Quantitative intra-short interspersed element PCR for species-specific DNA identification.
We have designed and evaluated four assays based upon PCR amplification of short interspersed elements (SINEs) for species-specific detection and quantitation of bovine, porcine, chicken, and ruminant DNA. The need for these types of approaches has increased drastically in response to the bovine spongiform encephalopathy epidemic. Using SYBR Green-based detection, the minimum effective quantitation levels were 0.1, 0.01, 5, and 1 pg of starting DNA template using our bovine, porcine, chicken, and ruminant species-specific SINE-based PCR assays, respectively. Background cross-amplification with DNA templates derived from 14 other species was negligible. Species specificity of the PCR amplicons was further demonstrated by the ability of the assays to accurately detect trace quantities of species-specific DNA from mixed (complex) sources. Bovine DNA was detected at 0.005% (0.5 pg), porcine DNA was detected at 0.0005% (0.05 pg), and chicken DNA was detected at 0.05% (5 pg) in a 10-ng mixture of bovine, porcine, and chicken DNA templates. We also tested six commercially purchased meat products using these assays. The SINE-based PCR methods we report here are species-specific, are highly sensitive, and will improve the detection limits for DNA sequences derived from these species.
Walker
Jerilyn A
JA
Department of Biological Sciences, Biological Computation and Visualization Center, Louisiana State University, 202 Life Sciences Building, Baton Rouge 70803, USA.
Hughes
David A
DA
Anders
Bridget A
BA
Shewale
Jaiprakash
J
Sinha
Sudhir K
SK
Batzer
Mark A
MA
D000328:Adult; D000818:Animals; D002417:Cattle; D002645:Chickens; D004247:DNA / Q000032:analysis*; D017931:DNA Primers; D006801:Humans; D008461:Meat Products / Q000032:analysis; D016133:Polymerase Chain Reaction / Q000379:methods*; D012418:Ruminants; D020086:Short Interspersed Nucleotide Elements / Q000235:genetics*; D013045:Species Specificity; D013552:Swine; D013698:Templates, Genetic
D017931:DNA Primers; D004247:DNA