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[Identification of the Mycobacterium tuberculosis complex by detecting recombinant secretory protein MPT64]
Zhong-hua Liu1, Lian-hua Qin, Yong-hong Feng
1Tuberculosis Key Laboratory of Shanghai, Shanghai Pulmonary Hospital, Affiliated to Tongji University, Shanghai, China.
Objective:
To identify the Mycobacterium tuberculosis complex by detecting the secretory protein MPT64.
Methods:
The gene mpt64 was amplified by polymerase chain reaction (PCR) from the genome of Mycobacterium tuberculosis H(37)Rv strain and cloned into expression vector. Immune sera from rabbits by recombinant proteins MPT64, were used to make enzyme-labeled antibodies and coated antibodies. A double antibody sandwich enzyme-linked immunosorbent assay (ELISA) was established to detect the secretory protein MPT64 of the culture supernatants in Mycobacterium strains. Results of ELISA were compared to those of the gene mpt64 amplified by PCR.
Results:
A recombinant vector was constructed. The minimum detectable concentration of MPT64 was 0.01 mg/L. A total of 27 reference strains and 170 clinical isolate strains were evaluated. PCR for Mycobacterium tuberculosis reference strain, Mycobacterium bovis reference strain and Mycobacterium africanum reference strain was positive, but that for other reference stains was negative, consistent with the results of ELISA. In the 170 clinical isolate stains, the positive result of PCR and ELISA was 98.2% (111/113) and 97.3% (110/113) respectively, while the specificity of PCR and ELISA was both 100%, no positive result in non-tuberculosis mycobacterium strains.
Conclusion:
Identification of the Mycobacterium tuberculosis complex by detecting secretory protein MPT64 is rapid, sensitive, and specific, which can be used routinely in clinical laboratories.
Insights
Detecting the secretory protein MPT64 accurately identifies the Mycobacterium tuberculosis complex. This enzyme-linked immunosorbent assay (ELISA) method is rapid, sensitive, and specific for clinical laboratories.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Context:
- Tuberculosis diagnosis relies on accurate identification of Mycobacterium tuberculosis complex.
- Current diagnostic methods can be time-consuming or lack sensitivity.
- The secretory protein MPT64 is a potential biomarker for M. tuberculosis.
Purpose:
- To develop and evaluate an enzyme-linked immunosorbent assay (ELISA) for detecting the secretory protein MPT64.
- To identify the Mycobacterium tuberculosis complex rapidly, sensitively, and specifically.
Summary:
- A double antibody sandwich ELISA was established using MPT64-specific antibodies.
- The assay detected MPT64 in culture supernatants with a minimum detectable concentration of 0.01 mg/L.
- ELISA results correlated well with PCR detection of the mpt64 gene, showing high sensitivity (97.3%) and specificity (100%) for clinical isolates.
Impact:
- This MPT64-based ELISA offers a rapid, sensitive, and specific method for identifying the Mycobacterium tuberculosis complex.
- The assay is suitable for routine use in clinical laboratories, potentially improving tuberculosis diagnosis turnaround times.
- High specificity ensures accurate differentiation from non-tuberculosis mycobacteria.
