Using droplet digital PCR in the detection of Mycobacterium tuberculosis DNA in FFPE samples

Abstract

BackgroundDroplet digital PCR (ddPCR) is a technology that has higher sensitivity than real-time PCR for the identification of trace DNA. However, the use of ddPCR for the detection of Mycobacterium tuberculosis DNA in pathological samples has not been fully studied. MethodsA total of 65 formalin-fixed and paraffin-embedded (FFPE) specimens were included in this study. Twenty samples with definite results for tuberculosis (TB) were used to establish the ddPCR system for TB detection. ddPCR was then conducted to detect TB DNA in the 45 patients who were classified as ‘possible TB’ (real-time PCR results in the gray area, Ziehl–Neelsen staining-negative, and hematoxylin and eosin staining showing morphology suspicious for TB). The clinical treatment and disease outcomes were followed to assess the accuracy of ddPCR in the detection of TB DNA. ResultsAmong the 45 possible TB samples, 26 were ddPCR-positive, 12 were ddPCR-negative, and seven were in the gray area. ddPCR improved the positive rate of 57.8% (26/45) for the samples that were in the gray area by real-time PCR. Moreover, several patients received anti-TB therapy, and the effective ratio of therapy for the ddPCR-positive, ddPCR-negative, and ddPCR-gray area cases was 61.9% (13/21), 50.0% (2/4), and 33.3% (1/3), respectively. ConclusionsddPCR is more sensitive for detecting mild TB via FFPE samples than real-time PCR. The ddPCR method is of additional value in the diagnosis of TB from pathological samples.