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Open Access Highly Accessed Research article

Combined rpoB duplex PCR and hsp65 PCR restriction fragment length polymorphism with capillary electrophoresis as an effective algorithm for identification of Mycobacterial species from clinical isolates

Chen-Cheng Huang19, Jiann-Hwa Chen2, Shiau-Ting Hu3, Chien-Shun Chiou4, Wei-Chang Huang5, Jeng-Yuan Hsu5, Jang-Jih Lu69 and Gwan-Han Shen578*

Author Affiliations

1 Department of internal medicine, Executive Yuan Department of health, Division of Respiratory and Critical Care Medicine, Taichung Hospital, Taichung, Taiwan

2 Institute of Molecular Biology, National Chung Hsing University, Taichung, 402, Taiwan

3 Institute of Microbiology and Immunology, National Yang-Ming University, Taipei, Taiwan

4 The Central Region Laboratory, Centers for Disease Control, Department of Health, Taichung, 408, Taiwan

5 Division of Respiratory and Critical Care Medicine, Department of Internal Medicine, Taichung Veterans General Hospital, Taichung, Taiwan

6 Department of Laboratory Medicine, Linkou Chang-Gung Memorial Hospital, Taoyuan, Taiwan

7 Department of Respiratory Therapy, College of Health Care, China Medical University, Taiwan

8 Institute of Nursing Care, Hungeuang University, Taichung, Taiwan

9 Graduate Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan

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BMC Microbiology 2012, 12:137  doi:10.1186/1471-2180-12-137

Published: 8 July 2012

Abstract

Background

Mycobacteria can be quickly and simply identified by PCR restriction-enzyme analysis (PRA), but misidentification can occur because of similarities in band sizes that are critical for discriminating among species. Capillary electrophoresis can provide computer-aided band discrimination. The aim of this research was to develop an algorithm for identifying mycobacteria by combined rpoB duplex PRA (DPRA) and hsp65 PRA with capillary electrophoresis.

Results

Three hundred and seventy-six acid-fast bacillus smear-positive BACTEC cultures, including 200 Mycobacterium tuberculosis complexes (MTC) and 176 non-tuberculous mycobacteria (NTM) were analyzed. With combined hsp65 and rpoB DPRA, the accuracy rate was 100% (200 isolates) for the MTC and 91.4% (161 isolates) for the NTM. Among the discordant results (8.6%) for the NTM, one isolate of Mycobacterial species and an isolate of M. flavescens were found as new sub-types in hsp65 PRA.

Conclusions

This effective and novel identification algorithm using combined rpoB DPRA and hsp65 PRA with capillary electrophoresis can rapidly identify mycobacteria and find new sub-types in hsp65 PRA. In addition, it is complementary to 16 S rDNA sequencing.

Keywords:
rpoB duplex polymerase chain reaction; hsp65 restriction fragment length polymorphism analysis,Capillary electrophoresis