Slots J, Liu Y B, DiRienzo J M, Chen C
Department of Periodontology, University of Southern California, School of Dentistry, Los Angeles.
Oral Microbiol Immunol. 1993 Dec;8(6):337-43. doi: 10.1111/j.1399-302x.1993.tb00608.x.
The arbitrary primer polymerase chain reaction (AP-PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogen A. actinomycetemcomitans. Total genomic DNA from 73 strains was extracted by conventional methods. Three random-sequence 10-base oligonucleotide primers were chosen for AP-PCR. The amplified DNA products were separated electrophoretically in a 1% agarose gel containing ethidium bromide and the banding patterns were compared among different strains. For RFLP analysis, DNA was digested with EcoRI, separated on a 0.8% agarose gel and transferred to a nylon membrane. The membrane was probed with a previously characterized 5.2 kilobases (kb) DNA fragment cloned from A. actinomycetemcomitans strain Y4. The probe was labeled with digoxigenin, and hybridized fragments were detected with anti-digoxigenin antibody. AP-PCR produced 4-10 DNA bands in the 0.5-5 kb regions and distinguished 9, 13 or 17 genotypes, depending on the specific primer used. Southern blot RFLP analysis revealed 12 hybridization patterns consisting of 1 or 2 DNA fragments (2-23 kb). The addition of the Southern blot analysis to the AP-PCR analysis gave rise to a total of 30 DNA profiles among the 73 A. actinomycetemcomitans study strains. The results indicate that both AP-PCR and Southern blot analysis are useful in clonal analysis of A. actinomycetemcomitans.
采用任意引物聚合酶链反应（AP-PCR）和Southern印迹限制性片段长度多态性（RFLP）技术对牙周病原菌伴放线放线杆菌进行基因分型。采用常规方法从73株菌株中提取总基因组DNA。选择3条随机序列的10碱基寡核苷酸引物用于AP-PCR。扩增的DNA产物在含溴化乙锭的1%琼脂糖凝胶中进行电泳分离，并比较不同菌株间的条带模式。对于RFLP分析，DNA用EcoRI酶切，在0.8%琼脂糖凝胶上分离并转移至尼龙膜上。用先前从伴放线放线杆菌Y4菌株克隆的一个5.2千碱基（kb）的DNA片段对膜进行杂交检测。该探针用地高辛标记，杂交片段用抗地高辛抗体进行检测。AP-PCR在0.5-5 kb区域产生4-10条DNA条带，根据所用的特定引物可区分出9、13或17种基因型。Southern印迹RFLP分析显示有12种杂交模式，由1条或2条DNA片段（2-23 kb）组成。在AP-PCR分析中加入Southern印迹分析后，73株伴放线放线杆菌研究菌株共产生30种DNA图谱。结果表明，AP-PCR和Southern印迹分析在伴放线放线杆菌的克隆分析中均有用。
