Use of polymerase chain reaction to detect swine influenza virus in nasal swab specimens.
Rapid and accurate detection of a virus in a population is a critical factor in the eventual treatment and/or control of the virus. In this study, we examined use of the polymerase chain reaction (PCR) to detect swine influenza virus in nasal swab specimens from infected pigs. This approach was first standardized, using viral RNA purified by guanidinium/phenol-chloroform extraction and placed in the same transport medium as the swabs. By using highly conserved primers for the swine H1 hemagglutinin, we amplified a 591-base pair fragment that was analyzed by use of agarose gel electrophoresis, Southern blot, and DNA sequencing. To evaluate PCR as a potential diagnostic tool for detection of swine influenza virus infection, we obtained nasal swab specimens from experimentally infected pigs. Amplification by PCR and reamplification of extracted samples with internal primers yielded detectable bands for an amount of virus less than that required to infect embryonating chicken eggs. We also tested swab specimens from pigs involved in 3 separate, natural episodes of swine influenza. These swab specimens were extracted, amplified and reamplified, producing visible bands on the gel and in Southern blots. We performed Southern blot analyses on all PCR products, to confirm that they were from viral H1 RNA. We also cloned and sequenced a 591-base pair product from 1 specimen and found that it was 100% identical to the hemagglutinin gene sequence of A/Sw/Ind/1726/88. Results indicate that PCR can be used to detect swine influenza virus, even in nasal swab specimens, the specimen typically collected for diagnosis of virus infection.
Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison 53706.
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Schorr
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Wentworth
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Hinshaw
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern / Q000379:methods; D002642:Chick Embryo; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D009980:Influenza A virus / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009297:Nasal Mucosa / Q000821:virology*; D009976:Orthomyxoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods; D013552:Swine; D013553:Swine Diseases
D017931:DNA Primers; D004279:DNA, Viral