TY - JOUR
T1 - Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen
AU - Christopher-Hennings, Jane
AU - Dammen, Matthew
AU - Nelson, Eric
AU - Rowland, Raymond
AU - Oberst, Richard
N1 - Funding Information:
Special thanks to an anonymous Journal of Clinical Microbiology reviewer, the Molecular Diagnostic Section at the Animal Disease Research and Diagnostic Laboratory, Gina Steinlicht, Jessica Zweibahmer, Rebecca Hewer and Shelly Weeks. Special thanks to the Kansas State University (KSU) Swine Teaching and Research Center (Mark Nelsen and Dr. Duane Davis), Dr. Steven Dritz and the KSU PRRSV laboratory (Paula Schneider, Jessica Rowland, Luna Aguirre). We are extremely grateful for funding from the National Pork Board (project #03-052), South Dakota State University, Veterinary Science Department, Kansas State University, College of Veterinary Medicine and the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2003-35204-13704 (RR).
Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2006/9
Y1 - 2006/9
N2 - 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 naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen.
AB - 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 naturally infected semen samples (cell associated virus) for comparing extraction protocols, but did identify a useful extraction technique for boar semen.
KW - Boar semen
KW - Extraction
KW - PCR
KW - PRRSV
KW - RNA
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U2 - 10.1016/j.jviromet.2006.03.012
DO - 10.1016/j.jviromet.2006.03.012
M3 - Article
C2 - 16621036
AN - SCOPUS:33745890208
SN - 0166-0934
VL - 136
SP - 248
EP - 253
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1-2
ER -