Proteins encoded by region of deletions (RD) of Mycobacterium tuberculosis are useful in development of vaccines and diagnostic reagents. In the present study, six M. tuberculosis genes from RD2 and RD11, rv1978, nrdf1, mpt64, cfp-21, ppe57 and ppe59, were cloned and overexpressed in Escherichia coli. All six purified recombinant proteins could distinguish tuberculosis (TB) patients and latent TB infected subjects (LTBI), or called subclinical TB infection, from BCG-vaccinated healthy controls by T-cell IFN-gamma releasing ELISPOT. ELISPOT of Rv1978, NrdF1, Mpt64, CFP-21, Ppe57 and Ppe59 achieved sensitivities of 59%, 60%, 82%, 48%, 59% and 47% respectively in the detection of active TB and specificities of 94%, 90%, 76%, 93%, 100% and 93% respectively in BCG-vaccinated healthy controls. Combination of Ppe57 or NrdF1 with early secreted antigen target 6 (ESAT-6) or 10-kDa culture filtrate protein (CFP-10) in the IFN-gamma releasing ESLIPOT assay could increase the sensitivities in detecting active TB, for ESAT-6 from 82.1% to 85.7% or 92.9% (P=0.5 or 0.03, respectively) and for CFP-10 from 67.9% to 78.6% or 83.9%, respectively (both P<0.05). The high sensitivities, specificities and promising antigenic combination of NrdF1 and Ppe57 in detection of TB in BCG-vaccinated controls suggest their potential application in TB diagnosis.