Rapid detection of SARS-CoV-2 RNA in saliva via Cas13

Chandrasekaran, Sita S.; Agrawal, Shreeya; Fanton, Alison; Jangid, Aditya R.; Charrez, Bérénice; Escajeda, Arturo M.; Son, Sungmin; McIntosh, Roger; Tran, Huyen; Bhuiya, Abdul; Derby, María Díaz de León; Switz, Neil A.; Armstrong, Maxim; Harris, Andrew R.; Prywes, Noam; Lukarska, Maria; Biering, Scott B.; Smock, Dylan C. J.; Mok, Amanda; Knott, Gavin J.; Dang, Qi; Dis, Erik Van; Dugan, Eli; Kim, Shin; Liu, Tina Y.; Hamilton, Jennifer; Lin-Shiao, Enrique; Stahl, Elizabeth C.; Tsuchida, Connor A.; Giannikopoulos, Petros; McElroy, Matthew; McDevitt, Shana L.; Zur, Arielle; Sylvain, Iman; Ciling, Alison; Zhu, Madeleine; Williams, Clara; Baldwin, Alisha; Moehle, Erica A.; Kogut, Katherine; Eskenazi, Brenda; Harris, Eva; Stanley, Sarah A.; Lareau, Liana F.; Tan, Ming X.; Fletcher, Daniel A.; Doudna, Jennifer A.; Savage, David F.; Hsu, Patrick

doi:10.1038/s41551-022-00917-y

Cited by 69 publications

(53 citation statements)

References 44 publications

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“…Moreover, Cas13a was found to be more active than Cas12a in terms of trans-cleavage/collateral cleavage, which had a larger linear range and a better signal-to-background ratio for detection [50]. A previous study indicated that the LbuCas13a detection was significantly faster than LbCas12a at low activator concentrations [51]. Additionally, combined with CRISPR type III effector nuclease Csm6, the trans-cleavage signals of Cas13a were enlarged by more than eight-fold [14].…”

Section: Discussionmentioning

confidence: 92%

An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter pylori

Wang

Liu

Liu³

et al. 2022

JPM

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The rapid and simple detection of Helicobacter pylori (H. pylori) is essential for its clinical eradication. Although various methods for detecting H. pylori have been well established, such as endoscopy in combination with histology or culture, rapid urease test (RUT) and molecular tests using clinical specimens, it is of great importance to develop an ultrasensitive and accurate nucleic acid detection platform and apply it to identify H. pylori. To meet these demands, a novel method based on PCR and CRISPR-Cas13a, called PCR-Cas13a, was developed and validated using the DNA of 84 clinical strains and 71 clinical specimens. PCR primers for the pre-amplification of conservative sequence and CRISPR RNA (crRNA) for the detection of specific sequence were designed according to the principle. The designed primers and crRNA were specific to H. pylori, and the assay showed a high degree of specificity compared with other common pathogens. Our detection system can screen H. pylori with a limit of 2.2 copies/μL within 30 mins after PCR amplification. Using a coincidence analysis with traditional methods, our method exhibited 100% accuracy for the detection of H. pylori. Furthermore, its diagnostic performance was compared, in parallel with a q-PCR. The PCR-Cas13a demonstrates 98% sensitivity and 100% specificity. Moreover, our approach had a lower limit of detection (LOD) than q-PCR. Herein, we present a diagnostic system for the highly sensitive screening of H. pylori and distinguish it from other pathogens. All the results demonstrated that this PCR-based CRISPR assay has wide application prospects for the detection of H. pylori and other slow-growth pathogens.

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Section: Discussionmentioning

confidence: 92%

An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter pylori

Wang

Liu

Liu³

et al. 2022

JPM

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“…Saliva is emerging as an attractive medium for POC diagnosis of COVID-19 in the current pandemic. The SARS-CoV-2 virus has a preferential tropism to human airway epithelial cells that express the cellular receptor angiotensin-converting enzyme 2 (ACE2) [69] . Besides, ACE2 was found to be higher in salivary glands compared to the lungs, suggesting that salivary glands could be a potential target for SARS-CoV-2 virus [70] .…”

Section: Discussionmentioning

confidence: 99%

Potential of vibrational spectroscopy coupled with machine learning as a non-invasive diagnostic method for COVID-19

Zhao

Zhai

Shao

et al. 2023

Computer Methods and Programs in Biomedicine

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“…Such a color variant could be used as part of a build-in control mechanism for appropriate sample collection to detect an endogenous NA that is amplified alongside the target NA in a duplex RPA reaction 46 The simple assay set-up, with only a brief heat inactivation step for in-sample viral NA detection, is expected to allow for relatively straightforward integration of one-pot (RT-)RPA-LUNAS in a cheap and portable diagnostic device, that can be readout using a smartphone camera. Making use of advances in microfluidics and miniature electronic or chemical heating devices, integrated sample preprocessing, and lyophilization of the reaction mixture could further streamline the assay 46,47 . In conclusion, RPA-LUNAS shows great potential for translation into a sensitive and rapid point-of-care nucleic acid diagnostic that can readily be reconfigured for a quick diagnostic response following an epidemic outbreak.…”

Section: Discussionmentioning

confidence: 99%

Fast bioluminescent nucleic acid detection using one-pot isothermal amplification and dCas9-based split luciferase complementation

Veer

Aalen

Michielsen

et al. 2022

Preprint

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Nucleic acid detection methods based on isothermal amplification techniques show great potential for point-of-care diagnostic applications. However, most current methods rely on fluorescent or lateral flow assay readout, requiring external excitation or post-amplification reaction transfer. Here, we developed a bioluminescent nucleic acid sensor (LUNAS) platform in which target dsDNA is sequence-specifically detected by a pair of dCas9-based probes mediating split NanoLuc luciferase complementation. Whereas LUNAS itself features a detection limit of ~1 pM for dsDNA targets, the LUNAS platform is easily integrated with recombinase polymerase amplification (RPA), providing attomolar sensitivity in a single-pot assay. We designed a one-pot RT-RPA-LUNAS assay for detecting SARS-CoV-2 RNA without the need for RNA isolation and demonstrated the diagnostic performance for COVID-19 patient nasopharyngeal swab samples using a digital camera to record the ratiometric signal. Detection of SARS-CoV-2 from samples with viral RNA loads of ~200 cp/microL was achieved within ~20 minutes, showing that RPA-LUNAS is attractive for point-of-care diagnostic applications.

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Rapid detection of SARS-CoV-2 RNA in saliva via Cas13

Cited by 69 publications

References 44 publications

An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter pylori

An Ultrasensitive PCR-Based CRISPR-Cas13a Method for the Detection of Helicobacter pylori

Potential of vibrational spectroscopy coupled with machine learning as a non-invasive diagnostic method for COVID-19

Fast bioluminescent nucleic acid detection using one-pot isothermal amplification and dCas9-based split luciferase complementation

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