Nascimento E R, Yamamoto R, Khan M I
Department of Epidemiology and Preventive Medicine, School of Veterinary Medicine, University of California, Davis 95616.
Avian Dis. 1993 Jan-Mar;37(1):203-11.
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.
开发并标准化了一种鸡毒支原体（MG）F疫苗株聚合酶链反应（PCR）（MGF-PCR）。引物来源于一个克隆（p08-M6#17），该克隆包含一个6.0千碱基对的MG F株特异性DNA片段，命名为fMGF-1。对fMGF-1的两端（BamHI和EcoRI）进行了测序，并选择了适合引物的区域。合成了7条25个碱基的引物，选择了靠近EcoRI端的两条引物（MGF-P1左[L]和右[R]）用于MGF-PCR，MGF-P1 L和R扩增出一个524碱基对（bp）的DNA产物，该产物仅针对与F株相关的MG。所检测的其他16种禽支原体均未产生MGF-PCR产物。MGF-PCR能够始终如一地检测到含有54个或更多细胞的F株样本，对于生物数量较少的样本则检测结果不一致（五次重复中至少有一次为阳性）。MGF-PCR产物通过凝胶电泳或与含有与MGF-PCR扩增的524-bp产物相同碱基序列的探针进行Southern印迹杂交来可视化。MGF-PCR比使用两种MG F株特异性探针的斑点印迹分析灵敏1000至10000倍。
