Development of a nucleic acid detection method based on the CRISPR-Cas13 for point-of-care testing of bovine viral diarrhea virus-1b
0
조회
0
다운로드
1
인용
0
참고문헌
키워드
초록
Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV- 1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5’ untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.
Abstract
Bovine viral diarrhea (BVD) is a single-stranded, positive-sense ribonucleic acid (RNA) virus belonging to the genus Pestivirus of the Flaviviridae family. BVD frequently causes economic losses to farmers. Among bovine viral diarrhea virus (BVDV) strains, BVDV-1b is predominant and widespread in Hanwoo calves. Reverse-transcription polymerase chain reaction (RT-PCR) is an essential method for diagnosing BVDV-1b and has become the gold standard for diagnosis in the Republic of Korea. However, this diagnostic method is time-consuming and requires expensive equipment. Therefore, Clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) systems have been used for point-of-care (POC) testing of viruses. Developing a sensitive and specific method for POC testing of BVDV-1b would be advantageous for controlling the spread of infection. Thus, this study aimed to develop a novel nucleic acid detection method using the CRISPR-Cas13 system for POC testing of BVDV- 1b. The sequence of the BVD virus was extracted from National Center for Biotechnology Information (NC_001461.1), and the 5’ untranslated region, commonly used for detection, was selected. CRISPR RNA (crRNA) was designed using the Cas13 design program and optimized for the expression and purification of the LwCas13a protein. Madin Darby bovine kidney (MDBK) cells were infected with BVDV-1b, incubated, and the viral RNA was extracted. To enable POC viral detection, the compatibility of the CRISPR-Cas13 system was verified with a paper-based strip through collateral cleavage activity. Finally, a colorimetric assay was used to evaluate the detection of BVDV-1b by combining the previously obtained crRNA and Cas13a protein on a paper strip. In conclusion, the CRISPR-Cas13 system is highly sensitive, specific, and capable of nucleic acid detection, making it an optimal system for the early point-of-care testing of BVDV-1b.
참고문헌 (30)
- Kim S, "Detection of diarrheagenic pathogens from feces and incidence of diarrhea in Korean calves", Korean J Vet Serv, 39, pp.187~192 (2016)[DOI]
- Jeong MH, "Detection of etiologic agents in diarrhea fecal samples from calves in Gyeongnam province, Korea", Korean J Vet Serv, 35, pp.339~342 (2012)[DOI]
- Cho Y, "An overview of calf diarrhea-infectious etiology, diagnosis, and intervention", J Vet Sci, 15, pp.1~17 (2014)[DOI]
- Chae JB, "The prevalence of causative agents of calf diarrhea in Korean native calves", J Anim Sci Technol, 63, pp.864~871 (2021)[DOI]
- Cho HM, "Real-time RT-PCR for quantitative detection of bovine viral diarrhoea virus during manufacture of biologics", Korean J Microbiol Biotechnol, 36, pp.34~42 (2008)
- Hou P, "Detection of bovine viral diarrhea virus genotype 1 in aerosol by a real time RT-PCR assay", BMC Vet Res, 16, pp.114~ (2020)[DOI]
- Adams MJ, "Tracking the changes in virus taxonomy", Arch Virol, 160, pp.1375~1383 (2015)[DOI]
- Lee W, "Application of the CRISPR", cas system for point-of-care diagnosis of cattle disease, 2020, pp.30, 313~319
- Yao R, "CRISPR-Cas13a-based detection for bovine viral diarrhea virus", Front Vet Sci, 8, pp.603919~ (2021)[DOI]
- Piepenburg O, "DNA detection using recombination proteins", PLOS Biol, 4, pp.e204~ (2006)[DOI]
- Notomi T, "Loopmediated isothermal amplification of DNA", Nucleic Acids Res, 28, pp.e63~ (2000)[DOI]
- Rabe BA, "SARS-CoV-2 detection using isothermal amplification reaction and a rapid, inexpensive protocol for sample inactivation and purification", Proc Natl Acad Sci USA, 117, pp.24450~24458 (2020)[DOI]
- Qian J, "An enhanced isothermal amplification assay for viral detection", Nat Commun, 11, pp.5920~ (2020)[DOI]
- Gootenberg JS, "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6", Science, 360, pp.439~444 (2018)[DOI]
- Gootenberg JS, "Nucleic acid detection with CRISPR-Cas13a", C2c2, 2017, pp.356, 438~442
- Myhrvold C, "Fielddeployable viral diagnostics using CRISPR-Cas13", Science, 360, pp.444~448 (2018)[DOI]
- Kellner MJ, "SHERLOCK : nucleic acid detection with CRISPR nucleases", Nat Protoc, 14, pp.2986~3012 (2019)[DOI]
- Deng R, "Molecular cloning and nucleotide sequence of a pestivirus genome, noncytopathic bovine viral diarrhea virus strain SD-1", Virology, 191, pp.867~879 (1992)[DOI]
- Collett MS, "Molecular cloning and nucleotide sequence of the pestivirus bovine viral diarrhea virus", Virology, 165, pp.191~199 (1988)[DOI]
- Oem JK, "Complete genome sequences of two bovine viral diarrhea viruses isolated from brain tissues of nonambulatory(downer)cattle", Genome Announc, 1, pp.e00733-13~ (2013)[DOI]
- Rümenapf T, "Processing of the envelope glycoproteins of pestiviruses", J Virol, 67, pp.3288~3294 (1993)[DOI]
- Tautz N, "Serine protease of pestiviruses : determination of cleavage sites", J Virol, 71, pp.5415~5422 (1997)[DOI]
- Lindenbach BD, "Molecular biology of flaviviruses", Adv Virus Res, 59, pp.23~61 (2003)[DOI]
- Gates, MC, "Not all cows are epidemiologically equal : quantifying the risks of bovine viral diarrhoea virus(BVDV)transmission through cattle movements", Vet Res, 45, pp.110~ (2014)[DOI]
- Pinior B, "A systematic review of financial and economic assessments of bovine viral diarrhea virus(BVDV)prevention and mitigation activities worldwide", Prev Vet Med, 137, pp.77~92 (2017)[DOI]
- Patchsung M, "Clinical validation of a Cas13-based assay for the detection of SARS-CoV-2 RNA", Nat Biomed Eng, 4, pp.1140~1149 (2020)[DOI]
- Kim H, "A new point-of-care test for the diagnosis of infectious diseases based on multiplex lateral flow immunoassays", Analyst, 144, pp.2460~2466 (2019)[DOI]
- Li H, "Sensitive and easy-read CRISPR strip for COVID-19 rapid point-of-care testing", CRISPR J, 4, pp.392~399 (2021)[DOI]
- Casati B, "Rapid, adaptable and sensitive Cas13-based COVID-19 diagnostics using ADESSO", Nat Commun, 13, pp.3308~ (2022)[DOI]
- Huang Z, "CRISPR-based assays for low-resource settings", Nat Rev Bioeng, 1, pp.230~231 (2023)[DOI]