Figure: Digital droplet PCR accurately quantifies SARS-CoV-2 viral load from crude lysate without nucleic acid purification | Scientific Reports

Bonolo S.P. Mathekga, ... Deepak B. Thimiri Govinda Raj

Digital Droplet PCR (ddPCR) is known as the third generation of quantitative PCR. It works by segmenting samples using water-in-oil emulsions to create droplets from which their genetic material can be identified and quantified.¹⁰⁵ Droplet digital polymerase chain reaction (ddPCR) is a third generation of polymerase chain reaction (PCR) that enables the exact quantification of nucleic acid targets within a sample. The capability of ddPCR to accurately detect and quantify low abundant targets has led to its fast-growing applications in detection of different pathogens. This review summarizes the ddPCR technology and its applications in tuberculosis diagnosis. From current studies including a total of nine publications on the applications of ddPCR in tuberculosis research, it is clear that ddPCR technology offers enormous advantages, such as unparalleled sensitivity, high precision, and absolute quantification without a standard curve, over common molecular diagnostic platforms like the real-time quantification PCR. The latest study also showed that rapid drug susceptibility test of Mycobacterium tuberculosis in sputa could be achieved within 4 days. However, the high cost is the main limitation for its wide applications, especially in developing countries. As we near the vision 2030 goal for sustainable development and ending the tuberculosis epidemic by 2030, ddPCR techniques may help achieve this objective and many more discussed in the UNGA-HLM-TB.¹⁰⁵ This is beneficial over conventional real-time (RT) PCR methods such as the GeneXpert (Cepheid, CA, USA) for the reasons that ddPCR is able to detect MTb DNA at limits of detection as low as 1.2 copies/μL.¹⁰⁶

In 2017 Yang et al.¹⁰⁷ was among the first to use the QX100 ddPCR (Bio-Rad, CA, USA) to diagnose TB in clinical samples. In their work, Yang et al. used whole blood samples from 84 patients (28 with pulmonary TB, 28 with EPTB and 28 healthy participants). They found that ddPCR could be used to diagnose TB in clinical samples with low levels of MTb DNA.

Since then, the use of ddPCR for MTb detection has grown in popularity. Several studies have explored the use of this technique, in detecting different MTb gene targets. Fig. 4 shows the performance of ddPCR reported in literature against RT-PCR and NCBA methods.

http://ciencedirect.com/topics/neuroscience/droplet-digital-polymerase-chain-reaction