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The Power of Digital Droplet Polymerase Chain Reaction (ddPCR) in TB Diagnosis and Drug Resistance Analysis

NextEdge Admin

15 Mar 2024

3 min 42 sec

Digital droplet polymerase chain reaction (ddPCR) has emerged as a groundbreaking method in molecular biology, addressing limitations associated with traditional quantitative polymerase chain reaction (qPCR). While qPCR has been the gold standard for nucleic acid quantification, its drawbacks, such as limited sensitivity for small differences and reliance on standard curves, have paved the way for the adoption of ddPCR.

DdPCR utilizes microfluidic technology combined with PCR to achieve absolute nucleic acid quantification. Its key innovation lies in the partitioning of samples into thousands of droplets, where independent PCR sub-reactions occur. This approach allows for the quantification of a single copy of DNA through Poisson statistical analysis of the positive droplets. The method offers distinct advantages over qPCR, such as the ability to amplify each target gene in separate compartments, ensuring higher specificity and sensitivity. Unlike qPCR, ddPCR does not necessitate a standard curve for quantification, relying instead on binomial statistics, making it inherently accurate. Various commercial microfluidic platforms based on ddPCR are available, such Bio-Rad QX200TM droplet digital system.

The application of ddPCR in diagnosing Mycobacterium tuberculosis (MTB) infections has garnered attention due to its potential to revolutionize tuberculosis (TB) diagnostics. Traditional methods like acid-fast staining and culture are time-consuming and may lack sensitivity. DdPCR provides a rapid, accurate alternative for diagnosing TB in low-copy samples. Its application extends to quantifying MTB in various biological samples, including plasma, whole blood, and CD34+ peripheral blood mononuclear cells. This capability has implications for diagnosing latent TB infections and monitoring treatment outcomes.

Additionally, ddPCR has proven valuable in analyzing MTB drug resistance, particularly in the context of multidrug-resistant TB (MDR-TB). Traditional drug susceptibility testing (DST) methods are often time-consuming and vary in reliability. DdPCR’s high sensitivity and precision in detecting copy number variations make it an effective tool for identifying drug-resistant mutants, even in cases of heteroresistance. The method has been applied to detect resistance mutations inducing rifampicin resistance and assess drug sensitivity for new drugs like bedaquiline and delamanid.

In essence, ddPCR stands as a powerful and promising tool in molecular diagnostics, offering advantages in sensitivity, specificity, and absolute quantification. Its applications in TB diagnosis and drug resistance analysis present opportunities to enhance patient care and address global health challenges associated with TB.