Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP.
OBJECTIVE
To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens.


RESULTS
Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively.


CONCLUSIONS
The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens.


CONCLUSIONS
This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden.
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Intorasoot
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Tharinjaroen
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Phunpae
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Butr-Indr
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Anukool
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Intachai
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Orrapin
School of Medicine, Mae Fah Luang University, Chiang Rai, Thailand.
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Apiratmateekul
Office of Disease Prevention and Control Region 1, Chiang Mai, Thailand.
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Arunothong
Office of Disease Prevention and Control Region 1, Chiang Mai, Thailand.
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Suthachai
Department of Clinical Pathology, Lampang Hospital, Lampang, Thailand.
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Saengsawang
Diagnostic Laboratory, Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand.
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Khamnoi
Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.|Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Pata
Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.|Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Kasinrerk
Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand.
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Tragoolpua
D017931:DNA Primers / Q000235:genetics; D003955:Diagnostic Tests, Routine / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D018189:Immunomagnetic Separation / Q000295:instrumentation / Q000379:methods*; D009163:Mycobacterium bovis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D009169:Mycobacterium tuberculosis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D014376:Tuberculosis / Q000175:diagnosis / Q000382:microbiology*
D017931:DNA Primers
Ag85B antigen; Mycobacterium bovis; Mycobacterium tuberculosis complex; PCR with confronting two-pair primers; immunomagnetic separation; tuberculosis