Development and application of Cas13a-based diagnostic assay for<i>Neisseria gonorrhoeae</i>detection and azithromycin resistance identification

Luo, Hongwei; Chen, Wentao; Z, Mai; Jiao, Yang; Lin, Xiaomian; Zeng, Li-Huan; Pan, Yuying; Xie, Qinghui; Xu, Qingqing; Li, Xiaoxiao; Liao, Yu-Cai; Zhao, Feng; Ou, Jiangli; Qin, Xiaolin; Zheng, Heping

doi:10.1093/jac/dkab447

Cited by 11 publications

(11 citation statements)

References 49 publications

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“…In these studies, gold nanoparticles were used as the colorimetric probe. Luo et al reported a similar detection limit of 10 copies µL −1 for a Cas13a-based specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay developed for NG porA and azithromycin resistance detection [ 38 ]. It may be noted that this assay was tested on only purified double-stranded DNA (dsDNA).…”

Section: Resultsmentioning

confidence: 99%

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

et al. 2023

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Herein, we report results of the studies relating to the development of an impedimetric, magnetic bead-assisted supersandwich DNA hybridization assay for ultrasensitive detection of Neisseria gonorrhoeae, the causative agent of a sexually transmitted infection (STI), gonorrhea. First, a conductive ink was formulated by homogenously dispersing carboxylated multiwalled carbon nanotubes (cMWCNTs) in a stable emulsion of terpineol and an aqueous suspension of carboxymethyl cellulose (CMC). The ink, labeled C5, was coated onto paper substrates to fabricate C5@paper conductive electrodes. Thereafter, a magnetic bead (MB)-assisted supersandwich DNA hybridization assay was optimized against the porA pseudogene of N. gonorrhoeae. For this purpose, a pair of specific 5′ aminated capture probes (SCP) and supersandwich detector probes (SDP) was designed, which allowed the enrichment of target gonorrheal DNA sequence from a milieu of substances. The SD probe was designed such that instead of 1:1 binding, it allowed the binding of more than one T strand, leading to a ‘ladder-like’ DNA supersandwich structure. The MB-assisted supersandwich assay was integrated into the C5@paper electrodes for electrochemical analysis. The C5@paper electrodes were found to be highly conductive by a four-probe conductivity method (maximum conductivity of 10.1 S·cm−1). Further, the biosensing assay displayed a wide linear range of 100 aM-100 nM (109 orders of magnitude) with an excellent sensitivity of 22.6 kΩ·(log[concentration])−1. The clinical applicability of the biosensing assay was assessed by detecting genomic DNA extracted from N. gonorrhoeae in the presence of DNA from different non-gonorrheal bacterial species. In conclusion, this study demonstrates a highly sensitive, cost-effective, and label-free paper-based device for STI diagnostics. The ink formulation prepared for the study was found to be highly thixotropic, which indicates that the paper electrodes can be screen-printed in a reproducible and scalable manner.

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Section: Resultsmentioning

confidence: 99%

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

et al. 2023

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“…Additionally, Yersinia pestis , the main pathogen causing plague, was detected by the RPA-SHERLOCK method with an LOD of as low as 700 zM (420 copies/ml; Schultzhaus et al, 2021 ). Using the RPA-SHERLOCK assay, Luo et al (2021) detected 10 copies/μl of the porA gene of Neisseria gonorrhoeae and successfully identified two azithromycin resistance mutations.…”

Section: Cas13a-based Detection Of Bacteria and Other Pathogensmentioning

confidence: 99%

“…Cas13a-based detection methods could recognize SNP in target gene via artificially adding mismatch between crRNA and target RNA, exhibiting the potential to detect AMR mutants. Following to this mechanism, several studies have achieved detection of AMR mutants in different pathogens ( Kiga et al, 2020 ; Cunningham et al, 2021 ; Luo et al, 2021 ; Bai et al, 2022 ). Cunningham et al demonstrated the capability of RPA-SHERLOCK assay for recognizing the A581G mutant in P. falciparum associated with sulfadoxine resistance ( Cunningham et al, 2021 ).…”

Section: Cas13a-based Detection Of Bacteria and Other Pathogensmentioning

confidence: 99%

“…Cunningham et al demonstrated the capability of RPA-SHERLOCK assay for recognizing the A581G mutant in P. falciparum associated with sulfadoxine resistance ( Cunningham et al, 2021 ). Similarly, another group utilized RPA-SHERLOCK assay to identify the A2059G and C2611T mutants in Neisseria gonorrhoeae associated with azithromycin resistance, in accordance with sequencing ( Luo et al, 2021 ). In addition, Bai et al used Cas13a system with synthetic mismatch to significantly distinguish the wild type strains of Mycobacterium tuberculosis from fluroquinolone resistance mutant strains (G88A, A90V, S91P, D94N, D94H, D94T, and D94G; Bai et al, 2022 ).…”

Section: Cas13a-based Detection Of Bacteria and Other Pathogensmentioning

confidence: 99%

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CRISPR-Cas13a system: A novel tool for molecular diagnostics

Zhao

Qiu

et al. 2022

Front. Microbiol.

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The clustered regularly interspaced short palindromic repeats (CRISPR) system is a natural adaptive immune system of prokaryotes. The CRISPR-Cas system is currently divided into two classes and six types: types I, III, and IV in class 1 systems and types II, V, and VI in class 2 systems. Among the CRISPR-Cas type VI systems, the CRISPR/Cas13a system has been the most widely characterized for its application in molecular diagnostics, gene therapy, gene editing, and RNA imaging. Moreover, because of the trans-cleavage activity of Cas13a and the high specificity of its CRISPR RNA, the CRISPR/Cas13a system has enormous potential in the field of molecular diagnostics. Herein, we summarize the applications of the CRISPR/Cas13a system in the detection of pathogens, including viruses, bacteria, parasites, chlamydia, and fungus; biomarkers, such as microRNAs, lncRNAs, and circRNAs; and some non-nucleic acid targets, including proteins, ions, and methyl groups. Meanwhile, we highlight the working principles of some novel Cas13a-based detection methods, including the Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) and its improved versions, Cas13a-based nucleic acid amplification-free biosensors, and Cas13a-based biosensors for non-nucleic acid target detection. Finally, we focus on some issues that need to be solved and the development prospects of the CRISPR/Cas13a system.

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“…Novel point-of-care diagnostics for N. gonorrhoeae are increasingly available. 18,19 20 Further, some of those tests appear to be both feasible and acceptable in low-resource settings. 21 However, few have met the World Health Organization (WHO) standards for point-of-care tests: real-time connectivity, ease of specimen collection, affordable, sensitive, specific, user-friendly, rapid and robust, equipment free/environmentally friendly, deliverable to end-users.…”

Section: Introductionmentioning

confidence: 99%

Clinical Performance of Cas13a-based Point-of-Care Lateral Flow Assay for DetectingNeisseria gonorrhoeae

Allan-Blitz,

Sanders,

Shah

et al. 2024

Preprint

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BackgroundDiagnosis ofNeisseria (N.) gonorrhoeaeis dependent on nucleic acid amplification testing (NAAT), which is not available in resource-limited settings where the prevalence of infection is highest. Recent advances in molecular diagnostics leveraging the high specificity of CRISPR enzymes can permit field-deployable, point-of-care lateral flow assays. We previously reported on the development andin vitroperformance of a lateral flow assay for detectingN. gonorrhoeae. Here we aimed to pair that assay with point-of-care DNA extraction techniques and assess the performance on clinical urine specimens.MethodsWe collected an additional urine specimen among individuals enrolling in an ongoing clinical trial at the Massachusetts General Hospital Sexual Health Clinic who presented with symptoms of urethritis or cervicitis (urethral or vaginal discharge, dysuria, or dyspareunia). We then assessed thermal, detergent, and combination DNA extraction conditions, varying the duration of heat at 95°C and concentration of Triton X. We assessed the efficacy of the various DNA extraction methods by quantitative polymerase chain reaction (qPCR). Once an extraction method was selected, we incubated samples for 90 minutes to permit isothermal recombinase polymerase amplification. We then assessed the performance of lateral flow Cas13a-based detection using our previously designedporA probe and primer system forN. gonorrhoeaedetection, comparing lateral flow results with NAAT results from clinical care.ResultsWe assessed DNA extraction conditions on 3 clinical urine specimens. There was no consistent significant difference in copies per microliter of DNA obtained using more or less heat. On average, we noted that 0.02% triton combined with 5 minutes of heating to 95°C resulted in the highest DNA yield, however, 0.02% triton alone resulted in a quantity of DNA that was above the previously determined analytic sensitivity of the assay. Given that detergent-based extraction is more easily deployable, we selected that as our method for extraction. We treated 23 clinical specimens with 0.02% triton, which we added to the Cas13a detection system. We ran all lateral flow detections in duplicate. The Cas13a-based assay detected 8 of 8 (100%) positive specimens, and 0 of 15 negative specimens.ConclusionUsing point-of-care DNA extraction, isothermal amplification, and Cas13a-based detection, our point-of-care lateral flowN. gonorrhoeaeassay correctly identified 23 clinical urine specimens as either positive or negative. Further evaluation of this assay among larger samples and more diverse sample types is warranted.

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Development and application of Cas13a-based diagnostic assay forNeisseria gonorrhoeaedetection and azithromycin resistance identification

Cited by 11 publications

References 49 publications

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

CRISPR-Cas13a system: A novel tool for molecular diagnostics

Clinical Performance of Cas13a-based Point-of-Care Lateral Flow Assay for DetectingNeisseria gonorrhoeae

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