Abstract
Porcine deltacoronavirus (PDCoV) is an enteropathogen that causes diarrhea in piglets and may undergo cross-species transmission. The prevention and control of PDCoV are complicated, and a sensitive, specific, and accessible method of diagnosis would be advantageous. Whereas qPCR is a standard approach for detecting PDCoV, it is not effectively sensitive. In the present study, we report such a strategy using an RT-PCR-based RspCas13d detection system and its efficacy in clinical sample diagnosis. The detection limit of this method was 4 copies/μL and no cross-reaction with other viruses such as the porcine epidemic diarrhea virus, classical swine fever virus, pseudorabies virus, porcine reproductive and respiratory syndrome virus, transmissible gastroenteritis virus and porcine rotavirus. The method was also effective in clinical samples. In summary, we demonstrate that RT-PCR-based RspCas13d detection system is an extremely sensitive and specific nucleic acid-based approach for detecting PDCoV.
Key points
• RspCas13d can be used as a candidate molecular diagnostic tool to diagnose viral genomes.
• A novel method is proposed using an RT-PCR-based RspCas13d detection system and its effectiveness in the detection of PDCoV.
• The RT-PCR-based RspCas13d detection system has excellent sensitivity and specificity.
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Data availability
The data presented in this study are available on request from the corresponding author.
References
Brogan DJ, Chaverra-Rodriguez D, Lin CP, Smidler AL, Yang T, Alcantara LM, Antoshechkin I, Liu J, Raban RR, Belda-Ferre P, Knight R, Komives EA, Akbari OS (2021) Development of a rapid and sensitive CasRx-based diagnostic assay for SARS-CoV-2. ACS Sens 6(11):3957–3966. https://doi.org/10.1021/acssensors.1c01088
Chang Y, Deng Y, Li T, Wang J, Wang T, Tan F, Li X, Tian K (2020) Visual detection of porcine reproductive and respiratory syndrome virus using CRISPR-Cas13a. Transbound Emerg Dis 67(2):564–571. https://doi.org/10.1111/tbed.13368
Chen JS, Ma E, Harrington LB, Da Costa M, Tian X, Palefsky JM, Doudna JA (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science (New York, NY) 360(6387):436–439. https://doi.org/10.1126/science.aar6245
Ding G, Fu Y, Li B, Chen J, Wang J, Yin B, Sha W, Liu G (2020) Development of a multiplex RT-PCR for the detection of major diarrhoeal viruses in pig herds in China. Transbound Emerg Dis 67(2):678–685. https://doi.org/10.1111/tbed.13385
Dong N, Fang L, Zeng S, Sun Q, Chen H, Xiao S (2015) Porcine deltacoronavirus in Mainland China. Emerg Infect Dis 21(12):2254–2255. https://doi.org/10.3201/eid2112.150283
Gootenberg JS, Abudayyeh OO, Lee JW, Essletzbichler P, Dy AJ, Joung J, Verdine V, Donghia N, Daringer NM, Freije CA, Myhrvold C, Bhattacharyya RP, Livny J, Regev A, Koonin EV, Hung DT, Sabeti PC, Collins JJ, Zhang F (2017) Nucleic acid detection with CRISPR-Cas13a/C2c2. Science (New York, NY) 356(6336):438–442. https://doi.org/10.1126/science.aam9321
Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA (2018) Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science (New York, NY) 362(6416):839–842. https://doi.org/10.1126/science.aav4294
Janetanakit T, Lumyai M, Bunpapong N, Boonyapisitsopa S, Chaiyawong S, Nonthabenjawan N, Kesdaengsakonwut S, Amonsin A (2016) Porcine deltacoronavirus, Thailand, 2015. Emerg Infect Dis 22(4):757–759. https://doi.org/10.3201/eid2204.151852
Jang G, Lee KK, Kim SH, Lee C (2017) Prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in South Korea, 2014–2016. Transbound Emerg Dis 64(5):1364–1370. https://doi.org/10.1111/tbed.12690
Lednicky JA, Tagliamonte MS, White SK, Elbadry MA, Alam MM, Stephenson CJ, Bonny TS, Loeb JC, Telisma T, Chavannes S, Ostrov DA, Mavian C, Beau De Rochars VM, Salemi M, Morris JG Jr (2021) Independent infections of porcine deltacoronavirus among Haitian children. Nature 600(7887):133–137. https://doi.org/10.1038/s41586-021-04111-z
Li G, Chen Q, Harmon KM, Yoon KJ, Schwartz KJ, Hoogland MJ, Gauger PC, Main RG, Zhang J (2014) Full-length genome sequence of porcine deltacoronavirus strain USA/IA/2014/8734. Genome Announc 2(2). https://doi.org/10.1128/genomeA.00278-14
Liu Y, Xu H, Liu C, Peng L, Khan H, Cui L, Huang R, Wu C, Shen S, Wang S, Liang W, Li Z, Xu B, He N (2019) CRISPR-Cas13a nanomachine based simple technology for avian influenza A (H7N9) virus on-site detection. J Biomed Nanotechnol 15(4):790–798. https://doi.org/10.1166/jbn.2019.2742
Lorsirigool A, Saeng-Chuto K, Temeeyasen G, Madapong A, Tripipat T, Wegner M, Tuntituvanont A, Intrakamhaeng M, Nilubol D (2016) The first detection and full-length genome sequence of porcine deltacoronavirus isolated in Lao PDR. Arch Virol 161(10):2909–2911. https://doi.org/10.1007/s00705-016-2983-8
Ma L, Zeng F, Huang B, Zhu Y, Wu M, Xu F, Xiao L, Huang R, Ma J, Cong F, Guo P (2019) Point-of-care diagnostic assay for rapid detection of porcine deltacoronavirus using the recombinase polymerase amplification method. Transbound Emerg Dis 66(3):1324–1331. https://doi.org/10.1111/tbed.13155
Mai K, Feng J, Chen G, Li D, Zhou L, Bai Y, Wu Q, Ma J (2018) The detection and phylogenetic analysis of porcine deltacoronavirus from Guangdong Province in southern China. Transbound Emerg Dis 65(1):166–173. https://doi.org/10.1111/tbed.12644
Marthaler D, Raymond L, Jiang Y, Collins J, Rossow K, Rovira A (2014b) Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus. Emerg Infect Dis 20(8):1347–1350. https://doi.org/10.3201/eid2008.140526
Marthaler D, Jiang Y, Collins J, Rossow K (2014a) Complete genome sequence of strain SDCV/USA/Illinois121/2014, a porcine deltacoronavirus from the United States. Genome Announc 2(2). https://doi.org/10.1128/genomeA.00218-14
Pan Z, Lu J, Wang N, He WT, Zhang L, Zhao W, Su S (2020) Development of a TaqMan-probe-based multiplex real-time PCR for the simultaneous detection of emerging and reemerging swine coronaviruses. Virulence 11(1):707–718. https://doi.org/10.1080/21505594.2020.1771980
Pérez-Rivera C, Ramírez-Mendoza H, Mendoza-Elvira S, Segura-Velázquez R, Sánchez-Betancourt JI (2019) First report and phylogenetic analysis of porcine deltacoronavirus in Mexico. Transbound Emerg Dis 66(4):1436–1441. https://doi.org/10.1111/tbed.13193
Qiao X, Gao Y, Li J, Wang Z, Qiao H, Qi H (2021) Sensitive analysis of single nucleotide variation by Cas13d orthologs, EsCas13d and RspCas13d. Biotechnol Bioeng 118(8):3037–3045. https://doi.org/10.1002/bit.27813
Qin P, Park M, Alfson KJ, Tamhankar M, Carrion R, Patterson JL, Griffiths A, He Q, Yildiz A, Mathies R, Du K (2019) Rapid and fully microfluidic Ebola virus detection with CRISPR-Cas13a. ACS Sens 4(4):1048–1054. https://doi.org/10.1021/acssensors.9b00239
Sun W, Wang L, Huang H, Wang W, Cao L, Zhang J, Zheng M, Lu H (2020) Genetic characterization and phylogenetic analysis of porcine deltacoronavirus (PDCoV) in Shandong Province, China. Virus Res 278:197869. https://doi.org/10.1016/j.virusres.2020.197869
Tang P, Cui E, Song Y, Yan R, Wang J (2021) Porcine deltacoronavirus and its prevalence in China: a review of epidemiology, evolution, and vaccine development. Arch Virol 166(11):2975–2988. https://doi.org/10.1007/s00705-021-05226-4
Wang L, Byrum B, Zhang Y (2014) Detection and genetic characterization of deltacoronavirus in pigs, Ohio, USA, 2014. Emerg Infect Dis 20(7):1227–1230. https://doi.org/10.3201/eid2007.140296
Wang J, Xia Q, Wu J, Lin Y, Ju H (2021) A sensitive electrochemical method for rapid detection of dengue virus by CRISPR/Cas13a-assisted catalytic hairpin assembly. Anal Chim Acta 1187:339131. https://doi.org/10.1016/j.aca.2021.339131
Woo PC, Lau SK, Lam CS, Lau CC, Tsang AK, Lau JH, Bai R, Teng JL, Tsang CC, Wang M, Zheng BJ, Chan KH, Yuen KY (2012) Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol 86(7):3995–4008. https://doi.org/10.1128/jvi.06540-11
Yan WX, Chong S, Zhang H, Makarova KS, Koonin EV, Cheng DR, Scott DA (2018) Cas13d Is a compact RNA-targeting type VI CRISPR effector positively modulated by a WYL-domain-containing accessory protein. Mol Cell 70(2):327-339.e5. https://doi.org/10.1016/j.molcel.2018.02.028
Zhou L, Chen Y, Fang X, Liu Y, Du M, Lu X, Li Q, Sun Y, Ma J, Lan T (2020) Microfluidic-RT-LAMP chip for the point-of-care detection of emerging and re-emerging enteric coronaviruses in swine. Anal Chim Acta 1125:57–65. https://doi.org/10.1016/j.aca.2020.05.034
Funding
This work was supported by the Sichuan Science and Technology Programs (2021ZDZX0010, 2020YFN0147, and 2021YFYZ0030).
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YZ, TZ, and CZ designed research and conducted experiments; PM, KG, HL, and WL analyzed data. XY and HW wrote the manuscript. All authors read and approved the manuscript.
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Zhao, Y., Zhang, T., Zhou, C. et al. Development of an RT-PCR-based RspCas13d system to detect porcine deltacoronavirus. Appl Microbiol Biotechnol 107, 5739–5747 (2023). https://doi.org/10.1007/s00253-023-12690-2
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DOI: https://doi.org/10.1007/s00253-023-12690-2