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Abstract
Introduction
Materials and Methods
Rpa and Crrna Primer Design and Synthesis
Results
Experimental Design and Assay Optimization
Discussion
Conclusion
References
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Biology
Virology

CRISPR IB

Profile image of Amjed TalibAmjed Talib
https://doi.org/10.33899/IJVS.2023.140335.3040
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Abstract

Infectious bronchitis is an acute respiratory disease of poultry associated with reduced egg production and heavy economic losses in chicken flocks. Rabid and accurate detection of IB virus (IBV) is essential for controlling and preventing the infection. In this study, we developed a rapid, accurate, and instrument less assay to detect IBV. For the first time, reverse transcription-Recombinase polymerase amplification (RT-RPA) coupled with CRISPR/Cas13 (SHERLOCK) was used to rapidly visualize IBV. The novel assay was tested in timing, sensitivity, and specificity. The spike gene (S gene) was used as a target gene for detecting the virus. Three samples were used to optimize the assay; sample form confirmed infected chickens with IB, positive sample (full synthesis of S gene), and negative sample from free IB infected chickens. The results show that the Sherlock-based Cas13 platform is a highly specificity and sensitivity assay for detecting infectious bronchitis virus. The assay detected ten copies per µL of the input RNA. No false positives or cross-reactions were seen when bovine coronavirus (BCV) was used instead of IBV in the tested sample. Readout of the results needs just fifty minutes, including RNA extraction. Furthermore, No instrument was used, and amplification of the virus's nucleic acid was performed at room temperature. Sherlock-based Cas13 should clinically use for rapid diagnosis of infectious bronchitis in chickens. However, further studies and experiments are needed to perform the assay at the sample base without extraction of RNA.

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References (41)

  1. Egaña-Labrin S, Hauck R, Figueroa A, Stoute S, Shivaprasad HL, Crispo M, Corsiglia C, Zhou H, Kern C, Crossley B, Gallardo RA. Genotypic characterization of emerging avian reovirus genetic variants in California. Sci Rep. 2019;9(1):9351. DOI: 10.1038/s41598-019- 45494-4
  2. Liu IL, Lin YC, Lin YC, Jian CZ, Cheng IC, Chen HW. A novel immunochromatographic strip for antigen detection of avian infectious bronchitis virus. Int J Mol Sci. 2019;20(9):2216. DOI: 10.3390/ijms20092216
  3. Icochea E, González R, Castro-Sanguinetti G, Maturrano L, Alzamora L, Sesti L, Chacón J, More-Bayona J. Genetic analysis of infectious bronchitis virus S1 gene reveals novel amino acid changes in the GI-16 lineage in Peru. Microorganisms. 2023;11(3):691. DOI: 10.3390/microorganisms11030691
  4. Thai TN, Yoo DS, Jang I, Kwon YK, Kim HR. Dynamics of the emerging genogroup of infectious bursal disease virus infection in broiler farms in South Korea: A nationwide study. Viruses. 2022;14(8):1604. DOI: 10.3390/v14081604
  5. Shirvani E, Paldurai A, Manoharan VK, Varghese BP, Samal SK. A Recombinant Newcastle disease virus (NDV) expressing S protein of infectious bronchitis virus (IBV) protects chickens against IBV and NDV. Sci Rep. 2020;10(1):762. DOI: 10.1038/s41598-018-30356-2
  6. Ulkarni AB, Resurreccion RS. Genotyping of newly isolated infectious bronchitis virus isolates from northeastern Georgia. Avian Dis Digest. 2010;5(4):e3-4. DOI: 10.1637/9543-935810-digest.1
  7. Gu K, Song Z, Ma P, Liao Z, Yang M, Zhou C, Li C, Zhao Y, Li H, Yang X, Lei C, Wang H. A novel nanobody-horseradish peroxidase fusion based-competitive ELISA to rapidly detect avian corona-virus- infectious bronchitis virus antibody in chicken serum. Int J Mol Sci. 2022;23(14):7589. DOI: 10.3390/ijms23147589
  8. Bóna M, Kiss I, Dénes L, Szilasi A, Mándoki M. Tissue tropism of H9N2 low-pathogenic avian influenza virus in broiler chickens by immunohistochemistry. Animals. 2023;13(6):1052. DOI: 10.3390/ani13061052
  9. Ma H, Shao Y, Sun C, Han Z, Liu X, Guo H, Liu X, Kong X, Liu S. Genetic diversity of avian infectious bronchitis coronavirus in recent years in China. Avian Dis Digest. 2012;7(1):e6-7. DOI: 10.1637/10000-980411-digest.1
  10. Lin SY, Chen HW. Infectious bronchitis virus variants: Molecular analysis and pathogenicity investigation. Int J Mol Sci. 2017;18(10):2030. DOI: 10.3390/ijms18102030
  11. Muhsen H, Alaraji F, Alhatami AO, khudhair YI. Real time PCR detection, sequencing, and phylogenetic tree analysis of Newcastle diseases virus isolated from an outbreak in layer flocks in Baghdad capital, Iraq. Indian J Public Health Res Dev. 2019;10(8):2077. DOI: 10.5958/0976-5506.2019.02162.4
  12. Srivastava S, Upadhyay DJ, Srivastava A. Next-generation molecular diagnostics development by CRISPR/Cas tool: Rapid detection and surveillance of viral disease outbreaks. Front Mol BioSci. 2020;23(7):582499. DOI: 10.3389/fmolb.2020.582499
  13. Al-Jameel W, Al-Mahmood SS. Similarities and differences of COVID-19 and avian infectious bronchitis from molecular pathologist and poultry specialist view point. Iraqi J Vet Sci. 2020;34(2):223-31. DOI: 10.33899/ijvs.2020.126984.1426
  14. Raquib A, Uddin A, Nurozzaman SM, Uddin MM, Ahsan G, Rahman MM, Rahman MM. Seroprevalence of Mycoplasma gallisepticum infection in layer chickens of Bangladesh. Iraqi J Vet Sci. 2022;36(1):9- 13. DOI: 10.33899/ijvs.2020.127511.1506
  15. Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DG. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT- PCR. Euro Surveill. 2020;25(3):2000045. DOI: 10.2807/1560- 7917.es.2020.25.3.2000045
  16. Palaz F, Kalkan AK, Can O, Demir AN, Tozluyurt A, Ozcan A, Ozsoz M. CRISPR-Cas13 system as a promising and versatile tool for cancer diagnosis, therapy, and research. ACS Synth Biol. 2021;10(6):1245-67. DOI: 10.1021/acssynbio.1c00107
  17. Xiang X, Qian K, Zhang Z, Lin F, Xie Y, Liu Y, Yang Z. CRISPR-Cas systems based molecular diagnostic tool for infectious diseases and emerging 2019 novel coronavirus (COVID-19) pneumonia. J Drug Target. 2020;28(7-8):727-31. DOI: 10.1080/1061186x.2020.1769637
  18. Esbin MN, Whitney ON, Chong S, Maurer A, Darzacq X, Tjian R. Overcoming the bottleneck to widespread testing: A rapid review of nucleic acid testing approaches for COVID-19 detection. RNA. 2020;26(7):771-83. DOI: 10.1261/rna.076232.120
  19. Myhrvold C, Freije CA, Gootenberg JS, Abudayyeh OO, Metsky HC, Durbin AF, Kellner MJ, Tan AL, Paul LM, Parham LA, Garcia KF. Field-deployable viral diagnostics using CRISPR-Cas13. Sci. 2018;360(6387):444-8. DOI: 10.1126/science.aas8836
  20. Shariq M, Khan MF, Raj R, Ahsan N, Singh R, Kumar P. CRISPR-based diagnostic approaches: Implications for rapid management of future pandemics (Review). Mol Med Rep. 2023;27(6):118. DOI: 10.3892/mmr.2023.13005
  21. Huang YY, Zhang XY, Zhu P, Ji L. Development of clustered regularly interspaced short palindromic repeats/CRISPR-associated technology for potential clinical applications. World J Clin Cases. 2022;10(18):5934-5945. DOI: 10.12998/wjcc.v10.i18.5934
  22. Hillary VE, Ceasar SA. A Review on the mechanism and applications of CRISPR/Cas9/Cas12/Cas13/Cas14 proteins utilized for genome engineering. Mol Biotechnol. 2023;65(3):311-325. DOI: 10.1007/s12033-022-00567-0
  23. Compton SR. PCR and RT-PCR in the diagnosis of laboratory animal infections and in health monitoring. J Am Assoc Lab Anim Sci. 2020;59(5):458-68. DOI: 10.30802/aalas-jaalas-20-000008
  24. Zhang X. Development of CRISPR-mediated nucleic acid detection technologies and their applications in the livestock industry. Genes. 2022;13(11):2007. DOI: 10.3390/genes13112007
  25. Chakraborty J, Chaudhary AA, Khan SU, Rudayni HA, Rahaman SM, Sarkar H. CRISPR/Cas-based biosensor as a new age detection method for pathogenic bacteria. ACS Omega. 2022;7(44):39562-39573. DOI: 10.1021/acsomega.2c04513
  26. Chavez M, Chen X, Finn PB, Qi LS. Advances in CRISPR therapeutics. Nat Rev Nephrol. 2023;19(1):9-22. DOI: 10.1038/s41581-022-00636- 2
  27. Vainionpää R, Leinikki P. Diagnostic techniques: Serological and molecular approaches. In: Mahy BJ, Van Regenmortel MV, editors. Encyclopaedia of virology. USA: Academic Press; 2008. 29-37 p. DOI: 10.1016/b978-012374410-4.00585-9
  28. Park HM, Park Y, Berani U, Bang E, Vankerschaver J, Van Messem A, De Neve W, Shim H. In silico optimization of RNA-protein interactions for CRISPR-Cas13-based antimicrobials. Biol Direct. 2022;17(1):27. DOI: 10.1186/s13062-022-00339-5
  29. Zhou Q, Chen Y, Wang R, Jia F, He F, Yuan F. Advances of CRISPR- Cas13 system in COVID-19 diagnosis and treatment. Genes Dis. 2022. DOI: 10.1016/j.gendis.2022.11.016
  30. Gao H, Shang Z, Chan SY, Ma D. Recent advances in the use of the CRISPR-Cas system for the detection of infectious pathogens. J Zhejiang Univ Sci B. 2022;23(11):881-898. DOI: 10.1631/jzus.B2200068
  31. Quansah E, Chen Y, Yang S, Wang J, Sun D, Zhao Y, Chen M, Yu L, Zhang C. CRISPR-Cas13 in malaria parasite: Diagnosis and prospective gene function identification. Front Microbiol. 2023;14:1076947. DOI: 10.3389/fmicb.2023.1076947
  32. Zhao L, Qiu M, Li X, Yang J, Li J. CRISPR-Cas13a system: A novel tool for molecular diagnostics. Front Microbiol. 2022;13:1060947. DOI: 10.3389/fmicb.2022.1060947
  33. Isa RH, Abdo JM, Al-Barzinji YM. Genotyping of avian infectious bronchitis virus in broiler farms in Duhok province, north of Iraq. Iraqi J Vet Sci. 2022;36(1):171-5. DOI: 10.33899/ijvs.2021.129635.1670
  34. Al-Jameel W, Al-Mahmood SS. Similarities and differences of COVID-19 and avian infectious bronchitis from molecular pathologist and poultry specialist view point. Iraqi J Vet Sci. 2020;34(2):223-31. DOI: 10.33899/ijvs.2020.126984.1426
  35. Yehia N, Salem HM, Mahmmod Y, Said D, Samir M, Mawgod SA, Sorour HK, AbdelRahman MA, Selim S, Saad AM, El-Saadony MT, El-Meihy RM, Abd El-Hack ME, El-Tarabily KA, Zanaty AM. Common viral and bacterial avian respiratory infections: An updated review. Poult Sci. 2023;102(5):102553. DOI: 10.1016/j.psj.2023.102553
  36. Zhao J, Zhao Y, Zhang G. Key aspects of coronavirus avian infectious bronchitis virus. Pathogens. 2023;12(5):698. DOI: 10.3390/pathogens12050698
  37. Yehia N, Salem HM, Mahmmod Y, Said D, Samir M, Mawgod SA, Sorour HK, AbdelRahman MA, Selim S, Saad AM, El-Saadony MT. Common viral and bacterial avian respiratory infections: An updated review. Poult Sci. 2023;102(5):102553. DOI: 10.1016/j.psj.2023.102553
  38. Ali Z, Mahas A, Mahfouz M. CRISPR/Cas13 as a tool for RNA interference. Trends Plant Sci. 2018;23(5):374-8. DOI: 10.1016/j.tplants.2018.03.003
  39. Bot JF, van der Oost J, Geijsen N. The double life of CRISPR-Cas13. Curr Opin Biotechnol. 2022;78:102789. DOI: 10.1016/j.copbio.2022.102789
  40. Lou J, Wang B, Li J, Ni P, Jin Y, Chen S, Xi Y, Zhang R, Duan G. The CRISPR-Cas system as a tool for diagnosing and treating infectious diseases. Mol Biol Rep. 2022;49(12):11301-11311. DOI: 10.1007/s11033-022-07752-z
  41. Abbas G, Yu J, Li G. Novel and alternative therapeutic strategies for controlling avian viral infectious diseases: Focus on infectious bronchitis and avian influenza. Front Vet Sci. 2022;9:933274. DOI: 10.3389/fvets.2022.933274

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NUZHAT AHSAN

Molecular Medicine Reports

Sudden viral outbreaks have increased in the early part of the 21st century, such as those of severe acute respiratory syndrome coronavirus (SarS-coV), Middle east respiratory syndrome corona virus, and SarS-coV-2, owing to increased human access to wildlife habitats. Therefore, the likelihood of zoonotic transmission of human-associated viruses has increased. The emergence of severe acute respiratory syndrome coronavirus 2 in china and its spread worldwide within months have highlighted the need to be ready with advanced diagnostic and antiviral approaches to treat newly emerging diseases with minimal harm to human health. The gold-standard molecular diagnostic approaches currently used are time-consuming, require trained personnel and sophisticated equipment, and therefore cannot be used as point-of-care devices for widespread monitoring and surveillance. clustered regularly interspaced short palindromic repeats (criSPr)-associated (cas) systems are widespread and have been reported in bacteria, archaea and bacteriophages. criSPr-cas systems are organized into criSPr arrays and adjacent cas proteins. The detection and in-depth biochemical characterization of class 2 type V and Vi criSPr-cas systems and orthologous proteins such as cas12 and cas13 have led to the development of criSPr-based diagnostic approaches, which have been used to detect viral diseases and distinguish between serotypes and subtypes. criSPr-based diagnostic approaches detect human single nucleotide polymorphisms in samples from patients with cancer and are used as antiviral agents to detect and destroy viruses that contain rna as a genome. criSPr-based diagnostic approaches are likely to improve disease detection methods in the 21st century owing to their ease of development, low cost, reduced turnaround time, multiplexing and ease of deployment. The present review discusses the biochemical properties of cas12 and cas13 orthologs in viral disease detection and other applications. The present review expands the scope of criSPr-based diagnostic approaches to detect diseases and fight viruses as antivirals. Contents 1. introduction 2. criSPr-cas systems provide tools for modern diagnostics 3. cas proteins recognize target double-stranded dna (dsdna) and rna mediated by single-stranded rna (ssrna)

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Development and evaluation of a real-time Taqman RT-PCR assay for the detection of infectious bronchitis virus from infected chickens
David Swayne

Journal of Virological Methods, 2006

It is important to rapidly differentiate infectious bronchitis virus (IBV) from disease agents like highly pathogenic avian influenza virus and exotic Newcastle disease virus, which can be extremely similar in the early stages of their pathogenesis. In this study, we report the development and testing of a real-time RT-PCR assay using a Taqman ® -labeled probe for early and rapid detection of IBV. The assay amplifies a 143-bp product in the 5 -UTR of the IBV genome and has a limit of detection and quantification of 100 template copies per reaction. All 15 strains of IBV tested as well as two Turkey coronavirus strains were amplified, whereas none of the other pathogens examined, tested positive. Evaluation of the assay was completed with 1329 tracheal swab samples. A total of 680 samples collected from IBV antibody negative birds were negative for IBV by the real-time RT-PCR assay. We tested 229 tracheal swabs submitted to two different diagnostic laboratories and found 79.04% of the tracheal swabs positive for IBV by real-time RT-PCR, whereas only 27.51% of the samples were positive by virus isolation, which is the reference standard test. We also collected a total of 120 tracheal swabs at six different time points from birds experimentally infected with different dosages of IBV and found that, independent of the dose given, the viral load in the trachea plateau at 5 days post-inoculation. In addition, an inverse relationship between the dose of virus given and the viral load at 14 days post-inoculation was observed. Finally, we tested 300 total tracheal swab samples, from a flock of commercial broilers spray vaccinated for IBV in the field. The percentage of birds infected with the IBV vaccine at 3, 7, and 14 days post-vaccination was 58%, 65%, and 83%, respectively, indicating that only slightly more than half the birds were initially infected then the vaccine was subsequently transmitted to other birds in the flock. This observation is significant because coronaviruses, which have a high mutation rate, can revert to pathogenicity when bird-to-bird transmission occurs. The real-time RT-PCR test described herein can be used to rapidly distinguish IBV from other respiratory pathogens, which is important for control of this highly infectious virus. The test was extremely sensitive and specific, and can be used to quantitate viral genomic RNA in clinical samples.

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A one-step, one-pot CRISPR nucleic acid detection platform (CRISPR-top): Application for the diagnosis of COVID-19
Logan Brown

Talanta

The existing CRISPR-mediated diagnostic tests require a two-step procedure (DNA or RNA amplification followed by CRISPR-mediated sequence-specific detection) for nucleic acid detection, which increases complexity and the risk of sample cross-contamination. Here, we report a new CRISPR-mediated test, called CRISPR-top (CRISPRmediated testing in one-pot), which integrates simultaneous target pre-amplification with CRISPR/cas12bmediated detection into a one-pot reaction mixture, performed at a constant temperature. The novel CRISPRtop assay was applied to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent for COVID-19 (coronavirus disease 2019). COVID-19 CRISPR-top targets the ORF1ab (opening reading frame 1a/b) and NP (nucleoprotein) genes of SARS-CoV-2, and operates at 59 • C for 40 min with minimal instrument. The COVID-19 CRISPR-top assay can return results within 60-min and is easily interpreted by visual fluorescence or lateral flow readouts. The analytical limit of detection (LoD) for COVID-19 CRISPR-top is 10 copies (for each detection target) per reaction with no cross-reactivity observed from non-SARS-CoV-2 templates. Among clinically collected non-COVID-19 samples, the assay's specificity was 100% (80/80 oropharynx swab samples). Among 52 COVID-19 positive clinical samples collected, the COVID-19 CRISPR-top assay yielded 38 (73.1%) positive results using fluorescence readout and 35 (67.3%) positive results with lateral-flow readout. These diagnostic results were similar to those obtained using RT-PCR (34 positive (65.4%)). These data indicate that COVID-19 CRISPR-top is a simple, rapid, accurate and highly sensitive method for SARS-CoV-2 detection which can be used in the clinic, field laboratories and primary care facilities in resource-challenged settings.

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CRISPR‐based point‐of‐care diagnostics incorporating Cas9, Cas12, and Cas13 enzymes advanced for SARS‐CoV‐2 detection
Kalyan Sethi

Journal of Biochemical and Molecular Toxicology

An outbreak of the novel beta coronavirus severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) first came to light in December 2019, which has unfolded rapidly and turned out to be a global pandemic. Early prognosis of viral contamination involves speedy intervention, disorder control, and good‐sized management of the spread of disease. Reverse transcription‐polymerase chain reaction, considered the gold standard test for detecting nucleic acids and pathogen diagnosis, provides high sensitivity and specificity. However, reliance on high‐priced equipped kits, associated reagents, and skilled personnel slow down sickness detection. Lately, the improvement of clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas (CRISPR‐associated protein)‐based diagnostic systems has reshaped molecular diagnosis due to their low cost, simplicity, speed, efficiency, high sensitivity, specificity, and versatility, which is vital for accomplishing point‐of‐care diagnostics. We rev...

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