Avila-Campos M J, Sacchi C T, Whitney A M, Steigerwalt A G, Mayer L W
Department of Microbiology, University of São Paulo, SP, Brazil.
J Periodontol. 1999 Oct;70(10):1202-8. doi: 10.1902/jop.1999.70.10.1202.
Fusobacterium nucleatum is the most frequently isolated bacterium in periodontal disease and plays an important role in serious infections in other parts of the body. Arbitrarily primed-polymerase chain reaction (AP-PCR) was used to construct primers for specific identification and subtyping of F. nucleatum. Subtypes may differ in virulence and, hence, are important as periodontal pathogens. Subtypes also may differ in antibiotic susceptibility; therefore, knowing the subtypes may influence choice of treatment.
We analyzed 70 DNA samples of F. nucleatum isolated from patients with periodontal disease (PD) (N = 32) or AIDS-related PD (N = 8) and from healthy carriers (N = 30). From 90 AP-PCR primers screened, five amplification products were selected, cloned in pCR II vector, and sequenced. These sequences were used to design new pairs of specific primers. Sequences were compared to GenBank entries with BLAST and showed no significant matches.
Three primer pairs produced bands of approximately 1 Kb (primer 5059S) or 0.5 Kb (primers FN5047 or M1211) with all F. nucleatum DNAs tested. PCR amplification using primer pair M8171 produced a 1 Kb band with isolates from 7 (22%) PD and 5 (63%) PD-AIDS patients and 9 (30%) healthy controls. Using the same primer pair, 2 other bands of approximately 0.5 Kb and 0.4 Kb were observed with DNA from isolates from 2 (6%) PD and all PD-AIDS patients, but were not observed with DNA samples from healthy controls (P<0.0001). All the primer pairs produced no or different amplicon profiles with DNA samples from bacterial species other than F. nucleatum.
Our results suggest that PCR primer pairs 5059S, FN5047 or M1211 can be used to specifically identify F. nucleatum isolates and distinguish them from other bacteria. The primer pair M8171 could also be used to differentiate F. nucleatum isolated from periodontal patients or healthy individuals. These specific primers can be used in PCR analysis for specific identification of F. nucleatum and to distinguish it from other bacteria associated with human periodontitis. These approaches appear promising in facilitating laboratory identification, molecular subtyping, and taxonomy of putative periodontopathogens.
具核梭杆菌是牙周疾病中最常分离出的细菌，在身体其他部位的严重感染中也起重要作用。任意引物聚合酶链反应（AP-PCR）被用于构建用于具核梭杆菌特异性鉴定和亚型分析的引物。亚型在毒力上可能有所不同，因此作为牙周病原体很重要。亚型在抗生素敏感性方面也可能不同；所以，了解亚型可能会影响治疗选择。
我们分析了从牙周疾病（PD）患者（N = 32）、艾滋病相关牙周疾病患者（N = 8）和健康携带者（N = 30）中分离出的70份具核梭杆菌DNA样本。从筛选的90条AP-PCR引物中，选择了5个扩增产物，克隆到pCR II载体中并进行测序。这些序列被用于设计新的特异性引物对。将序列与GenBank条目进行BLAST比较，未发现显著匹配。
三对引物对所有测试的具核梭杆菌DNA产生了约1 Kb（引物5059S）或0.5 Kb（引物FN5047或M1211）的条带。使用引物对M8171进行PCR扩增，从7名（22%）牙周疾病患者和5名（63%）艾滋病相关牙周疾病患者以及9名（30%）健康对照者的分离株中产生了1 Kb的条带。使用同一引物对，在2名（6%）牙周疾病患者和所有艾滋病相关牙周疾病患者的分离株DNA中观察到另外两条约0.5 Kb和0.4 Kb的条带，但在健康对照者的DNA样本中未观察到（P<0.0001）。所有引物对与具核梭杆菌以外的细菌DNA样本均未产生条带或产生不同的扩增产物图谱。
我们的结果表明，PCR引物对5059S、FN5047或M1211可用于特异性鉴定具核梭杆菌分离株，并将其与其他细菌区分开来。引物对M8171也可用于区分从牙周病患者或健康个体中分离出的具核梭杆菌。这些特异性引物可用于PCR分析，以特异性鉴定具核梭杆菌，并将其与其他与人类牙周炎相关的细菌区分开来。这些方法在促进假定牙周病原体的实验室鉴定、分子亚型分析和分类学方面似乎很有前景。
