Long PCR-RFLP of 16S-ITS-23S rRNA genes: a high-resolution molecular tool for bacterial genotyping
Article first published online: 20 DEC 2012
© 2012 The Society for Applied Microbiology
Journal of Applied Microbiology
Volume 114, Issue 2, pages 433–447, February 2013
How to Cite
Zeng, Y.H., Koblížek, M., Li, Y.X., Liu, Y.P., Feng, F.Y., Ji, J.D., Jian, J.C. and Wu, Z.H. (2013), Long PCR-RFLP of 16S-ITS-23S rRNA genes: a high-resolution molecular tool for bacterial genotyping. Journal of Applied Microbiology, 114: 433–447. doi: 10.1111/jam.12057
- Issue published online: 14 JAN 2013
- Article first published online: 20 DEC 2012
- Accepted manuscript online: 5 NOV 2012 10:38AM EST
- Manuscript Accepted: 25 OCT 2012
- Manuscript Revised: 19 OCT 2012
- Manuscript Received: 13 AUG 2012
- NSFC. Grant Number: 30900045
- NSF. Grant Numbers: 9452408801002444, MELRS0923
- National Key Lab of Marine Environmental Science
- Czech project Algatech. Grant Number: CZ.1.05/2.1.00/03.0110
- bacterial genome;
- computer simulation;
- Huguangyan Maar Lake;
- long PCR;
- RFLP ;
- rRNA operon
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.
Methods and Results
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.
Significance and Impact of the Study
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.