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Titolo:
ESTABLISHMENT OF CONDITIONS FOR THE DETECTION OF BOVINE HERPESVIRUS-1BY POLYMERASE CHAIN-REACTION USING PRIMERS IN THE THYMIDINE KINASE REGION
Autore:
YASON CV; HARRIS LM; MCKENNA PK; WADOWSKA D; KIBENGE FSB;
Indirizzi:
UNIV PRINCE EDWARD ISL,ATLANTIC VET COLL,DEPT PATHOL & MICROBIOL CHARLOTTETOWN PE C1A 4P3 CANADA
Titolo Testata:
Canadian journal of veterinary research
fascicolo: 2, volume: 59, anno: 1995,
pagine: 94 - 101
SICI:
0830-9000(1995)59:2<94:EOCFTD>2.0.ZU;2-9
Fonte:
ISI
Lingua:
ENG
Soggetto:
PCR; SEQUENCES; DIAGNOSIS; CATTLE; ANEMIA; VIRUS; DNA;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
25
Recensione:
Indirizzi per estratti:
Citazione:
C.V. Yason et al., "ESTABLISHMENT OF CONDITIONS FOR THE DETECTION OF BOVINE HERPESVIRUS-1BY POLYMERASE CHAIN-REACTION USING PRIMERS IN THE THYMIDINE KINASE REGION", Canadian journal of veterinary research, 59(2), 1995, pp. 94-101

Abstract

Polymerase chain reaction (PCR) for detection of bovine herpesvirus-1(BHV-1) was developed and optimized using 22 bp sense and 20 bp antisense primers in the thymidine kinase (TK) coding region. The amplification product is 183 bp long. The PCR optimization was done using BHV-1tissue culture supernate (BHV-1TCS), concentrated BHV-1 tissue culture supernate (cBHV-1TCS) and sucrose gradient purified BHV-1 (pBHV-1). The sensitivity of four methods of sample preparation which are standard DNA extraction, modified proteinase K (PK) digestion, GeneReleaser(TM) + 34 cycles or + 44 cycles, and boiling were compared with virus isolation (VI) using BHV-1TCS. The incorporation of 10% glycerol in thereaction mixture, the incubation in PK for 18 hours and predenaturation of samples and cooling in ice prior to PCR were essential for the amplification of BHV-1 DNA for samples prepared by standard DNA extraction and modified PK digestion. The preparation of samples by GeneReleaser(TM), a proprietary nucleic acid releasing cocktail, showed 10 to 1,000-fold increase in sensitivity compared to standard DNA extraction and modified PK digestion. No amplification was observed in samples prepared by boiling. The sample preparation of BHV-1 LA strain by GeneReleaser(TM) showed sensitivity equivalent to virus isolation. The BHV-1TK PCR using GeneReleaser(TM) has a detection limit of 1 picogram and10 fentograms of purified BHV-1 DNA using ethidium bromide stained gel and Southern blot hybridization, respectively. It could detect viralDNA in 1,000 infected cells in a total suspension of 10,000 cells using either ethidium bromide stained gel or Southern blot hybridization.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 26/05/20 alle ore 23:41:30