Evaluating two methods for fingerprinting genomes of Actinobacillus actinomycetemcomitans.
Evaluating two methods for fingerprinting genomes of Actinobacillus actinomycetemcomitans.
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评估两种放线放线杆菌基因组指纹识别方法。
DOI：
10.1111/j.1399-302x.1993.tb00608.x
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发表时间：
1993
影响因子：
--
通讯作者：
Chen,C
中科院分区：
文献类型：
--
作者：
Slots,J;Liu,YB;DiRienzo,JM;Chen,C
采用随机引物聚合酶链反应（AP-PCR）和Southern印迹限制性片段长度多态性（RFLP）对牙周致病菌A进行基因分型。用常规方法提取73株放线菌的总基因组DNA。选择三种随机序列10碱基寡核苷酸引物用于AP-PCR。扩增产物经1%溴化乙锭琼脂糖凝胶电泳分离，并对不同菌株的带型进行比较。对于RFLP分析，DNA用EcoRI消化，在0.8%琼脂糖凝胶上分离并转移到尼龙膜上。用先前表征的从A克隆的5.2 kb DNA片段探测膜。放线菌共生菌株Y 4.用地高辛标记探针，并用抗地高辛抗体检测杂交片段。AP-PCR在0.5-5 kb区域产生4-10条DNA条带，并根据所用的特异性引物区分9、13或17种基因型。Southern印迹RFLP分析显示12种杂交模式，由1或2个DNA片段（2-23 kb）组成。在AP-PCR分析中增加Southern印迹分析，在73 A中总共产生了30个DNA图谱。放线菌共生体研究菌株。结果表明，AP-PCR和Southern blot分析均适用于A.伴放线菌
The arbitrary primer polymerase chain reaction (AP‐PCR) and Southern blot restriction fragment length polymorphism (RFLP) were used to genotype the periodontal pathogenA. 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 withEcoRI, 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 fromA. actinomycetemcomitansstrain 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 73A. actinomycetemcomitansstudy strains. The results indicate that both AP‐PCR and Southern blot analysis are useful in clonal analysis ofA. actinomycetemcomitans.