Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen

Journal of Virological Methods
J Christopher-HenningsRichard Oberst

Abstract

To detect Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in semen, various RNA extraction techniques have been utilized for RT-PCR, but rarely compared, to determine an optimized extraction protocol. Due to the viscosity, non-homogeneity, high cellularity and large volume of boar semen produced, difficulties can be encountered in obtaining RNA from the seminal cell fraction. This study compared six RNA extractions, five which used a commercially available kit (RNeasy, Qiagen Inc.) for use on highly cellular samples and a traditional phenol/chloroform procedure. All extractions were compared on serially diluted PRRSV "spiked" seminal cell fractions. The two methods resulting in recovery of the highest amount of RNA, which included a Qiashredder (Qiagen Inc.) (protocol 1) or cell lysis/centrifugation technique (protocol 3) preceding the RNeasy procedure were then compared using naturally infected semen samples from experimentally infected boars. Both protocols detected similar amounts of virus in "spiked" samples, but protocol 1 detected eight additional PRRSV-positive semen samples in naturally infected semen. This study demonstrated that semen "spiked" with PRRSV (cell-free virus) may not be representative of natur...Continue Reading

References

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Oct 1, 1995·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·J Christopher-HenningsD A Benfield
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Citations

Feb 18, 2012·Transboundary and Emerging Diseases·A Burgara-EstrellaJ Hernández
Jan 12, 2013·Veterinary Microbiology·Ahmed AliChang-Won Lee
Oct 28, 2008·Journal of Virological Methods·Thomas Bruun RasmussenDonald P King
Sep 8, 2007·Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc·Albert RoviraClaudia Muñoz-Zanzi
Dec 19, 2018·The American Journal of Tropical Medicine and Hygiene·Rodion GorchakovKristy O Murray
Jul 7, 2007·Archives of Andrology·Robert GoodrichStephen A Krawetz

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