Comparison of RNA extraction methods for the detection of porcine reproductive and respiratory syndrome virus from boar semen.
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.
Christopher-Hennings
Jane
J
Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD 57007-1396, USA. Jane.Hennings@sdstate.edu
Dammen
Matthew
M
Nelson
Eric
E
Rowland
Raymond
R
Oberst
Richard
R
D000818:Animals; D019316:Porcine respiratory and reproductive syndrome virus / Q000235:genetics* / Q000302:isolation & purification; D012367:RNA, Viral / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012661:Semen / Q000821:virology*; D034421:Sus scrofa / Q000821:virology*
D012367:RNA, Viral