Journal of Developmental Medicine(Electronic Version)  2013, Vol. 1 Issue (3): 129-135 DOI: |
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| 16S rDNA-based analysis of bacterial diversity of COnjunctitis by DGGE and sequence |
| LAN He-kui,CHEN Li-shan, MA Xiao-xia, et al
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1.Zhujiang Hospital,Southern Medical Universitj4,Guangzhou 510282,China；2. Insdtuw ofGenetics,Fudan University,Shanghai
200433,China) |
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Abstract 【Abstract】objectives To study bacterial diversity and pathogen in ocular environment by PCR—DGGE and sequencing．Methods A new molecular biology technique for identifying multiple bacteria from the ocular environment developed in this paper．From 1 3 1 human conjunctivae(58 with purulent，39 with nonpurulent conjunctivitis and 34 normal contr01)，swabs were taken and DNAs were extracted．V3 region of the 16S rDNA was amplified by polymerase chain reaction(PCR)and separated by denaturing gradient gel electrophoresis (DGGE)．Then DGGE bands were excised and directly sequenced，or cloned to pGEM—T vector and sequenced．Vailable segments of each sequence were compared with the sequences in GenBank．Furthermore，the results were compared with those obtained from the eonventional cultivation．Results Targeted fragments could be amplified from 54 of 59 cases in purulent conjunctiovitis eyes，from 1 5 of 39 in nonpurulent conjunctivitis
eyes and from 6 of 34 in normal controls．The following genera ordinal were detected：Staphylococcus，Corynebaeterium，Streptococcus，Acinetobacter，Bacillus，Pseudomonas，Propionibacterium，and pantoea．Three sequences could not be identified to genus level．The sequences had the highest similarity to enterobacteria，uncultured eubacterium and uncultured beta proteobacterium．Cuhures were only positive for Staphylococcus，Corynebaeterium，Streptococcus，Pseudomonas，and Klebsiella oxytoca．Some bacteria need commensal culture or co—culture．Conclusion Our data suggest that combination of 16S rDNA sequence analysis and DGGE fingerprinting is a rapid alternative to identify pathogens in patients with bacterial conjunctivitis and an appropriate method for detection and identification of monomicrobiaYpolymicrobial ocular infection which might not be detected by conventional cultivation．
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Received: 19 May 2013
Published: 06 September 2019
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