Development of non-radioactive DNA probe and its clinical use

伴放线放线杆菌（Actinobacillus actinomycetemcomitans（A.a.）是牙周病的重要致病菌之一。近年来，人们提出了一种无需事先知道DNA序列就能检测遗传多态性的引物聚合酶链反应（AP-PCR）。在这项研究中，我们研究了实用的AP-PCR的基因分型A.a.。采用改良的货车Steenberen法从8个实验室菌株中提取全基因组DNA。用人工合成的10-mer随机寡核苷酸作为引物进行AP-PCR，从Operon 10-mer试剂盒A的20条引物中筛选出一条（GAAACGGGTG）可用于鉴别这些菌株。结果表明，AP-PCR技术能够区分不同的A.a.此外，我们还评估了非放射性DNA探针法（Affirm DP^<TM>）在临床诊断和疗效评价中的应用潜力。比较细菌培养法检测牙龈卟啉单胞菌、牙龈拟杆菌和伴放线放线杆菌的敏感性和临界值（10^4）的特异性。结果表明，对于B、牙龈卟啉单胞菌，以10^4为界值的DNA探针法的检出率与培养法相当或高于培养法。提示该试剂盒可作为临床诊断和疗效评价的辅助工具。

Actinobacillus actinomycetemcomitans (A.a..) is one of the important pathogens in the periodontal diseases. Recently, Arbitrarily Primed Polymerase Chain Reaction (AP-PCR) has been proposed for detecting genetic polymorphism without prior knowledge of the DNA sequence. In this study, we examined the utility of AP-PCR for genotyping of A.a.. Whole genomic DNA from 8 laboratory strains was extracted by a modification of the method of Van Steenberen. A single 10-mer, synthetic, oligonucleotide of random sequence was used as primer in AP-PCR.One (GAAACGGGTG) of the 20 primers (Operon 10-mer kit A) was found useful in differentiating these strains. The result indicates that AP-PCR is capable of distinguishing different strains of A.a... In addition, we evaluated the potential of application of non-radioactive DNA probe method (Affirm DP^<TM>) in clinical diagnosis and evaluation of treatment efficacy. Sensitivity and specificity of cut-off level (10^4) of the method with bacterial culture in detection of Bacteroide forsythus, Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans were compared. The result showed that detection percentage of DNA probe method with 10^4 cut-off was equal to or higher than culture method for B.forsythus and P.gingivalis. It is suggested to be a useful chair-side DNA probe kit as an aid in clinical diagnosis and evaluation of treatment efficacy.