CRISPR-BASED MOLECULAR DETECTION METHODS FOR RNA VIRUSES: APPLICATIONS IN THE DIAGNOSIS AND TREATMENT OF COVID-19 VARIANTS
Main Article Content
Keywords
CRISPR-Cas system, DETECTR assay, SARS-CoV-2, RNA viruses, molecular detection, COVID-19 variants
Abstract
Introduction: The COVID-19 pandemic caused by the SARS-CoV-2 virus has affected millions worldwide. In this study, we investigate the potential of CRISPR systems Cas12 and Cas13 for diagnosing and treating RNA viruses, particularly SARS-CoV-2.
Aim: This study aims to explore the role and application of CRISPR-system-based methods in detecting and treating RNA viruses, especially SARS-CoV-2, using Cas12 and Cas13 proteins.
Materials and Methodology: CRISPR enzymes, types V and VI, can target RNA or DNA through processes known as RNA and DNA targeting. Cas12 and Cas13 enzymes are specific for single-stranded RNA viruses and play a crucial role in diagnosing and treating these viruses. We used the DETECTR assay to detect mutations in SARS-CoV-2, including circulating and rare or new variants, in 304 respiratory swab samples collected from patients.
References
1. Chen JS, Ma E, Harrington LB, et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science. 2018;360(6387):436-439.
2. Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438-442.
3. Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv. 2020. Doi: 10.1101/2020.05.04.20091231.
4. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
5. Broughton JP, Deng X, Yu G, et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874.
6. Li SY, Cheng QX, Li XJ, et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20.
7. Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018;360(6387):444-448.
8. Patchsung M, Jantarug K, Pattama A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
9. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019;14(10):2986-3012.
10. Zhang F. CRISPR-based genome editing and diagnostics: the applications of CRISPR technology in disease detection and treatment. J Med Genet. 2020;57(12):771-776.
11. Abbott TR, Dhamdhere G, Liu Y, et al. Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Cell. 2020;181(4):865-876.e12.
12. Chen JS, Doudna JA. The chemistry of Cas9 and its CRISPR colleagues. Nat Rev Chem. 2017;1(7):0078.
13. Zhang F, Cong L, Lodato S, et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol. 2011;29(2):149-153.
14. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med. 2015;21(2):121-131.
15. Dever DP, Bak RO, Reinisch A, et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature. 2016;539(7629):384-389.
16. Komor AC, Badran AH, Liu DR. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017;168(1-2):20-36.
17. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.
18. Liang P, Xu Y, Zhang X, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363-372.
19. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826.
20. Wang H, Yang H, Shivalila CS, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910-918.
21. Zhang Y, Liang P, Chen T, et al. Efficient and transgene-free genome editing in human pluripotent stem cells using CRISPR-Cas9 ribonucleoproteins. Cell Stem Cell. 2014;15(4):433-440.
22. Ma H, Marti-Gutierrez N, Park SW, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;548(7668):413-419.
23. Liang X, Potter J, Kumar S, et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015;208:44-53.
24. Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA editing with CRISPR-Cas13. Science. 2017;358(6366):1019-1027.
25. Shmakov S, Smargon A, Scott D, et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol. 2017;15(3):169-182.
26. Gootenberg JS, Abudayyeh OO, Kellner MJ, et al. Multiplexed and portable nucleic acid detection platform.
27. Ackerman CM, Myhrvold C, Thakku SG, et al. Massively multiplexed nucleic acid detection with Cas13. Nature. 2020;582(7811):277-282.
28. Patchsung M, Jantarug K, Pattama A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
29. Vogels CBF, Brito AF, Wyllie AL, et al. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets. Nat Microbiol. 2020;5(10):1299-1305.
30. Zhang F, Abudayyeh OO, Gootenberg JS. A protocol for detection of COVID-19 using CRISPR diagnostics. Broad Institute. https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf. Published April 28, 2020.
31. Li SY, Cheng QX, Li XJ, et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20.
32. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
33. Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874.
34. Lalli MA, Jang JS, Park JC, et al. Rapid, extraction-free detection of SARS-CoV-2 from saliva by combined use of CRISPR-based detection and fluorescence imaging. BioRxiv. Preprint. Doi: 10.1101/2020.10.28.359454.
35. Huang Z, Tian D, Liu Y, et al. Ultra-sensitive and high-throughput CRISPR-powered COVID-19 diagnosis. Biosens Bioelectron. 2021;175:112887.
36. Wang M, Fu A, Hu B, et al. Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses. Small. 2021;17(12):e2002169.
37. Sun S, Chen J, Li W, et al. A CRISPR-based assay for the detection of opportunistic fungal pathogens. Nat Commun. 2020;11(1):2938.
38. Lu X, Wang Y, Xu C, et al. CRISPR-SHERLOCK-based method for rapid detection of SARS-CoV-2 infection. Transbound Emerg Dis. 2021;68(2):470-475.
39. Joung J, Ladha A, Saito M, et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. N Engl J Med. 2020;383(15):1492-1494.
40. Arizti-Sanz J, Freije CA, Stanton AC, et al. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nat Commun. 2020;11(1):5921.
41. Abbott TR, Dhamdhere G, Liu Y, et al. Development and validation.
42. Patchsung M, Jantarug K, Leelahavanichkul A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
43. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
44. Hou T, Zeng W, Yang M, et al. Development and evaluation of a rapid CRISPR-based diagnostic platform for detection of SARS-CoV-2. PLoS Pathog. 2020;16(8):e1008705.
45. Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874.
46. Lu R, Wu X, Wan Z, et al. A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2. Int J Mol Sci. 2020;21(8):2826.
47. Zhang Y, Odiwuor N, Xiong J, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv. Preprint. Doi: 10.1101/2020.02.26.20028373.
48. Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv. Preprint. Doi: 10.1101/2020.05.04.20091231.
49. Liang Y, Zhang C, Cai Y, et al. CRISPR-Cas12a coupled with platinum nanoparticles for the visual detection of SARS-CoV-2 nucleic acids. Chem Commun (Camb). 2020;56(70):10015-10018.
50. Lucira COVID-19 All-In-One Test Kit. Lucira Health. https://www.lucirahealth.com/products/lucira-covid-19-all-in-one-test-kit. Accessed June 7, 2023.
51. Ding X, Yin K, Li Z, et al. MeCas12a, a highly sensitive and specific system for COVID-19 detection. Adv Sci (Weinh). 2021;8(1):2003464.
52. Ding X, Yin K, Li Z, et al. Ultrafast and visual COVID-19 detection via CRISPR/Cas12a. Anal Chem. 2020;92(6):3961-3964.
53. Wang X, Yao H, Xu X, et al. A CRISPR-based platform for COVID-19 testing using saliva samples. medRxiv. Preprint. Doi: 10.1101/2020.06.15.20132027.
54. Patchsung M, Jantarug K, Pattama A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
55. Wee SK, Sivalingam SP, Yap EPH. Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler. Genes.
56. Broughton JP, Deng X, Yu G, et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biomed Eng. 2020;4(7):675-687.
57. Huang Z, Tian D, Liu Y, et al. Ultra-sensitive and high-throughput CRISPR-powered COVID-19 diagnosis. Biosens Bioelectron. 2021;175:112887.
58. Wang M, Fu A, Hu B, et al. Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses. Small. 2021;17(12):e2002169.
59. Sun S, Chen J, Li W, et al. A CRISPR-based assay for the detection of opportunistic fungal pathogens. Nat Commun. 2020;11(1):2938.
60. Lu X, Wang Y, Xu C, et al. CRISPR-SHERLOCK-based method for rapid detection of SARS-CoV-2 infection. Transbound Emerg Dis. 2021;68(2):470-475.
61. Joung J, Ladha A, Saito M, et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. N Engl J Med. 2020;383(15):1492-1494.
62. Arizti-Sanz J, Freije CA, Stanton AC, et al. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nat Commun. 2020;11(1):5921.
63. Patchsung M, Jantarug K, Leelahavanichkul A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
64. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
65. Hou T, Zeng W, Yang M, et al. Development and evaluation of a rapid CRISPR-based diagnostic platform for detection of SARS-CoV-2. PLoS Pathog. 2020;16(8):e1008705.
66. Lu R, Wu X, Wan Z, et al. A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2. Int J Mol Sci. 2020;21(8):2826.
67. Zhang Y, Odiwuor N, Xiong J, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv. Preprint. Doi: 10.1101/2020.02.26.20028373.
68. Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv. Preprint. Doi: 10.1101/2020.05.04.20091231.
69. Liang Y, Zhang C, Cai Y, et al. CRISPR-Cas12a coupled with platinum nanoparticles for the visual detection of SARS-CoV-2 nucleic acids. Chem Commun (Camb). 2020;56(70):10015-10018.
70. Shen M, Zhou Y, Ye J, et al. Recent advances and perspectives of nucleic acid detection for coronavirus. J Pharm Anal. 2020;10(2):97-101.
71. Wang X, Yao H, Xu X, et al. A CRISPR-based platform for COVID-19 testing using saliva samples. medRxiv. Preprint. Doi: 10.1101/2020.06.15.20132027.
72. Amanat F, Nguyen T, Chromikova V, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat Med. 2020;26(7):1033-1036.
73. Jiang Y, Ying W, Wu L, et al. Clinical evaluation of a rapid colloidal gold immunochromatography assay for SARS-CoV-2 IgM/IgG. Am J Transl Res. 2020;12(4):1348-1354.
74. Xiang J, Yan M, Li H, et al. Evaluation of enzyme-linked immunoassay and colloidal gold-immunochromatographic assay kit for detection of novel coronavirus (SARS-Cov-2) causing an outbreak of pneumonia (COVID-19). medRxiv. Preprint. Doi: 10.1101/2020.02.27.20028787.
75. Liu R, Liu X, Yuan L, et al. Analysis of adjunctive serological detection to nucleic acid test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection diagnosis. Int Immunopharmacol. 2020;86:106746.
76. Li L, Wu H, Liu C, et al. Detection of SARS-CoV-2 antigen using a lateral flow immunoassay for rapid screening of COVID-19 patients. medRxiv. Preprint. Doi: 10.1101/2020.03.06.20031856.
77. Padoan A, Cosma C, Sciacovelli L, et al. Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics. Clin Chem Lab Med. 2020;58(7):1081-1088.
78. Ben-David A, Sui H, Sauer MM, et al. Single-virus genomics reveals hidden cosmopolitan and abundant viruses. Nat Commun. 2018;9(1):1229.
79. Chen Y, Liu Q, Guo D. Emerging coronaviruses: genome structure, replication, and pathogenesis. J Med Virol. 2020;92(4):418-423.
80. Liu DX, Liang JQ, Fung TS. Human coronavirus-229E, -OC43, -NL63, and -HKU1. Reference Module in Biomedical Sciences. Elsevier; 2020:213-218.
81. Perlman S, Netland J. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol. 2009;7(6):439-450.
82. Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367(6483):1260-1263.
83. Tai W, He L, Zhang X, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020;17(6):613-620.
84. Walls AC, Park YJ, Tortorici MA, et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020;181(2):281-292.e6.
85. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181(2):271-280.e8.
86. Wang C, Horby PW, Hayden FG, et al. A novel coronavirus outbreak of global health concern. Lancet. 2020;395(10223):470-473.
87. Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579(7798):270-273.
88. Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579(7798):265-269.
89. Zhu N, Zhang D, Wang W, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020;382(8):727-733.
90. Yan R, Zhang Y, Li Y, et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020;367(6485):1444-1448.
91. Zhang H, Penninger JM, Li Y, et al. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med. 2020;46(4):586-590.
92. Wang Q, Zhang Y, Wu L, et al. Structural and functional basis of SARS-CoV-2 entry by using human ACE2. Cell. 2020;181(4):894-904.e9.
93. Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367(6483):1260-1263.
94. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181(2):271-280.e8.
95. Tai W, He L, Zhang X, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020;17(6):613-620.
96. Walls AC, Park YJ, Tortorici MA, et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020;181(2):281-292.e6.
97. Khan S, Siddique R, Shereen MA, et al. The emergence of a novel coronavirus (SARS-CoV-2), their biology and therapeutic options. J Clin Microbiol. 2020;58(5):e00187-20.
98. Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):2000045.
99. Chu DK, Pan Y, Cheng SM, et al. Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clin Chem. 2020;66(4):549-555.
100. WHO. Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases: interim guidance, 17 January 2020. World Health Organization. https://www.who.int/publications/i/item/10665-331501. Accessed June 7, 2023.
101. CDC. Interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (PUIs) for coronavirus disease 2019 (COVID-19). Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html. Accessed June 7, 2023.
102. European Centre for Disease Prevention and Control. Guidance for managing and testing suspected cases of COVID-19. https://www.ecdc.europa.eu/en/publications-data/testing-strategies-rapidly-detecting-sars-cov-2-cases. Accessed June 7, 2023.
103. Sethuraman N, Jeremiah SS, Ryo A. Interpreting diagnostic tests for SARS-CoV-2. JAMA. 2020;323(22):2249-2251.
104. FDA. Coronavirus (COVID-19) update: FDA authorizes additional COVID-19 combination diagnostic test ahead of flu season. U.S. Food and Drug Administration. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-covid-19-combination-diagnostic-test-ahead. Accessed June 7, 2023.
105. FindDiagnostic. COVID-19 diagnostics tracker. FindDx. https://www.finddx.org/covid-19/pipeline/. Accessed June 7, 2023.
106. Medina-Moreno S, Zapata-Zúñiga M, Valdovinos-Chávez SB, et al. SARS-CoV-2 diagnostics: a review of types, methods, and importance of molecular and serological techniques for early detection. Diagnostics (Basel). 2021;11(5):827.
107. Nalla AK, Casto AM, Huang MW, et al. Comparative performance of SARS-CoV-2 detection assays using seven different primer-probe sets and one assay kit. J Clin Microbiol. 2020;58(6):e00557-20.
108. Baek YH, Um J, Antigua KJC, et al. Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2. Emerg Microbes Infect. 2020;9(1):998-1007.
109. Yu L, Wu S, Hao X, et al. Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform. Clin Chem. 2020;66(7):975-977.
110. Nagura-Ikeda M, Imai-Nishiya H, Ohnishi K, et al. Clinical evaluation of self-collected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19. J Clin Microbiol. 2021;59(3):e02828-20.
111. Kumar S, Pandey AK, Kapoor RK, et al. Molecular techniques for diagnosis of COVID-19. Critical Reviews in Analytical Chemistry. 2021;51(4):315-328.
112. Wang X, Yao H, Xu X, et al. Limits of detection of 6 approved RT-PCR kits for the novel SARS-CoV-2 coronavirus. J Clin Microbiol. 2020;58(8):e00722-20.
113. Udugama B, Kadhiresan P, Kozlowski HN, et al. Diagnosing COVID-19: the disease and tools for detection. ACS Nano. 2020;14(4):3822-3835.
114. Zhang Y, Odiwuor N, Xiong J, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv. 2020. Doi: 10.1101/2020.02.26.20028373.
115. Yan C, Cui J, Huang L, et al. Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay. Clin Microbiol Infect. 2020;26(6):773-779.
116. Zhang Y, Ren H, Shen J, et al. The diagnostic value and key techniques of the Lab-on-a-Chip systems for detection of COVID-19: a review. Sensors (Basel). 2021;21(1):285.
117. Wang R, Zhang J, Peng Y, et al. Recent advances in the detection of respiratory virus infection in humans. J Med Virol. 2021;93(3):1274-1283.
118. Lundgren, Magnus; Charpentier, Emmanuelle; Fineran, Peter C. (2015). [Methods in Molecular Biology] CRISPR Volume 1311 || Annotation and Classification of CRISPR-Cas Systems. 10.1007/978-1-4939-2687-9(Chapter 4), 47–75.
119. Escalona-Noguero C, López-Valls M, Sot B. CRISPR/Cas technology as a promising weapon to combat viral infections. Bioessays. 2021;43(4):e2000315.
2. Gootenberg JS, Abudayyeh OO, Lee JW, et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science. 2017;356(6336):438-442.
3. Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv. 2020. Doi: 10.1101/2020.05.04.20091231.
4. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
5. Broughton JP, Deng X, Yu G, et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874.
6. Li SY, Cheng QX, Li XJ, et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20.
7. Myhrvold C, Freije CA, Gootenberg JS, et al. Field-deployable viral diagnostics using CRISPR-Cas13. Science. 2018;360(6387):444-448.
8. Patchsung M, Jantarug K, Pattama A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
9. Kellner MJ, Koob JG, Gootenberg JS, Abudayyeh OO, Zhang F. SHERLOCK: nucleic acid detection with CRISPR nucleases. Nat Protoc. 2019;14(10):2986-3012.
10. Zhang F. CRISPR-based genome editing and diagnostics: the applications of CRISPR technology in disease detection and treatment. J Med Genet. 2020;57(12):771-776.
11. Abbott TR, Dhamdhere G, Liu Y, et al. Development of CRISPR as an antiviral strategy to combat SARS-CoV-2 and influenza. Cell. 2020;181(4):865-876.e12.
12. Chen JS, Doudna JA. The chemistry of Cas9 and its CRISPR colleagues. Nat Rev Chem. 2017;1(7):0078.
13. Zhang F, Cong L, Lodato S, et al. Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription. Nat Biotechnol. 2011;29(2):149-153.
14. Cox DBT, Platt RJ, Zhang F. Therapeutic genome editing: prospects and challenges. Nat Med. 2015;21(2):121-131.
15. Dever DP, Bak RO, Reinisch A, et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature. 2016;539(7629):384-389.
16. Komor AC, Badran AH, Liu DR. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017;168(1-2):20-36.
17. Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346(6213):1258096.
18. Liang P, Xu Y, Zhang X, et al. CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes. Protein Cell. 2015;6(5):363-372.
19. Mali P, Yang L, Esvelt KM, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823-826.
20. Wang H, Yang H, Shivalila CS, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153(4):910-918.
21. Zhang Y, Liang P, Chen T, et al. Efficient and transgene-free genome editing in human pluripotent stem cells using CRISPR-Cas9 ribonucleoproteins. Cell Stem Cell. 2014;15(4):433-440.
22. Ma H, Marti-Gutierrez N, Park SW, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;548(7668):413-419.
23. Liang X, Potter J, Kumar S, et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J Biotechnol. 2015;208:44-53.
24. Cox DBT, Gootenberg JS, Abudayyeh OO, et al. RNA editing with CRISPR-Cas13. Science. 2017;358(6366):1019-1027.
25. Shmakov S, Smargon A, Scott D, et al. Diversity and evolution of class 2 CRISPR-Cas systems. Nat Rev Microbiol. 2017;15(3):169-182.
26. Gootenberg JS, Abudayyeh OO, Kellner MJ, et al. Multiplexed and portable nucleic acid detection platform.
27. Ackerman CM, Myhrvold C, Thakku SG, et al. Massively multiplexed nucleic acid detection with Cas13. Nature. 2020;582(7811):277-282.
28. Patchsung M, Jantarug K, Pattama A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
29. Vogels CBF, Brito AF, Wyllie AL, et al. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets. Nat Microbiol. 2020;5(10):1299-1305.
30. Zhang F, Abudayyeh OO, Gootenberg JS. A protocol for detection of COVID-19 using CRISPR diagnostics. Broad Institute. https://www.broadinstitute.org/files/publications/special/COVID-19%20detection%20(updated).pdf. Published April 28, 2020.
31. Li SY, Cheng QX, Li XJ, et al. CRISPR-Cas12a-assisted nucleic acid detection. Cell Discov. 2018;4:20.
32. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
33. Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874.
34. Lalli MA, Jang JS, Park JC, et al. Rapid, extraction-free detection of SARS-CoV-2 from saliva by combined use of CRISPR-based detection and fluorescence imaging. BioRxiv. Preprint. Doi: 10.1101/2020.10.28.359454.
35. Huang Z, Tian D, Liu Y, et al. Ultra-sensitive and high-throughput CRISPR-powered COVID-19 diagnosis. Biosens Bioelectron. 2021;175:112887.
36. Wang M, Fu A, Hu B, et al. Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses. Small. 2021;17(12):e2002169.
37. Sun S, Chen J, Li W, et al. A CRISPR-based assay for the detection of opportunistic fungal pathogens. Nat Commun. 2020;11(1):2938.
38. Lu X, Wang Y, Xu C, et al. CRISPR-SHERLOCK-based method for rapid detection of SARS-CoV-2 infection. Transbound Emerg Dis. 2021;68(2):470-475.
39. Joung J, Ladha A, Saito M, et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. N Engl J Med. 2020;383(15):1492-1494.
40. Arizti-Sanz J, Freije CA, Stanton AC, et al. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nat Commun. 2020;11(1):5921.
41. Abbott TR, Dhamdhere G, Liu Y, et al. Development and validation.
42. Patchsung M, Jantarug K, Leelahavanichkul A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
43. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
44. Hou T, Zeng W, Yang M, et al. Development and evaluation of a rapid CRISPR-based diagnostic platform for detection of SARS-CoV-2. PLoS Pathog. 2020;16(8):e1008705.
45. Broughton JP, Deng X, Yu G, et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol. 2020;38(7):870-874.
46. Lu R, Wu X, Wan Z, et al. A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2. Int J Mol Sci. 2020;21(8):2826.
47. Zhang Y, Odiwuor N, Xiong J, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv. Preprint. Doi: 10.1101/2020.02.26.20028373.
48. Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv. Preprint. Doi: 10.1101/2020.05.04.20091231.
49. Liang Y, Zhang C, Cai Y, et al. CRISPR-Cas12a coupled with platinum nanoparticles for the visual detection of SARS-CoV-2 nucleic acids. Chem Commun (Camb). 2020;56(70):10015-10018.
50. Lucira COVID-19 All-In-One Test Kit. Lucira Health. https://www.lucirahealth.com/products/lucira-covid-19-all-in-one-test-kit. Accessed June 7, 2023.
51. Ding X, Yin K, Li Z, et al. MeCas12a, a highly sensitive and specific system for COVID-19 detection. Adv Sci (Weinh). 2021;8(1):2003464.
52. Ding X, Yin K, Li Z, et al. Ultrafast and visual COVID-19 detection via CRISPR/Cas12a. Anal Chem. 2020;92(6):3961-3964.
53. Wang X, Yao H, Xu X, et al. A CRISPR-based platform for COVID-19 testing using saliva samples. medRxiv. Preprint. Doi: 10.1101/2020.06.15.20132027.
54. Patchsung M, Jantarug K, Pattama A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
55. Wee SK, Sivalingam SP, Yap EPH. Rapid direct nucleic acid amplification test without RNA extraction for SARS-CoV-2 using a portable PCR thermocycler. Genes.
56. Broughton JP, Deng X, Yu G, et al. CRISPR–Cas12-based detection of SARS-CoV-2. Nat Biomed Eng. 2020;4(7):675-687.
57. Huang Z, Tian D, Liu Y, et al. Ultra-sensitive and high-throughput CRISPR-powered COVID-19 diagnosis. Biosens Bioelectron. 2021;175:112887.
58. Wang M, Fu A, Hu B, et al. Nanopore target sequencing for accurate and comprehensive detection of SARS-CoV-2 and other respiratory viruses. Small. 2021;17(12):e2002169.
59. Sun S, Chen J, Li W, et al. A CRISPR-based assay for the detection of opportunistic fungal pathogens. Nat Commun. 2020;11(1):2938.
60. Lu X, Wang Y, Xu C, et al. CRISPR-SHERLOCK-based method for rapid detection of SARS-CoV-2 infection. Transbound Emerg Dis. 2021;68(2):470-475.
61. Joung J, Ladha A, Saito M, et al. Detection of SARS-CoV-2 with SHERLOCK one-pot testing. N Engl J Med. 2020;383(15):1492-1494.
62. Arizti-Sanz J, Freije CA, Stanton AC, et al. Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nat Commun. 2020;11(1):5921.
63. Patchsung M, Jantarug K, Leelahavanichkul A, et al. Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA. Nat Biomed Eng. 2020;4(12):1140-1149.
64. Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun. 2020;11(1):4711.
65. Hou T, Zeng W, Yang M, et al. Development and evaluation of a rapid CRISPR-based diagnostic platform for detection of SARS-CoV-2. PLoS Pathog. 2020;16(8):e1008705.
66. Lu R, Wu X, Wan Z, et al. A novel reverse transcription loop-mediated isothermal amplification method for rapid detection of SARS-CoV-2. Int J Mol Sci. 2020;21(8):2826.
67. Zhang Y, Odiwuor N, Xiong J, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv. Preprint. Doi: 10.1101/2020.02.26.20028373.
68. Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv. Preprint. Doi: 10.1101/2020.05.04.20091231.
69. Liang Y, Zhang C, Cai Y, et al. CRISPR-Cas12a coupled with platinum nanoparticles for the visual detection of SARS-CoV-2 nucleic acids. Chem Commun (Camb). 2020;56(70):10015-10018.
70. Shen M, Zhou Y, Ye J, et al. Recent advances and perspectives of nucleic acid detection for coronavirus. J Pharm Anal. 2020;10(2):97-101.
71. Wang X, Yao H, Xu X, et al. A CRISPR-based platform for COVID-19 testing using saliva samples. medRxiv. Preprint. Doi: 10.1101/2020.06.15.20132027.
72. Amanat F, Nguyen T, Chromikova V, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat Med. 2020;26(7):1033-1036.
73. Jiang Y, Ying W, Wu L, et al. Clinical evaluation of a rapid colloidal gold immunochromatography assay for SARS-CoV-2 IgM/IgG. Am J Transl Res. 2020;12(4):1348-1354.
74. Xiang J, Yan M, Li H, et al. Evaluation of enzyme-linked immunoassay and colloidal gold-immunochromatographic assay kit for detection of novel coronavirus (SARS-Cov-2) causing an outbreak of pneumonia (COVID-19). medRxiv. Preprint. Doi: 10.1101/2020.02.27.20028787.
75. Liu R, Liu X, Yuan L, et al. Analysis of adjunctive serological detection to nucleic acid test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection diagnosis. Int Immunopharmacol. 2020;86:106746.
76. Li L, Wu H, Liu C, et al. Detection of SARS-CoV-2 antigen using a lateral flow immunoassay for rapid screening of COVID-19 patients. medRxiv. Preprint. Doi: 10.1101/2020.03.06.20031856.
77. Padoan A, Cosma C, Sciacovelli L, et al. Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics. Clin Chem Lab Med. 2020;58(7):1081-1088.
78. Ben-David A, Sui H, Sauer MM, et al. Single-virus genomics reveals hidden cosmopolitan and abundant viruses. Nat Commun. 2018;9(1):1229.
79. Chen Y, Liu Q, Guo D. Emerging coronaviruses: genome structure, replication, and pathogenesis. J Med Virol. 2020;92(4):418-423.
80. Liu DX, Liang JQ, Fung TS. Human coronavirus-229E, -OC43, -NL63, and -HKU1. Reference Module in Biomedical Sciences. Elsevier; 2020:213-218.
81. Perlman S, Netland J. Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol. 2009;7(6):439-450.
82. Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367(6483):1260-1263.
83. Tai W, He L, Zhang X, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020;17(6):613-620.
84. Walls AC, Park YJ, Tortorici MA, et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020;181(2):281-292.e6.
85. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181(2):271-280.e8.
86. Wang C, Horby PW, Hayden FG, et al. A novel coronavirus outbreak of global health concern. Lancet. 2020;395(10223):470-473.
87. Zhou P, Yang XL, Wang XG, et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020;579(7798):270-273.
88. Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature. 2020;579(7798):265-269.
89. Zhu N, Zhang D, Wang W, et al. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med. 2020;382(8):727-733.
90. Yan R, Zhang Y, Li Y, et al. Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science. 2020;367(6485):1444-1448.
91. Zhang H, Penninger JM, Li Y, et al. Angiotensin-converting enzyme 2 (ACE2) as a SARS-CoV-2 receptor: molecular mechanisms and potential therapeutic target. Intensive Care Med. 2020;46(4):586-590.
92. Wang Q, Zhang Y, Wu L, et al. Structural and functional basis of SARS-CoV-2 entry by using human ACE2. Cell. 2020;181(4):894-904.e9.
93. Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science. 2020;367(6483):1260-1263.
94. Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell. 2020;181(2):271-280.e8.
95. Tai W, He L, Zhang X, et al. Characterization of the receptor-binding domain (RBD) of 2019 novel coronavirus: implication for development of RBD protein as a viral attachment inhibitor and vaccine. Cell Mol Immunol. 2020;17(6):613-620.
96. Walls AC, Park YJ, Tortorici MA, et al. Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein. Cell. 2020;181(2):281-292.e6.
97. Khan S, Siddique R, Shereen MA, et al. The emergence of a novel coronavirus (SARS-CoV-2), their biology and therapeutic options. J Clin Microbiol. 2020;58(5):e00187-20.
98. Corman VM, Landt O, Kaiser M, et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25(3):2000045.
99. Chu DK, Pan Y, Cheng SM, et al. Molecular diagnosis of a novel coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clin Chem. 2020;66(4):549-555.
100. WHO. Laboratory testing for 2019 novel coronavirus (2019-nCoV) in suspected human cases: interim guidance, 17 January 2020. World Health Organization. https://www.who.int/publications/i/item/10665-331501. Accessed June 7, 2023.
101. CDC. Interim guidelines for collecting, handling, and testing clinical specimens from patients under investigation (PUIs) for coronavirus disease 2019 (COVID-19). Centers for Disease Control and Prevention. https://www.cdc.gov/coronavirus/2019-ncov/lab/guidelines-clinical-specimens.html. Accessed June 7, 2023.
102. European Centre for Disease Prevention and Control. Guidance for managing and testing suspected cases of COVID-19. https://www.ecdc.europa.eu/en/publications-data/testing-strategies-rapidly-detecting-sars-cov-2-cases. Accessed June 7, 2023.
103. Sethuraman N, Jeremiah SS, Ryo A. Interpreting diagnostic tests for SARS-CoV-2. JAMA. 2020;323(22):2249-2251.
104. FDA. Coronavirus (COVID-19) update: FDA authorizes additional COVID-19 combination diagnostic test ahead of flu season. U.S. Food and Drug Administration. https://www.fda.gov/news-events/press-announcements/coronavirus-covid-19-update-fda-authorizes-additional-covid-19-combination-diagnostic-test-ahead. Accessed June 7, 2023.
105. FindDiagnostic. COVID-19 diagnostics tracker. FindDx. https://www.finddx.org/covid-19/pipeline/. Accessed June 7, 2023.
106. Medina-Moreno S, Zapata-Zúñiga M, Valdovinos-Chávez SB, et al. SARS-CoV-2 diagnostics: a review of types, methods, and importance of molecular and serological techniques for early detection. Diagnostics (Basel). 2021;11(5):827.
107. Nalla AK, Casto AM, Huang MW, et al. Comparative performance of SARS-CoV-2 detection assays using seven different primer-probe sets and one assay kit. J Clin Microbiol. 2020;58(6):e00557-20.
108. Baek YH, Um J, Antigua KJC, et al. Development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel SARS-CoV-2. Emerg Microbes Infect. 2020;9(1):998-1007.
109. Yu L, Wu S, Hao X, et al. Rapid detection of COVID-19 coronavirus using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform. Clin Chem. 2020;66(7):975-977.
110. Nagura-Ikeda M, Imai-Nishiya H, Ohnishi K, et al. Clinical evaluation of self-collected saliva by RT-qPCR, direct RT-qPCR, RT-LAMP, and a rapid antigen test to diagnose COVID-19. J Clin Microbiol. 2021;59(3):e02828-20.
111. Kumar S, Pandey AK, Kapoor RK, et al. Molecular techniques for diagnosis of COVID-19. Critical Reviews in Analytical Chemistry. 2021;51(4):315-328.
112. Wang X, Yao H, Xu X, et al. Limits of detection of 6 approved RT-PCR kits for the novel SARS-CoV-2 coronavirus. J Clin Microbiol. 2020;58(8):e00722-20.
113. Udugama B, Kadhiresan P, Kozlowski HN, et al. Diagnosing COVID-19: the disease and tools for detection. ACS Nano. 2020;14(4):3822-3835.
114. Zhang Y, Odiwuor N, Xiong J, et al. Rapid molecular detection of SARS-CoV-2 (COVID-19) virus RNA using colorimetric LAMP. medRxiv. 2020. Doi: 10.1101/2020.02.26.20028373.
115. Yan C, Cui J, Huang L, et al. Rapid and visual detection of 2019 novel coronavirus (SARS-CoV-2) by a reverse transcription loop-mediated isothermal amplification assay. Clin Microbiol Infect. 2020;26(6):773-779.
116. Zhang Y, Ren H, Shen J, et al. The diagnostic value and key techniques of the Lab-on-a-Chip systems for detection of COVID-19: a review. Sensors (Basel). 2021;21(1):285.
117. Wang R, Zhang J, Peng Y, et al. Recent advances in the detection of respiratory virus infection in humans. J Med Virol. 2021;93(3):1274-1283.
118. Lundgren, Magnus; Charpentier, Emmanuelle; Fineran, Peter C. (2015). [Methods in Molecular Biology] CRISPR Volume 1311 || Annotation and Classification of CRISPR-Cas Systems. 10.1007/978-1-4939-2687-9(Chapter 4), 47–75.
119. Escalona-Noguero C, López-Valls M, Sot B. CRISPR/Cas technology as a promising weapon to combat viral infections. Bioessays. 2021;43(4):e2000315.
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Jul 31, 2023
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Myle Akshay Kiran, Muhammed A. Bakhrebah, Muhammad A. Halwani, Bashayer M. AlShehail, , A. A. O., , A. J. A., Fahd A. Alshehri, Ahmad A. Alshehri, Mohammed Alissa, & Ali A. Rabaan. (2023). CRISPR-BASED MOLECULAR DETECTION METHODS FOR RNA VIRUSES: APPLICATIONS IN THE DIAGNOSIS AND TREATMENT OF COVID-19 VARIANTS. Journal of Population Therapeutics and Clinical Pharmacology, 30(16), 477–490. https://doi.org/10.53555/jptcp.v30i16.2415
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Author Biographies
Myle Akshay Kiran
Department of Clinical Research, Clinical practise and Pharmacology, General and Integrative Medicine, Clinical Research, Health care and Hospital Administration Department.. National Institute of Medicine Sciences, Andhra Pradesh State, India
Muhammed A. Bakhrebah
Advanced Diagnostics and Therapeutics Institutes, Health sector, King Abdulaziz City for Science and Technology (KACST) Riyadh 11442, Saudi Arabia
Muhammad A. Halwani
Department of Medical Microbiology, Faculty of Medicine, Al Baha University. Al Baha 4781, Saudi Arabia
Bashayer M. AlShehail
Pharmacy Practice Department, College of Clinical Pharmacy, Imam Abdulrahman Bin Faisal University, Dammam 31441, Saudi Arabia
Abdulrazzag Abdulaziz Othman
Department of clinical laboratory Sciences, College of Applied Medical Sciences, Shaqra University, Shaqra 11911, Saudi Arabia
Abdullah Jaber AlShahrani
Public Health Department, Health affairs, Asir region, Abha 62523, Saudi Arabia.
Fahd A. Alshehri
Nursing administration, East Jeddah Hospital, Jeddah 22253, Saudi Arabia.
Ahmad A. Alshehri
Department of Clinical Laboratory Sciences, Faculty of Applied Medical Sciences, Najran University, Najran 61441, Saudi Arabia
Mohammed Alissa
Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Prince Sattam bin Abdulaziz University, Al-Kharj 11942, Saudi Arabia
Ali A. Rabaan
Molecular Diagnostic Laboratory, Johns Hopkins Aramco Healthcare, Dhahran 31311, Saudi ArabiaCollege of Medicine, Alfaisal University, Riyadh 11533, Saudi ArabiaDepartment of Public Health and Nutrition, The University of Haripur, Haripur 22610, Pakistan