Tang G, Yip H K, Luo G, Cheung B P K, Shen S, Samaranayake L P
Oral Bio-Sciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China.
Oral Dis. 2003 Jul;9(4):203-9. doi: 10.1034/j.1601-0825.2003.02926.x.
The traditional, biochemical and enzymatic methods of identifying Actinomyces species are frequently confounded by the similar phenotypic characteristics shared by the different members of this genus. Therefore, we developed novel species-specific oligonucleotide probes to accurately speciate seven pathogenic Actinomyces species, namely, Actinomyces bovis, A. gerencseriae, A. israelii, A. meyeri, A. naeslundii, A. odontolyticus and A. viscosus.
A pair of universal primers and seven 15- to 19-base oligonucleotide probes with a tail of 20 thymidines on the 5' end were developed. The variable regions of 16S ribosomal DNA of 36 strains of Actinomyces belonging to the above species were amplified and labeled with digoxigenin, and an oligonucleotide-DNA hybridization assay was performed to examine the specificity and sensitivity of these probes.
All seven, newly developed probes were specific and sensitive, and accurately detected 36 reference and wild type strains belonging to Actinomyces species, without cross-reactions. The probe for A. naeslundii detected all strains belonging to the genospecies 1 (12 strains) and catalase-negative genospecies 2 (four strains); it failed to detect catalase-positive A. naeslundii genospecies 2 (previous A. viscosus serotype II) (two strains). However, the latter strains of catalase-positive A. naeslundii genospecies 2 were correctly detected by the probe developed for A. viscosus. The new probes were then field tested using supragingival plaque samples from 28 healthy preschool children. Whilst A. odontolyticus was detected in almost all samples (96.4%), A. gerencseriae, A. meyeri, catalase-negative A. naeslundii and catalase-positive A. naeslundii genospecies 2 were detected in < 50% samples.
We conclude that the developed oligonucleotide probes, complementary to the variable regions of 16S rDNA, would be of potential value for differentiating Actinomyces spp. in clinical samples from the oral cavity and other ecosystems where such species may abound.
放线菌属不同成员具有相似的表型特征，这常常使传统的、生化及酶学方法在鉴定放线菌菌种时产生混淆。因此，我们开发了新型的种特异性寡核苷酸探针，以准确区分七种致病性放线菌，即牛放线菌、杰氏放线菌、衣氏放线菌、迈氏放线菌、内氏放线菌、溶齿放线菌和黏性放线菌。
设计了一对通用引物以及七个15至19个碱基的寡核苷酸探针，这些探针在5'端带有20个胸腺嘧啶的尾巴。对属于上述菌种的36株放线菌的16S核糖体DNA可变区进行扩增并用洋地黄毒苷标记，然后进行寡核苷酸-DNA杂交试验，以检测这些探针的特异性和敏感性。
新开发的所有七个探针均具有特异性和敏感性，能准确检测36株属于放线菌属的参考菌株和野生型菌株，无交叉反应。内氏放线菌探针检测到了所有属于基因种1（12株）和过氧化氢酶阴性基因种2（4株）的菌株；未能检测到过氧化氢酶阳性的内氏放线菌基因种2（以前的黏性放线菌血清型II）（2株）。然而，过氧化氢酶阳性的内氏放线菌基因种2的后一类菌株被为黏性放线菌开发的探针正确检测到。然后使用来自28名健康学龄前儿童的龈上菌斑样本对新探针进行现场测试。虽然几乎在所有样本（96.4%）中都检测到了溶齿放线菌，但在<50%的样本中检测到了杰氏放线菌、迈氏放线菌、过氧化氢酶阴性的内氏放线菌和过氧化氢酶阳性的内氏放线菌基因种2。
我们得出结论，所开发的与16S rDNA可变区互补的寡核苷酸探针，对于区分来自口腔和其他可能存在此类菌种的生态系统的临床样本中的放线菌属菌种具有潜在价值。
