In a significant stride toward revolutionizing tuberculosis diagnostics, researchers at Tulane University have engineered a novel CRISPR-based assay that dramatically enhances the detection of Mycobacterium tuberculosis using a simple, non-invasive tongue swab. This innovation holds immense potential to transform tuberculosis screening, especially in resource-limited settings where access to conventional diagnostic infrastructure is scarce. Unlike traditional methods, which rely heavily on sputum samples fraught with practical collection challenges, the newly developed assay significantly lowers barriers to widespread community testing.
Historically, tuberculosis (TB) diagnosis has depended on sputum specimens, a viscous mucus originating from the lungs and lower respiratory tract. While sputum samples are ideal because they harbor ample quantities of TB bacteria necessary for assay sensitivity, their collection is cumbersome, often requiring trained personnel and patient cooperation. Such collection challenges lead to inefficiencies in large-scale screening programs. More critically, sputum testing is not feasible in approximately 25% of symptomatic patients and nearly 90% of those without symptoms, creating a diagnostic void that contributes to an estimated four million undiagnosed TB cases annually on a global scale.
Addressing these critical gaps, the Tulane research team leveraged prior CRISPR diagnostic platforms and refined them to amplify detection sensitivity in samples with low bacterial loads—specimens traditionally considered unsuitable for reliable TB detection, such as stool, cerebrospinal fluid, and notably, tongue swabs. These biologically diverse matrices pose significant sensitivity challenges due to dilute bacterial DNA concentrations and the presence of PCR inhibitors, necessitating a more robust molecular detection approach.
Published in the prestigious journal Nature Communications, the study showcases the tailored CRISPR assay’s performance across several clinical sample types. Most notably, tongue swabs—non-invasive, painless, and easily collectible specimens—yielded a TB detection sensitivity of 74%, markedly surpassing traditional methods which hovered around 56%. This enhancement represents a paradigm shift, pointing toward a feasible, scalable, and patient-friendly approach to TB screening that can function outside conventional healthcare facilities.
Furthermore, the assay demonstrated exceptional sensitivity when applied to other challenging sample types. Respiratory samples exhibited a detection sensitivity of 93%, pediatric stool samples showed 83%, and adult cerebrospinal fluid samples also achieved 93% sensitivity. These figures underscore the assay’s versatility across a spectrum of patient groups, including children, individuals living with HIV, and patients with extrapulmonary TB—all populations that commonly face sputum collection difficulties.
At the core of this breakthrough is a CRISPR diagnostic platform termed ActCRISPR-TB, designed to enhance the amplification and detection of pathogen-associated DNA via a multi-guide RNA Cas12a system. This system exploits the unique trans-cleavage activity of Cas12a endonuclease, which, upon activation by target DNA binding via multiple guide RNAs, indiscriminately cleaves single-stranded DNA reporters, generating a measurable signal. By preferentially favoring trans-cleavage over cis-cleavage activity, the assay attains heightened sensitivity, effectively detecting even trace amounts of TB bacterial DNA.
To facilitate decentralized testing, the researchers innovated a streamlined “one-pot” diagnostic procedure. This entails combining the patient-collected tongue swab directly with a preloaded reaction tube containing freeze-dried reagents and a lateral flow test strip. The tube is then incubated under controlled conditions for roughly 45 minutes, after which the test strip visually indicates TB presence via colorimetric bands. Notably, this workflow emulates the user-friendliness of rapid COVID-19 antigen tests, eliminating the necessity for elaborate laboratory infrastructure or highly trained personnel.
The advantages of a non-sputum, point-of-care test extend beyond accessibility. Conventional nucleic acid amplification tests for TB sputum samples often require lengthy processing times and specialized equipment that challenge widespread deployment. Conversely, the ActCRISPR-TB assay drastically reduces turnaround time, delivering definitive results in under an hour. This rapidity is poised to accelerate clinical decision-making, enabling prompt initiation of treatment regimens and curbing transmission within communities.
Tulane’s research represents an integral component of a broader vision led by Dr. Tony Hu, whose laboratory is pioneering portable TB diagnostics that integrate advanced molecular biology techniques with user-centric design. Beyond sample versatility and assay sensitivity, Hu’s team has developed handheld devices, comparable in size to smartphones, and even electricity-free units, tailored for environments lacking reliable power sources. Additionally, their integration of artificial intelligence algorithms to assess drug resistance profiles from genetic data ensures patients receive customized therapies swiftly, a critical step in combating multidrug-resistant tuberculosis strains.
The persistent global burden of tuberculosis—still one of the world’s deadliest infectious diseases—necessitates innovative diagnostic tools that transcend traditional clinical settings. As Dr. Hu emphasized, over 10 million individuals develop active TB annually, yet a staggering 40% remain undiagnosed due to the inadequacies of current detection paradigms. Non-invasive, efficient, and accessible tests such as ActCRISPR-TB hold promise to bridge this gap by enabling large-scale community screenings that identify hidden reservoirs of infection.
Equally promising is the potential impact on vulnerable populations who traditionally face diagnostic neglect. Children, people with HIV, and those with extrapulmonary TB often cannot produce sputum, rendering existing diagnostic options ineffective or inaccessible. The validated performance of the new CRISPR assay in pediatric stool and spinal fluid samples reflects a critical advance toward inclusive testing strategies that do not discriminate based on patient-specific sample availability constraints.
From a molecular standpoint, the strategic use of multiple guide RNAs in the Cas12a system enhances target recognition fidelity and signal amplification, mitigating false negatives commonly associated with low-copy-number pathogens. This technical refinement elevates confidence in test results and aligns with stringent clinical standards. Moreover, the entire assay’s simplification into a single-tube format not only conserves reagents and reduces contamination risks but also minimizes procedural errors, a frequent hindrance in decentralized testing environments.
While additional validation and regulatory approvals lie ahead, the revolutionary prospects introduced by this research signal a new chapter in tuberculosis control. By democratizing TB diagnostics through technology that fits into the palm of a hand and can be deployed in the most remote settings, the pathway to ending TB becomes clearer and more attainable.
As the study’s lead author, Dr. Zhen Huang, aptly noted, the painless and straightforward nature of tongue swabs effectively removes traditional barriers to sample collection. This innovation opens the gateway for widescale testing campaigns that were previously unimaginable in under-resourced regions. Combined with rapid turnaround times and high diagnostic accuracy, such advances could dramatically reshape public health responses to tuberculosis worldwide.
Ultimately, the Tulane team’s dual focus on molecular innovation and pragmatic deployment tools epitomizes the future of infectious disease diagnostics. By focusing on community-reaching formats rather than centralized labs, they offer a tangible solution to one of the most stubborn public health challenges of our time. With continued development, the ActCRISPR-TB assay and accompanying technologies could establish a new global standard for TB detection, cost-effectiveness, and accessibility, bringing us markedly closer to a world free of tuberculosis.
Subject of Research: Tuberculosis diagnostics, CRISPR-based pathogen detection
Article Title: Sensitive pathogen DNA detection by a multi-guide RNA Cas12a assay favoring trans- versus cis-cleavage
News Publication Date: 17-Sep-2025
Web References: 10.1038/s41467-025-63094-x
Keywords: Tuberculosis, Respiratory disorders, Infectious diseases, Medical diagnosis, Biotechnology, Clinical medicine, Respiratory system, Epidemiology
Tags: CRISPR technology in medicineCRISPR tuberculosis diagnostic testimproved TB diagnosis methodsinnovative assay for TBlarge-scale TB screening programsMycobacterium tuberculosis detectionnon-invasive tuberculosis screeningpublic health tuberculosis testingresource-limited TB diagnosticssputum sample challengestongue swab for TBundiagnosed tuberculosis cases
