ORIGINAL ARTICLE Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP S. Intorasoot 1 , C.S. Tharinjaroen 1 , P. Phunpae 1 , B. Butr-Indr 1 , U. Anukool 1 , K. Intachai 1 , S. Orrapin 1 , N. Apiratmateekul 2 , S. Arunothong 3 , V. Suthachai 3 , K. Saengsawang 4 , P. Khamnoi 5 , S. Pata 6,7 , W. Kasinrerk 6,7 and K. Tragoolpua 1 1 Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand 2 School of Medicine, Mae Fah Luang University, Chiang Rai, Thailand 3 Office of Disease Prevention and Control Region 1, Chiang Mai, Thailand 4 Department of Clinical Pathology, Lampang Hospital, Lampang, Thailand 5 Diagnostic Laboratory, Maharaj Nakorn Chiang Mai Hospital, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand 6 Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand 7 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 Keywords Ag85B antigen, immunomagnetic separation, Mycobacterium bovis, Mycobacterium tuberculosis complex, PCR with confronting two-pair primers, tuberculosis. Correspondence Khajornsak Tragoolpua, Division of Clinical Microbiology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. E-mail: khajornsak.tr@cmu.ac.th The first two authors have contributed equally. 2016/0268: received 16 October 2015, revised 3 February 2016 and accepted 19 April 2016 doi:10.1111/jam.13157 Abstract Aims: 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. Methods and 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. Significance and Impact of the Study: 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. Introduction Tuberculosis (TB) is a chronic and highly contagious dis- ease caused by the acid-fast bacilli, Mycobacterium tuber- culosis complex. TB has been spreading worldwide and has become one of the gravest major public health concerns. World Health Organization (WHO) has recently estimated new TB cases to be approx. 9 million, with 15 million TB patients dead worldwide (WHO 2014). Cur- rently, multidrug-resistant tuberculosis (MDR-TB) has become a major threat worldwide since the first-line drugs, isoniazid and rifampicin, have been unable to cure TB. Journal of Applied Microbiology 121, 528--538 © 2016 The Society for Applied Microbiology 528 Journal of Applied Microbiology ISSN 1364-5072