TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis

Sam, Ikuan; Chen, Yingying; Ma, Jiguang; Li, Shi‐Yuan; Ying, Ruoyan; Li, Lin-xian; Ji, Ping; Wang, Shujun; Xu, Jie; Bao, Yu-Jie; Zhao, Guoping; Zheng, Huajun; Wang, Jin; Wang, Sha; Wang, Ying

doi:10.1016/j.jinf.2021.04.032

Cited by 49 publications

(34 citation statements)

References 33 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, previous studies have determined that Mtb -cfDNA circulates at very low concentrations, 22 resulting in poor and highly variable diagnostic sensitivity, 9 – 11 which was not improved by use of a CRISPR assay with a high LOD. 14 Here we provide evidence that the combination of an assay optimised for serum cfDNA extraction and sensitive and specific target amplification and cleavage can sensitively detect serum Mtb -cfDNA in most patients with tuberculosis, including those missed by sputum-based diagnostics. This assay analyses cfDNA isolated from about 200 μL of serum, has a 2 h sample-to-answer time, detects its Mtb -cfDNA target across its full reported concentration range, and can be adapted to a portable format suitable for resource-limited settings ( appendix p 20).…”

Section: Discussionmentioning

confidence: 81%

“… 9 – 11 Clustered regularly-interspaced short palindromic repeats (CRISPR)-Cas12a cleavage activity can be used to enhance detection sensitivity, given that binding of a Cas12a guide RNA (gRNA) complex to its target DNA sequence can be used to cleave a quenched fluorescent probe, causing an enzymatic signal amplification that can markedly enhance the detection of low-concentration target sequences. This approach has been applied to detect M tuberculosis -derived DNA targets in sputum, 12 , 13 but had poor performance when applied to detect circulating Mtb -cfDNA, 14 because of the high cfDNA limit of detection (LOD) of the employed diagnostic assay. We adapted an ultrasensitive CRISPR-Cas12a-powered fluorescence assay 15 to detect Mtb -cfDNA in blood (CRISPR-TB), and evaluated its diagnostic performance for tuberculosis using cryopreserved samples from adults and children with presumed tuberculosis and their asymptomatic household contacts, and samples from a diagnostically challenging cohort of children living with HIV (CLHIV) who were symptomatic and immunocompromised, which included longitudinal serum obtained during tuberculosis treatment.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study

et al. 2022

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 81%

Section: Introductionmentioning

confidence: 99%

CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study

et al. 2022

View full text Add to dashboard Cite

“…TB-QUICK is a recent ultrasensitive MTB detection platform which combines loop-mediated isothermal amplification and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12b reaction for M TB detection. It is highly sensitive (with a near single-copy sensitivity), requires less sample input and offers even a shorter turnaround time than Gene-Xpert for RIF resistance[ 24 ].…”

Section: Diagnosis Of Tbmentioning

confidence: 99%

Tuberculosis conundrum - current and future scenarios: A proposed comprehensive approach combining laboratory, imaging, and computing advances

Merchant¹,

Shaikh²,

Nadkarni³

2022

WJR

View full text Add to dashboard Cite

Tuberculosis (TB) remains a global threat, with the rise of multiple and extensively drug resistant TB posing additional challenges. The International health community has set various 5-yearly targets for TB elimination: mathematical modelling suggests that a 2050 target is feasible with a strategy combining better diagnostics, drugs, and vaccines to detect and treat both latent and active infection. The availability of rapid and highly sensitive diagnostic tools (Gene-Xpert, TB-Quick) will vastly facilitate population-level identification of TB (including rifampicin resistance and through it, multi-drug-resistant TB). Basic-research advances have illuminated molecular mechanisms in TB, including the protective role of Vitamin D. Also, Mycobacterium tuberculosis impairs the host immune response through epigenetic mechanisms (histone-binding modulation). Imaging will continue to be key, both for initial diagnosis and follow-up. We discuss advances in multiple imaging modalities to evaluate TB tissue changes, such as molecular imaging techniques (including pathogen-specific positron emission tomography imaging agents), non-invasive temporal monitoring, and computing enhancements to improve data acquisition and reduce scan times. Big data analysis and Artificial Intelligence (AI) algorithms, notably in the AI sub-field called “Deep Learning”, can potentially increase the speed and accuracy of diagnosis. Additionally, Federated learning makes multi-institutional/multi-city AI-based collaborations possible without sharing identifiable patient data. More powerful hardware designs - e.g. , Edge and Quantum Computing- will facilitate the role of computing applications in TB. However, “Artificial Intelligence needs real Intelligence to guide it!” To have maximal impact, AI must use a holistic approach that incorporates time tested human wisdom gained over decades from the full gamut of TB, i.e. , key imaging and clinical parameters, including prognostic indicators, plus bacterial and epidemiologic data. We propose a similar holistic approach at the level of national/international policy formulation and implementation, to enable effective culmination of TB’s endgame, summarizing it with the acronym “TB - REVISITED”.

show abstract

“…CRISPR-based methods have been developed and applied to detect pathogens of public health importance, including bacteria [e.g. Mycobacterium tuberculosis (Ai et al, 2019; Sam et al, 2021)], viruses [e.g. Zika virus, dengue virus (Gootenberg et al, 2017; Gootenberg et al, 2018; Myhrvold et al, 2018), human papillomavirus (Chen et al, 2018; Gong et al, 2021), SARS-CoV-2 (Broughton et al, 2020; Fozouni et al, 2021; Alcántara et al, 2021a)], fungi (Huang et al, 2021), and protozoa [with a major focus on parasites belonging to the phylum Apicomplexa, such as Plasmodium spp.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Novel CRISPR-based detection of Leishmania species

Dueñas

Nakamoto

Cabrera-Sosa

et al. 2022

Preprint

View full text Add to dashboard Cite

Tegumentary leishmaniasis, a disease caused by protozoan parasites of the genus Leishmania, is a major public health problem in many regions of Latin America. Its diagnosis is difficult given other conditions resembling leishmaniasis lesions and co-occurring in the same endemic areas. A combination of parasitological and molecular methods lead to accurate diagnosis, with the latter being traditionally performed in centralized reference and research laboratories as they require specialized infrastructure and operators. CRISPR-Cas systems have recently driven innovative tools for nucleic acid detection that combine high specificity, sensitivity and speed and are readily adaptable for point-of-care testing. Here, we harnessed the CRISPR-Cas12a system for molecular detection of Leishmania spp., emphasizing medically relevant parasite species circulating in Peru and other endemic areas in Latin America, with L. (Viannia) braziliensis being the main etiologic agent of cutaneous and mucosal leishmaniasis. We developed two assays targeting multi-copy targets commonly used in the molecular diagnosis of leishmaniasis: the 18S ribosomal RNA gene (18S rDNA), highly conserved across Leishmania species, and a region of kinetoplast DNA (kDNA) minicircles conserved in the L. (Viannia) subgenus. Our CRISPR-based assays were capable of detecting down to 5 × 10−2 (kDNA) or 5 × 100 (18S rDNA) parasite genome equivalents/reaction with PCR preamplification. The 18S PCR/CRISPR assay achieved pan-Leishmania detection, whereas the kDNA PCR/CRISPR assay was specific for L. (Viannia) detection. No cross-reaction was observed with Trypanosoma cruzi strain Y or human DNA. We evaluated the performance of the assays using 49 clinical samples compared to a kDNA real-time PCR assay as the reference test. The kDNA PCR/CRISPR assay performed equally well as the reference test, with positive and negative percent agreement of 100%. The 18S PCR/CRISPR assay had high positive and negative percent agreement of 82.1% and 100%, respectively. The findings support the potential applicability of the newly developed CRISPR-based molecular tools for first-line diagnosis of Leishmania infections at the genus and L. (Viannia) subgenus levels.

show abstract

TB-QUICK: CRISPR-Cas12b-assisted rapid and sensitive detection of Mycobacterium tuberculosis

Cited by 49 publications

References 33 publications

CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study

CRISPR detection of circulating cell-free Mycobacterium tuberculosis DNA in adults and children, including children with HIV: a molecular diagnostics study

Tuberculosis conundrum - current and future scenarios: A proposed comprehensive approach combining laboratory, imaging, and computing advances

Novel CRISPR-based detection of Leishmania species

Contact Info

Product

Resources

About