu pa je zelo primerna kot potrditveni test v primerih, ko je virusni titer zelo nizek ali ko dobimo dvomljive rezultate pri testih ELISA.
Descriptors		BOVINE VIRUS DIARRHEA-MUCOSAL DISEASE DIARRHEA VIRUS, BOVINE VIRAL POLYMERASE CHAIN REACTION CATTLE