Exploring the diagnostic synergy of isothermal amplification-integrated CRISPR technology for tuberculosis: a systematic review

Abstract

To address the problems linked with Mycobacterium tuberculosis (MTB) detection, we need an accurate, sensitive, and rapid detection method for efficient epidemiological management of tuberculosis (TB). Nucleic acid-based diagnosis of TB is more sensitive and specific but primarily requires trained workers and costly infrastructure. Isothermal amplification methods have paved the way for efficient and rapid diagnosis of TB due to their negligible infrastructure requirements; however, they sometimes suffer from drawbacks such as false-positive results and challenges in primer design. With progress in clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas)-integrated nucleic acid detection methods, the above limitations are being overcome in pathogen detection. The combination of CRISPR with any suitable isothermal amplification techniques such as recombinase polymerase amplification (RPA) or loop-mediated isothermal amplification (LAMP) offers several advantages due to its higher sensitivity, specificity, versatility and reproducibility as a point-of-care detection technique. Thus, in this systematic review, we aimed to provide a comprehensive overview of the various isothermal amplification methods coupled with CRISPR-based TB diagnostic studies that are reported in the literature. About 12 articles were included in this review using predefined selection criteria. Data were extracted for detailed review from PubMed, Google Scholar and ScienceDirect, and diagnostic efficiency was evaluated. The data uncovered that most of the studies were conducted in China, with IS6110 and IS6108 as the major target genes employed. The most used detection methods were based on fluorescence and lateral flow. Analytical sensitivity, defined by the limit of detection, ranged between 10 and 20 copies per μL. Diagnostic sensitivity and specificity were consistently high, ranging from 95 to 100%. Taken together, the synergy between isothermal amplification methods and CRISPR-Cas technique could serve as a potential alternative to qPCR, GeneXpert, and conventional acid-fast staining, particularly in low-resource regions for easy and rapid TB diagnosis.