Abstract
Mycobacterium tuberculosis (M. tb) is emerging as a more serious pathogen due to the increased multidrug-resistant TB and co-infection of human immunodeficiency virus (HIV). The development of an effective and sensitive detection method is urgently needed for bacterial load evaluation in vaccine development, early TB diagnosis, and TB treatment. Droplet digital polymerase chain reaction (ddPCR) is a newly developed sensitive PCR method for the absolute quantification of nucleic acid concentrations. Here, we used ddPCR to quantify the circulating virulent M. tb-specific CFP10 (10-kDa culture filtrate protein, Rv3874) and Rv1768 DNA copy numbers in the blood samples from Bacille Calmette-Guerin (BCG)-vaccinated and/or virulent M. tb H37Rv-challenged rhesus monkeys. We found that ddPCR was more sensitive compared to real-time fluorescence quantitative PCR (qPCR), as the detection limits of CFP10 were 1.2 copies/μl for ddPCR, but 15.8 copies/μl for qPCR. We demonstrated that ddPCR could detect CFP10 and Rv1768 DNA after 3 weeks of infection and at least two weeks earlier than qPCR in M.tb H37Rv-challenged rhesus monkey models. DdPCR could also successfully quantify CFP10 and Rv1768 DNA copy numbers in clinical TB patients’ blood samples (active pulmonary TB, extrapulmonary TB (EPTB), and infant TB). To our knowledge, this study is the first to demonstrate that ddPCR is an effective and sensitive method of measuring the circulating CFP10 and Rv1768 DNA for vaccine development, bacterial load evaluation in vivo, and early TB (including EPTB and infant TB) diagnosis as well.
Highlights
Tuberculosis (TB) is an emerging health problem with high morbidity and mortality, in developing countries
The fluorescence signal intensity demonstrated that 350 nmol/L concentration was optimal for CFP10 and ESAT6, and 300 nmol/L concentration for Rv1768, by quantitative polymerase chain reaction (PCR) (qPCR), respectively
The probe concentration in the PCR reaction was selected as 350 nmol/L for CFP10 and ESAT6, 300 nmol/ L for Rv1768 (Fig. 1a)
Summary
Tuberculosis (TB) is an emerging health problem with high morbidity and mortality, in developing countries. DdPCR has not been reported to detect virulent M. tbspecific DNA copy numbers in the blood samples, for infant TB, and has not been used to detect bacterial loads at different time courses in M. tb- infected animal models and evaluate the vaccine protective efficacy in vivo.
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