SARS-CoV-2 detection with CRISPR diagnostics

Guo, Lu; Sun, Xuehan; Wang, Xinge; Chen, Liang; Jiang, Haiping; Gao, Qingqin; Dai, Moyu; Qu, Bin; Fang, Sen; Mao, Yihuan; Chen, Yangcan; Feng, Guihai; Gu, Qi; Wang, Liu; Wang, Ruiqi Rachel; Zhou, Qi; Li, Wei

doi:10.1101/2020.04.10.023358

Cited by 9 publications

(13 citation statements)

References 8 publications

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“…During the early period of the COVID-19 outbreak, we developed a SHERLOCK-based test for SARS-CoV-2 ( SHERLOCK-Covid ). Additional CRISPR-based tests have also been recently (v.20200505) Page 2 of 21 developed (Broughton et al, 2020;Ding et al, 2020;Guo et al, 2020;Lucia et al, 2020) , but similar to the SHERLOCK-Covid test, all involve two separate reaction steps, liquid handling, and opening of tubes. These steps not only add complexity but also significantly increase the likelihood of sample cross-contamination, prohibiting their use outside well-controlled laboratory environments.…”

Section: Introductionmentioning

confidence: 99%

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

Joung

Ladha

Saito

et al. 2020

Preprint

282

284

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The recent outbreak of the novel coronavirus SARS-CoV-2, which causes COVID-19, can be diagnosed using RT-qPCR, but inadequate access to reagents and equipment has slowed disease detection and impeded efforts to mitigate viral spread. Alternative approaches based on combinations of isothermal amplification and CRISPR-mediated detection, such as the SHERLOCK ( S pecific H igh S ensitivity E nzymatic R eporter Un LOCK ing) technique, offer reduced dependence on RT-qPCR equipment, but previously reported methods required multiple fluid handling steps, complicating their deployment outside clinical labs. Here we developed a simple test chemistry called STOP (SHERLOCK Testing in One Pot) for detecting SARS-CoV-2 in one hour that is suitable for point-of-care use. This simplified test, STOPCovid, provides sensitivity comparable to RT-qPCR-based SARS-CoV-2 tests and has a limit of detection of 100 copies of viral genome input in saliva or nasopharyngeal swabs per reaction. Using lateral flow readout, the test returns result in 70 minutes, and using fluorescence readout, the test returns result in 40 minutes. Moreover, we validated STOPCovid using nasopharyngeal swabs from COVID-19 patients and were able to correctly diagnose 12 positive and 5 negative patients out of 3 replicates. We envision that implementation of STOPCovid will significantly aid "test-trace-isolate" efforts, especially in low-resource settings, which will be critical for long-term public health safety and effective reopening of the society.

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Section: Introductionmentioning

confidence: 99%

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

Joung

Ladha

Saito

et al. 2020

Preprint

282

284

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“…DECTECTR is Cas12a based detection method that involves Cas12a activation after binding with COVID-cDNA and cuts reporter probes (oligonucleotide sequences labelled with the reporter molecules 6-carboxyfluorescein (FAM) and biotin) which confirms detection of virus. Similarly, another Cas12a-based technique CASdetect has been successfully developed, where gRNA has been modified to improve the accuracy further.DECTECTR AND CASdetec were reported to detect the viral RNA genome samples at a level of 10 4 -10 5 copies per ml of sample [21]. In addition to SARS-CoV-2 CASdetect has been successfully used for detection of other coronaviruses (SARS-CoV, MERS-CoV, CoV-HKU1, CoV-229E, CoV-OC43, and CoV-NL63) known to cause respiratory diseases with high sensitivity and specificity [21].…”

Section: Crispr Based Methods For Detection Of Virus and Pathogensmentioning

confidence: 99%

“…Similarly, another Cas12a-based technique CASdetect has been successfully developed, where gRNA has been modified to improve the accuracy further.DECTECTR AND CASdetec were reported to detect the viral RNA genome samples at a level of 10 4 -10 5 copies per ml of sample [21]. In addition to SARS-CoV-2 CASdetect has been successfully used for detection of other coronaviruses (SARS-CoV, MERS-CoV, CoV-HKU1, CoV-229E, CoV-OC43, and CoV-NL63) known to cause respiratory diseases with high sensitivity and specificity [21]. Other CRISPR-assisted nucleases such as fnCAS9, Cas12b have also been used for COVID-19 detection, and of these fnCAS9 with its higher mismatch specificity exhibited very high level of accuracy for faster detection [21,22].…”

Section: Crispr Based Methods For Detection Of Virus and Pathogensmentioning

confidence: 99%

“…In addition to SARS-CoV-2 CASdetect has been successfully used for detection of other coronaviruses (SARS-CoV, MERS-CoV, CoV-HKU1, CoV-229E, CoV-OC43, and CoV-NL63) known to cause respiratory diseases with high sensitivity and specificity [21]. Other CRISPR-assisted nucleases such as fnCAS9, Cas12b have also been used for COVID-19 detection, and of these fnCAS9 with its higher mismatch specificity exhibited very high level of accuracy for faster detection [21,22]. Table 3 shows genes of Covid-19 amplified during different CRISPR-based detection assays.…”

Section: Crispr Based Methods For Detection Of Virus and Pathogensmentioning

confidence: 99%

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CRISPR-Based Assays for Rapid Detection of SARS-CoV-2

Javalkote

Kancharla

Bhadra

et al. 2020

Preprint

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COVID-19 represents an unprecedented threat to global public health and economies. Assays are urgently needed for rapid detection and diagnosis of SARS-CoV-2-infected patients in order to inform treatment and quarantine strategies. Establishing globally accepted easy-to-access diagnostic tests is extremely important to understanding the epidemiology of the present pandemic. While nucleic acid-based tests are considered to be more sensitive with respect to serological tests, but the present gold standard RT-PCR-based assays possess limitations such as low sample throughput, requirement for sophisticated reagents and instrumentation. To overcome these shortcomings, recent efforts of incorporating LAMP-based isothermal detection, and minimizing the number of reagents required are on rise. Novel CRISPR- and other nuclease-based techniques, when merge with isothermal and allied technologies, promises to provide sensitive and rapid detection of SARS-CoV-2 nucleic acids. Here we discuss and present compilation of state-of-the-art CRISPR based detection techniques for use in COVID diagnosis and epidemiology.

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“…Recently developed CRISPR-based diagnostics have the potential to transform infectious disease diagnosis. Both CRISPR-Cas13-and Cas12-based assays have been developed for SARS-CoV-2 detection using extracted nucleic acids as input (17)(18)(19)(20)(21)(22). One such CRISPR-based diagnostic, SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing), involves two separate steps, starting with extracted nucleic acid: (1) isothermal RPA and (2) T7 transcription and Cas13-mediated collateral cleavage of a single-stranded RNA reporter (23) (Fig.…”

Section: Introductionmentioning

confidence: 99%

Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2

Arizti-Sanz

Freije

Stanton

et al. 2020

Preprint

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The COVID-19 pandemic has highlighted that new diagnostic technologies are essential for controlling disease transmission. Here, we develop SHINE (SHERLOCK and HUDSON Integration to Navigate Epidemics), a sensitive and specific integrated diagnostic tool that can detect SARS-CoV-2 RNA from unextracted samples. We combine the steps of SHERLOCK into a single-step reaction and optimize HUDSON to accelerate viral inactivation in nasopharyngeal swabs and saliva. SHINE's results can be visualized with an in-tube fluorescent readout -reducing contamination risk as amplification reaction tubes remain sealed -and interpreted by a companion smartphone application. We validate SHINE on 50 nasopharyngeal patient samples, demonstrating 90% sensitivity and 100% specificity compared to RT-PCR with a sample-to-answer time of 50 minutes. SHINE has the potential to be used outside of hospitals and clinical laboratories, greatly enhancing diagnostic capabilities.

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SARS-CoV-2 detection with CRISPR diagnostics

Cited by 9 publications

References 8 publications

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

Point-of-care testing for COVID-19 using SHERLOCK diagnostics

CRISPR-Based Assays for Rapid Detection of SARS-CoV-2

Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2

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