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Infectious diseases of poverty
Dec 10, 2024;13 (1): 94.

A novel single-tube LAMP-CRISPR/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment.

Yao Liang, Shi-Chen Xie, Yi-Han Lv, Yuan-Hui He, Xiao-Nan Zheng, Wei Cong, Hany M Elsheikha, Xing-Quan Zhu
Author Information
  1. Yao Liang: Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, 030801, Shanxi, People's Republic of China.
  2. Shi-Chen Xie: Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, 030801, Shanxi, People's Republic of China.
  3. Yi-Han Lv: Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, 030801, Shanxi, People's Republic of China.
  4. Yuan-Hui He: Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, 030801, Shanxi, People's Republic of China.
  5. Xiao-Nan Zheng: Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, 030801, Shanxi, People's Republic of China.
  6. Wei Cong: Marine College, Shandong University, Weihai, 264209, Shandong, People's Republic of China.
  7. Hany M Elsheikha: Faculty of Medicine and Health Sciences, School of Veterinary Medicine and Science, University of Nottingham, Sutton Bonington Campus, Loughborough, LE12 5RD, UK. hany.elsheikha@nottingham.ac.uk.
  8. Xing-Quan Zhu: Laboratory of Parasitic Diseases, College of Veterinary Medicine, Shanxi Agricultural University, Taigu, 030801, Shanxi, People's Republic of China. xingquanzhu1@hotmail.com. ORCID
PMID: 39654027 DOI: 10.1186/s40249-024-01266-5

Abstract

BACKGROUND: Toxoplasma gondii oocysts, excreted in cat feces, pose a significant health risk to humans through contaminated soil and water. Rapid and accurate detection of T. gondii in environmental samples is essential for public health protection.
METHODS: We developed a novel, single-tube detection method that integrates loop-mediated isothermal amplification (LAMP), the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12b system, and lateral flow immunoassay strips for rapid, visual identification of T. gondii. This method targets the T. gondii B1 gene, initially amplifies it with LAMP, directed by a single-guide RNA (sgRNA). It then recognizes the amplified target gene and activates trans-cleavage, cutting nearby single-stranded DNA (ssDNA) reporters. Fluorescence detection was performed using a 6-Carboxyfluorescein (FAM)-12N-Black Hole Quencher-1 (BHQ1) reporter, while Fluorescein Isothiocyanate (FITC)-12N-Biotin enabled visual detection on lateral flow strips. The method was tested for its ability to detect various T. gondii genotypes and related parasites, assessing its specificity and broad-spectrum applicability. It was further applied to real-world environmental samples to evaluate its practicality.
RESULTS: The LAMP-CRISPR/Cas12b method exhibited high specificity and broad-spectrum detection capability, successfully identifying nine T. gondii genotypes and distinguishing them from 11 other parasitic species. Sensitivity testing at both molecular (plasmid) and practical (oocyst) levels showed detection limits of 10  copies/μL and 0.1 oocyst, respectively. When applied to 112 environmental samples (soil, water, and cat feces), the method demonstrated 100% sensitivity, accurately reflecting known infection rates.
CONCLUSIONS: This LAMP-CRISPR/Cas12b single-tube method offers a robust, innovative approach for monitoring zoonotic T. gondii in environmental samples, with significant implications for public health surveillance.

Keywords

Toxoplasma gondii CRISPR/Cas12b Environmental detection Lateral flow immunoassay Loop-mediated isothermal amplification Molecular diagnostics Zoonotic parasites

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Grants

  1. 2021YFC2300800/National Key Research and Development Program of China
  2. 2021YFC2300802/National Key Research and Development Program of China
  3. 2021YFC2300804/National Key Research and Development Program of China
  4. LXXMsxnd202101/Shanxi Provincial Agricultural and Rural Research Program
  5. RFSXIHLT202101/the Research Fund of Shanxi Province for Introduced High-level Leading Talents
  6. 2021XG001/the Special Research Fund of Shanxi Agricultural University for High-level Talents

MeSH Term

Toxoplasma
Nucleic Acid Amplification Techniques
CRISPR-Cas Systems
Animals
Molecular Diagnostic Techniques
Humans
Sensitivity and Specificity
Cats
Zoonoses
Feces
Toxoplasmosis
Journal Article
Article has an altmetric score of 1

Word Cloud

Created with Highcharts 10.0.0gondiidetectionmethodTenvironmentalsamplesToxoplasmahealthsingle-tubeflowvisualLAMP-CRISPR/Cas12bcatfecessignificantsoilwaterpublicnovelisothermalamplificationLAMPlateralimmunoassaystripsrapidgenegenotypesparasitesspecificitybroad-spectrumappliedoocystzoonoticBACKGROUND:oocystsexcretedposeriskhumanscontaminatedRapidaccurateessentialprotectionMETHODS:developedintegratesloop-mediatedclusteredregularlyinterspacedshortpalindromicrepeatsCRISPR/Cas12bsystemidentificationtargetsB1initiallyamplifiesdirectedsingle-guideRNAsgRNArecognizesamplifiedtargetactivatestrans-cleavagecuttingnearbysingle-strandedDNAssDNAreportersFluorescenceperformedusing6-CarboxyfluoresceinFAM-12N-BlackHoleQuencher-1BHQ1reporterFluoresceinIsothiocyanateFITC-12N-Biotinenabledtestedabilitydetectvariousrelatedassessingapplicabilityreal-worldevaluatepracticalityRESULTS:exhibitedhighcapabilitysuccessfullyidentifyingninedistinguishing11parasiticspeciesSensitivitytestingmolecularplasmidpracticallevelsshowedlimits10 copies/μL01respectively112demonstrated100%sensitivityaccuratelyreflectingknowninfectionratesCONCLUSIONS:offersrobustinnovativeapproachmonitoringimplicationssurveillanceenvironmentCRISPR/Cas12bEnvironmentalLateralLoop-mediatedMoleculardiagnosticsZoonotic

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