Mycoplasma gallisepticum F-vaccine strain-specific polymerase chain reaction.
A Mycoplasma gallisepticum (MG) F-vaccine strain polymerase chain reaction (PCR) (MGF-PCR) was developed and standardized. The origin of the primers was a clone (p08-M6#17) that contained an MG F-strain-specific DNA fragment of 6.0 kilobase pairs designated fMGF-1. Both ends of fMGF-1 (BamHI and EcoRI) were sequenced, and regions adequate for the primers were chosen. Seven 25-base primers were synthesized, and two near the EcoRI end (MGF-P1 left [L] and right [R]) were selected for MGF-PCR, MGF-P1 L and R amplified a DNA product of 524 base pairs (bp) that was directed at F-strain-related MG only. None of 16 other species of avian mycoplasmas that were tested yielded MGF-PCR product. MGF-PCR was able to consistently detect F-strain samples containing 54 cells or more and inconsistently (at least one positive out of five replicates) in samples with fewer organisms. The MGF-PCR products were visualized either by gel electrophoresis or Southern blot hybridization with a probe containing an identical base sequence as the 524-bp product amplified by MGF-PCR. The MGF-PCR was 1000 to 10,000 times more sensitive than dot-blot assays using two MG F-strain-specific probes.
Nascimento
E R
ER
Department of Epidemiology and Preventive Medicine, School of Veterinary Medicine, University of California, Davis 95616.
Yamamoto
R
R
Khan
M I
MI
D001428:Bacterial Vaccines / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D009174:Mycoplasma / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D013045:Species Specificity
D001428:Bacterial Vaccines; D004269:DNA, Bacterial