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A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas
Published on: June 5, 2014
[Establishment and application of a standard IS6110-RFLP method in the study of molecular genotyping analysis on
Jing-hua Liu1, Kristin Kremer, Christine Pourcel
1State Key Laboratory for Infectious Diseases Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.
Objective:
To develop a standardized IS6110-restriction fragment length polymorphism (RFLP) method, used for evaluating the capacity of genotyping.
Methods:
IS6110-RFLP of 78 Mycobacterium (M.) tuberculosis strains were studied by bio-molecular techniques including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis, together with data analysis by software Gel-Pro analyzer 3.1 and BioNumerics (Version 5.0).
Results:
IS6110-RFLP method was established and standardized successfully, including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis and usage of the analysis software with standard parameters. By this method, 78 M. tuberculosis isolates were classified into 75 genotypes which belonged to 11 different clusters. Of all the isolates, 66.7% (52/78) belonged to a main cluster.
Conclusion:
Standard IS6110-RFLP method was established successfully. This method had powerful capacity for genotyping and strain level identification and could be used for the surveillance on pathogens of M. tuberculosis in China.
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