Use of Digital Droplet PCR to Detect Mycobacterium tuberculosis DNA in Whole Blood-Derived DNA Samples from Patients with Pulmonary and Extrapulmonary Tuberculosis.

Xiyuan Bai,Shi Feng,Jiaru Yang,Aihua Liu,Lvyan Tao,Xinlin Han,Fukai Bao,Cuiping Xu,Mingbiao Ma,Yun Peng

doi:10.3389/fcimb.2017.00369

Abstract

Tuberculosis (TB) is a chronic infectious disease that has been threatening public health for many centuries. The clinical diagnostic procedure for TB is time-consuming and laborious. In the last 20 years, real-time fluorescence-based quantitative PCR (real-time PCR) has become a better alternative for TB diagnosis in clinics due to its sensitivity and specificity. Recently, digital droplet PCR (ddPCR) has been developed, and it might be an ideal alternative to conventional real-time PCR for microorganism detection. In this study, we aimed to assess the capacity of ddPCR and real-time PCR for detecting low levels of circulating Mycobacterium tuberculosis (MTB) DNA. The study involved testing whole blood samples for an MTB DNA target (known as IS6110). Blood samples were obtained from 28 patients with pulmonary TB, 28 patients with extrapulmonary TB, and 28 healthy individuals. The results show that ddPCR could be used to measure low levels of MTB DNA, and it has the potential to be used to diagnose pulmonary and extrapulmonary TB based on clinical samples.

Highlights

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (MTB) infection, is a chronic infectious disease that has been threatening public health for many years
The lack of contamination during our study helps to ensure the reliability of the results and, according to the digital droplet PCR (ddPCR) Detecting Tuberculosis-Causing Bacteria results for the MTB-positive controls, IS6110 could be detected in the tissue infected with MTB
The ddPCR results showed that a large number of IS6110positive microdroplets were detected in the DNA samples from the patients with pulmonary TB, extrapulmonary TB, new cases of TB, and cases of TB involving retreatment, as shown in Figures 1B,C,E,F, respectively

Summary

Introduction

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (MTB) infection, is a chronic infectious disease that has been threatening public health for many years. About 8 million new infections and 2 million TB-causing deaths occur annually (Baumann et al, 2006). Recent studies have shown that 20% of TB cases are extrapulmonary forms, including TB affecting the lymph nodes, pleura, and osteoarticular areas. This remains especially challenging for diagnosis because of the small amount of MTB present at the sites of the disease and the difficulty of obtaining clinical specimens from deep-seated organs (Golden and Vikram, 2005; Noussair and Bert, 2009)

Objectives

Methods

Results

Conclusion