1887

Journal of Medical Microbiology logo

Volume 72, Issue 12

Engineering of the LAMP-CRISPR/Cas12b platform for detection Open Access

Abstract

. () is a zoonotic infection, that causes psittacosis (parrot fever) in humans, leading to severe clinical manifestations, including severe pneumonia, adult respiratory distress syndrome, and, in rare cases, death.

. Rapid, sensitive and specific detection of facilitates timely diagnosis and treatment of patients.

. This study aimed to engineer the LAMP-CRISPR/Cas12b platform for detection.

. The loop-mediated isothermal amplification (LAMP) technique and clustered regularly interspaced short palindromic repeats-CRISPR associated protein 12b (CRISPR-Cas12b) assay were combined to establish two-step and one-tube LAMP-CRISPR/Cas12b reaction systems, respectively, for rapidly detecting .

. The two-step and one-tube LAMP-CRISPR/Cas12b assay could complete detection within 1 h. No cross-reactivity was observed from non- templates with specific LAMP amplification primers and single-guide RNA (sgRNA) targeting the highly conserved short fragment CPSIT_0429 gene of . The detection limits of the two-step and one-tube LAMP-CRISPR/Cas12b reaction were 10 aM and 10 aM, respectively. The results were consistent with qPCR for nucleic acid detection in 160 clinical samples, including 80 suspected samples, kept in the laboratory.

. The LAMP-CRISPR/Cas12b assay developed in this study provides a sensitive and specific method for rapidly detecting and offers technical support for its rapid diagnosis.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Chlamydia psittaci , CRISPR-Cas12b , detection and LAMP
Funding
This study was supported by the:
  • Key Research and Development Project of Jiangsu Province (Award BE2023694)
    • Principle Award Recipient: FengmingWang
  • Changzhou Science and Technology Plan Project (Award CJ20220114)
    • Principle Award Recipient: JianXu
  • Changzhou Science and Technology Plan Project (Award CE20225041)
    • Principle Award Recipient: QiongLi
  • Leading Talent of Changzhou “The 14th Five-Year Plan” High-Level Health Talents Training Project (Award 2022CZLJ025)
    • Principle Award Recipient: FengmingWang
  • Key Laboratory Project of Changzhou City (Award CM20223016)
    • Principle Award Recipient: FengmingWang
  • Zhejiang Provincial Ten Thousand Plan for Young Top Talents (Award CZQM2022023)
    • Principle Award Recipient: JingyiJiang
  • Young Talent Development Plan of Changzhou Health Commission (Award CZQM2020113)
    • Principle Award Recipient: XujianMao
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2023-12-06
2024-12-12
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References

  1. Harkinezhad T, Geens T, Vanrompay D. Chlamydophila psittaci infections in birds: a review with emphasis on zoonotic consequences. Vet Microbiol 2009; 135:68–77 [View Article] [PubMed]
     [Google Scholar]
  2. Rodolakis A, Yousef Mohamad K. Zoonotic potential of Chlamydophila. Vet Microbiol 2010; 140:382–391 [View Article] [PubMed]
     [Google Scholar]
  3. Bommana S, Polkinghorne A. Mini review: antimicrobial control of Chlamydial infections in animals: current practices and issues. Front Microbiol 2019; 10:113 [View Article] [PubMed]
     [Google Scholar]
  4. Knittler MR, Sachse K. Chlamydia psittaci: update on an underestimated zoonotic agent. Pathog Dis 2015; 73:1–15 [View Article] [PubMed]
     [Google Scholar]
  5. Hogerwerf L, Deg B, Baan B, Vdh W. Chlamydia psittaci (psittacosis) as a cause of community-acquired pneumonia: a systematic review and meta-analysis. Epidemiol Infect 2017; 145:3096–3105 [View Article] [PubMed]
     [Google Scholar]
  6. Wallensten A, Fredlund H, Runehagen A. Multiple human-to-human transmission from a severe case of psittacosis, Sweden, January–February 2013. Euro Surveill 2014; 19: [View Article] [PubMed]
     [Google Scholar]
  7. Zhang Z, Zhou H, Cao H, Ji J, Zhang R et al. Human-to-human transmission of Chlamydia psittaci in China, 2020: an epidemiological and aetiological investigation. Lancet Microbe 2022; 3:e512–e520 [View Article] [PubMed]
     [Google Scholar]
  8. Metlay JP, Waterer GW, Long AC, Anzueto A, Brozek J et al. Diagnosis and treatment of adults with community-acquired pneumonia. An official clinical practice guideline of the American thoracic society and infectious diseases society of America. Am J Respir Crit Care Med 2019; 200:e45–e67 [View Article] [PubMed]
     [Google Scholar]
  9. Qin X-C, Huang J, Yang Z, Sun X, Wang W et al. Severe community-acquired pneumonia caused by Chlamydia psittaci genotype E/B strain circulating among geese in Lishui city, Zhejiang province, China. Emerg Microbes Infect 2022; 11:2715–2723 [View Article] [PubMed]
     [Google Scholar]
  10. Shaw KA, Szablewski CM, Kellner S, Kornegay L, Bair P et al. Psittacosis outbreak among workers at chicken slaughter plants, Virginia and Georgia, USA, 2018. Emerg Infect Dis 2018; 25:2143–2145 [View Article]
     [Google Scholar]
  11. Yin Q, Li Y, Pan H, Hui T, Yu Z et al. Atypical pneumonia caused by Chlamydia psittaci during the COVID-19 pandemic. Int J Infect Dis 2022; 122:622–627 [View Article] [PubMed]
     [Google Scholar]
  12. Nieuwenhuizen AA, Dijkstra F, Notermans DW, van der Hoek W. Laboratory methods for case finding in human psittacosis outbreaks: a systematic review. BMC Infect Dis 2018; 18:442 [View Article] [PubMed]
     [Google Scholar]
  13. Gu L, Liu W, Ru M, Lin J, Yu G et al. The application of metagenomic next-generation sequencing in diagnosing Chlamydia psittaci pneumonia: a report of five cases. BMC Pulm Med 2020; 20:65 [View Article] [PubMed]
     [Google Scholar]
  14. Moehling TJ, Choi G, Dugan LC, Salit M, Meagher RJ. LAMP diagnostics at the point-of-care: emerging trends and perspectives for the developer community. Expert Rev Mol Diagn 2021; 21:43–61 [View Article] [PubMed]
     [Google Scholar]
  15. Bonney LC, Watson RJ, Slack GS, Bosworth A, Wand NIV et al. A flexible format LAMP assay for rapid detection of Ebola virus. PLoS Negl Trop Dis 2020; 14:e0008496 [View Article] [PubMed]
     [Google Scholar]
  16. Wei S, Suryawanshi H, Djandji A, Kohl E, Morgan S et al. Field-deployable, rapid diagnostic testing of saliva for SARS-CoV-2. Sci Rep 2021; 11:5448 [View Article] [PubMed]
     [Google Scholar]
  17. Wong Y-P, Othman S, Lau Y-L, Radu S, Chee H-Y. Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms. J Appl Microbiol 2018; 124:626–643 [View Article] [PubMed]
     [Google Scholar]
  18. Broughton JP, Deng X, Yu G, Fasching CL, Servellita V et al. CRISPR-Cas12-based detection of SARS-CoV-2. Nat Biotechnol 2020; 38:870–874 [View Article] [PubMed]
     [Google Scholar]
  19. Mukama O, Wu J, Li Z, Liang Q, Yi Z et al. An ultrasensitive and specific point-of-care CRISPR/Cas12 based lateral flow biosensor for the rapid detection of nucleic acids. Biosens Bioelectron 2020; 159:112143 [View Article] [PubMed]
     [Google Scholar]
  20. Mojica FJM, Díez-Villaseñor C, García-Martínez J, Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol 2005; 60:174–182 [View Article] [PubMed]
     [Google Scholar]
  21. Marraffini LA, Sontheimer EJ. CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat Rev Genet 2010; 11:181–190 [View Article] [PubMed]
     [Google Scholar]
  22. Chen JS, Ma E, Harrington LB, Da Costa M, Tian X et al. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 2018; 360:436–439 [View Article] [PubMed]
     [Google Scholar]
  23. East-Seletsky A, O’Connell MR, Knight SC, Burstein D, Cate JHD et al. Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection. Nature 2016; 538:270–273 [View Article] [PubMed]
     [Google Scholar]
  24. Kaminski MM, Abudayyeh OO, Gootenberg JS, Zhang F, Collins JJ. CRISPR-based diagnostics. Nat Biomed Eng 2021; 5:643–656 [View Article] [PubMed]
     [Google Scholar]
  25. Swarts DC, Jinek M. Mechanistic insights into the cis- and trans-acting DNase activities of Cas12a. Mol Cell 2019; 73:589–600 [View Article] [PubMed]
     [Google Scholar]
  26. Liu L, Chen P, Wang M, Li X, Wang J et al. C2c1-sgRNA complex structure reveals RNA-Guided DNA cleavage mechanism. Mol Cell 2017; 65:310–322 [View Article] [PubMed]
     [Google Scholar]
  27. Teng F, Guo L, Cui T, Wang X-G, Xu K et al. CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity. Genome Biol 2019; 20:132 [View Article] [PubMed]
     [Google Scholar]
  28. Joung J, Ladha A, Saito M, Segel M, Bruneau R et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. medRxiv 20202020.05.04.20091231 [View Article] [PubMed]
     [Google Scholar]
  29. Qiu X, Liu X, Ma X, Wang R, Chen S et al. One-Pot isothermal LAMP-CRISPR-based assay for Klebsiella pneumoniae detection. Microbiol Spectr 2022; 10:e0154522 [View Article] [PubMed]
     [Google Scholar]
  30. Li L, Li S, Wu N, Wu J, Wang G et al. HOLMESv2: a CRISPR-Cas12b-assisted platform for nucleic acid detection and DNA methylation quantitation. ACS Synth Biol 2019; 8:2228–2237 [View Article] [PubMed]
     [Google Scholar]
  31. Voigt A, Schöfl G, Saluz HP. The Chlamydia psittaci genome: a comparative analysis of intracellular pathogens. PLoS One 2012; 7:e35097 [View Article] [PubMed]
     [Google Scholar]
  32. Lu S, Tong X, Han Y, Zhang K, Zhang Y et al. Fast and sensitive detection of SARS-CoV-2 RNA using suboptimal protospacer adjacent motifs for Cas12a. Nat Biomed Eng 2022; 6:286–297 [View Article] [PubMed]
     [Google Scholar]
  33. Lin M, Yue H, Tian T, Xiong E, Zhu D et al. Glycerol additive boosts 100-fold sensitivity enhancement for One-Pot RPA-CRISPR/Cas12a assay. Anal Chem 2022; 94:8277–8284 [View Article] [PubMed]
     [Google Scholar]
  34. Yin K, Ding X, Li Z, Zhao H, Cooper K et al. Dynamic aqueous multiphase reaction system for one-pot CRISPR-Cas12a-based ultrasensitive and quantitative molecular diagnosis. Anal Chem 2020; 92:8561–8568 [View Article] [PubMed]
     [Google Scholar]
  35. McGovern OL, Kobayashi M, Shaw KA, Szablewski C, Gabel J et al. Use of real-time PCR for Chlamydia psittaci detection in human specimens during an outbreak of Psittacosis - Georgia and Virginia, 2018. MMWR Morb Mortal Wkly Rep 2021; 70:505–509 [View Article] [PubMed]
     [Google Scholar]
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Engineering of the LAMP-CRISPR/Cas12b platform for Chlamydia psittaci detection
J. Med. Microbiol. 72, 001781 (2024); https://doi.org/10.1099/jmm.0.001781
/content/journal/jmm/10.1099/jmm.0.001781
/content/journal/jmm/10.1099/jmm.0.001781
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