A duplex real-time RT-PCR for the detection of bluetongue virus in bovine semen

被引：33

作者：

Vanbinst, Tine ^{[1
]}

Vandenbussche, Frank

Dernelle, Eric ^{[2
]}

De Clercq, Kris ^{[1
]}

机构：

[1] Vet & Agrochem Res Ctr, Dept Virol, Sect Vesicular & Exot Dis, B-1180 Brussels, Belgium

[2] Ctr Inseminat Artificielle, Assoc Wallonne Elevage, B-5590 Ciney, Belgium

来源：

JOURNAL OF VIROLOGICAL METHODS | 2010年 / 169卷 / 01期

关键词：

Artificial insemination; Bluetongue virus; Bovine semen; Extraction method; Real-time RT-PCR; Validation; MESSENGER-RNA; BELGIUM; SEROTYPE-8; INFECTION; EPIDEMIC; FLUID; BLOOD;

D O I：

10.1016/j.jviromet.2010.07.019

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The control measures prescribed by the World Organization for Animal Health (OIE) for international trade in extended semen implicate repeated free testing of the donor's blood for bluetongue virus (BTV). The aim of this study was to validate a real-time RT-PCR for the direct testing of semen for artificial insemination (AI). The amplification of the BTV target was combined with an internal control target in duplex format. Optimal RNA recovery and efficient removal of PCR inhibitors was established using Trizol-based RNA extraction. The total assay was highly repeatable, the preliminary analysis of the specificity was 100% (95% Cl: 92-100%) and the limit of detection was 0.36 log(10) TCID50 ml(-1) (95% CI: -0.53 to -0.18 log(10) TCID50 ml(-1)) in BTV-8 spiked extended semen. The protocol was evaluated using 89 extended semen samples from 19 bulls showing typical clinical signs of a natural BTV-8 infection. Forty-eight samples were positive, 30 were doubtful and 11 were negative. Infectious BTV-8 was isolated. Based on varying real-time RT-PCR results of additional straws from cut-off samples it is highly recommended to analyse at least five straws per semen batch before declaring semen free of BTV. In conclusion, the partially validated assay presented has the potential to be used for the control of semen for international trade through direct testing of the semen. (C) 2010 Elsevier B.V. All rights reserved.

引用

页码：162 / 168

页数：7

共 41 条

[1] DETECTION OF BLUETONGUE VIRUS IN CLINICAL-SAMPLES BY POLYMERASE CHAIN-REACTION [J].

AKITA, GY ;

GLENN, J ;

CASTRO, AE ;

OSBURN, BI .

JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION, 1993, 5 (02) :154-158

[2]

BOWEN RA, 1983, AM J VET RES, V44, P2268

[3] THE PATHOGENESIS OF BLUETONGUE VIRUS-INFECTION OF BOVINE BLOOD-CELLS IN-VITRO - ULTRASTRUCTURAL CHARACTERIZATION [J].

BREWER, AW ;

MACLACHLAN, NJ .

ARCHIVES OF VIROLOGY, 1994, 136 (3-4) :287-298

[4]

Burns M, 2007, EUR FOOD RES TECHNOL, V226, P7, DOI [10.1007/s00217-006-0502-y, 10.1007/s00217-007-0683-z]

[5]

DARCEL CLQ, 1976, CAN VET J, V17, P318

[6] Transplacental Transmission of Bluetongue Virus 8 in Cattle, UK [J].

Darpel, Karin E. ;

Batten, Carrie A. ;

Veronesi, Eva ;

Williamson, Susanna ;

Anderson, Peter ;

Dennison, Mike ;

Clifford, Stuart ;

Smith, Ciaran ;

Philips, Lucy ;

Bidewell, Cornelia ;

Bachanek-Bankowska, Katarzyna ;

Sanders, Anna ;

Bin-Tarif, Abid ;

Wilson, Anthony J. ;

Gubbins, Simon ;

Mertens, Peter P. C. ;

Oura, Chris A. ;

Mellor, Philip S. .

EMERGING INFECTIOUS DISEASES, 2009, 15 (12) :2025-2028

[7] Transplacental infection and apparently immunotolerance induced by a wild-type bluetongue virus serotype 8 natural infection [J].

De Clercq, K. ;

De Leeuw, I. ;

Verheyden, B. ;

Vandemeulebroucke, E. ;

Vanbinst, T. ;

Herr, C. ;

Meroc, E. ;

Bertels, G. ;

Steurbaut, N. ;

Miry, C. ;

De Bleecker, K. ;

Maquet, G. ;

Bughin, J. ;

Saulmont, M. ;

Lebrun, M. ;

Sustronck, B. ;

De Deken, R. ;

Hooyberghs, J. ;

Houdart, P. ;

Raemaekers, M. ;

Mintiens, K. ;

Kerkhofs, P. ;

Goris, N. ;

Vandenbussche, F. .

TRANSBOUNDARY AND EMERGING DISEASES, 2008, 55 (08) :352-359

[8] Field observations during the Bluetongue serotype 8 epidemic in 2006 - II. Morbidity and mortality rate, case fatality and clinical recovery in sheep and cattle in the Netherlands [J].

Elbers, Armin R. W. ;

Backx, Anoek ;

Mintiens, Koen ;

Gerbier, Guillaume ;

Staubach, Christoph ;

Hendrickx, Guy ;

van der Spek, Arco .

PREVENTIVE VETERINARY MEDICINE, 2008, 87 (1-2) :31-40

[9] INVESTIGATIONS OF BLUETONGUE AND OTHER ARBOVIRUSES IN THE BLOOD AND SEMEN OF NATURALLY INFECTED BULLS [J].

GARD, GP ;

MELVILLE, LF ;

SHORTHOSE, JE .

VETERINARY MICROBIOLOGY, 1989, 20 (04) :315-322

[10] Validation of two real-time RT-PCR methods for foot-and-mouth disease diagnosis: RNA-extraction, matrix effect, uncertainty of measurement and precision [J].

Goris, Nesya ;

Vandenbussche, Frank ;

Herr, Cecile ;

Villers, Jerome ;

Van der Stede, Yves ;

De Clercq, Kris .

JOURNAL OF VIROLOGICAL METHODS, 2009, 160 (1-2) :157-162

← 1 2 3 4 5 →