Guangdong Provincial Key Laboratory of Pathogenic Biology and Epidemiology for Aquatic Economic Animals, Guangdong Ocean University, Zhanjiang, China.
J Appl Microbiol. 2013 Feb;114(2):433-47. doi: 10.1111/jam.12057. Epub 2012 Dec 20.
To perform a systematic evaluation of the applicability, validity and reliability of the long PCR-RFLP of 16S-ITS-23S rRNA genes for bacterial genotyping using both sequences retrieved from public genome databases and the experimental data obtained on bacterial cultures.
3301 Full-length sequences of 16S-ITS-23S rRNA genes were retrieved from 885 published bacterial genomes. Copy numbers of the whole set of 16S-ITS-23S rRNA genes per genome ranged from 1 (n = 161) to 14 (n = 4) with an average of 3.71. Their length varied greatly, from 4319 to 6568 bp with an average of 4952 bp. Computer-simulated RFLP analyses of the 16S-ITS-23S fragments flanked by the conserved primers 27F and 2241R suggested MspI, RsaI, HhaI and TaqI as the most appropriate enzymes for long PCR-RFLP analysis of the 16S-ITS-23S sequence. MspI was used to screen over 900 bacterial cultures isolated from the Huguangyan Maar Lake in southern China. An experimental sequencing of 16S rRNA genes of the isolates possessing a unique RFLP band pattern proved the broad applicability and high resolution of this approach.
These results indicate that long PCR-RFLP of 16S-ITS-23S rRNA genes is a potentially universal and reliable bacterial genotyping tool with a high resolution.
The methodology of long PCR-RFLP of 16S-ITS-23S rRNA genes will facilitate the exploration and tracing of cultivable microbial diversity in natural environments.
通过使用从公共基因组数据库中检索到的序列和从细菌培养物中获得的实验数据，对 16S-ITS-23S rRNA 基因的长 PCR-RFLP 进行系统评估，以评估其在细菌基因分型中的适用性、有效性和可靠性。
从 885 个已发表的细菌基因组中检索到 3301 条全长 16S-ITS-23S rRNA 基因序列。每个基因组中整套 16S-ITS-23S rRNA 基因的拷贝数范围从 1（n = 161）到 14（n = 4），平均值为 3.71。它们的长度差异很大，从 4319 到 6568 bp，平均值为 4952 bp。使用保守引物 27F 和 2241R 侧翼的 16S-ITS-23S 片段的计算机模拟 RFLP 分析表明，MspI、RsaI、HhaI 和 TaqI 是 16S-ITS-23S 序列长 PCR-RFLP 分析的最合适酶。使用 MspI 筛选了来自中国南方黄羊岩玛珥湖的 900 多个细菌培养物。对具有独特 RFLP 带型的分离物 16S rRNA 基因进行实验测序证明了该方法的广泛适用性和高分辨率。
这些结果表明，16S-ITS-23S rRNA 基因的长 PCR-RFLP 是一种具有高分辨率的潜在通用和可靠的细菌基因分型工具。
16S-ITS-23S rRNA 基因长 PCR-RFLP 方法将有助于探索和追踪自然环境中可培养微生物的多样性。
