Application of a rapid and sensitive RPA-CRISPR/Cas12a assay for naked-eye detection of Haemophilus parasuis.

Bacterial pathogen detection Haemophilus parasuis Naked-eye visualization RPA-CRISPR/Cas12a Rapid on-site detection

Journal

Analytica chimica acta
ISSN: 1873-4324
Titre abrégé: Anal Chim Acta
Pays: Netherlands
ID NLM: 0370534

Informations de publication

Date de publication:
25 Jan 2024
Historique:
received: 08 08 2023
revised: 27 11 2023
accepted: 29 11 2023
medline: 6 1 2024
pubmed: 6 1 2024
entrez: 5 1 2024
Statut: ppublish

Résumé

Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions. In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min. We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.

Sections du résumé

BACKGROUND BACKGROUND
Haemophilus parasuis (H. parasuis) is a gram-negative bacterial pathogen that causes severe infections in swine, resulting in substantial economic losses. Currently, the majority of H. parasuis detection methods are impractical for on-site application due to their reliance on large instruments or complex procedures. Thus, there is an urgent need to develop a rapid, visually detectable, and highly sensitive detection method, especially under resource-limited environments and field conditions.
RESULTS RESULTS
In this study, we established a naked eye assay for highly sensitive detection by combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a technology. Positive samples exhibited a clear red color visible to the naked eye, while negative samples appeared blue. We achieved a remarkable sensitivity, detecting H. parasuis down to a single copy, with no cross-reactivity with other bacteria. In a mouse model, our assay detected H. parasuis infection nearly 8 h earlier than traditional PCR. Compared to qPCR, our detection results were 100 % accurate. To enhance point-of-care applicability and mitigate the risk of aerosol contamination from uncapping, we consolidated RPA and CRISPR/Cas12a cleavage into a single-tube reaction system. This integrated approach was validated with 20 clinical lung samples, yielding results consistent with those obtained from qPCR. The entire procedure, from DNA extraction to detection, was completed in 35 min.
SIGNIFICANCE CONCLUSIONS
We present an RPA-CRISPR/Cas12a assay suitable for the early and resource-efficient diagnosis of H. parasuis infections. Its simplicity and visual detection are advantageous for field diagnostics, representing a substantial develpoment in the diagnosis of H. parasuis.

Identifiants

DOI: 10.1016/j.aca.2023.342101 PMID: 38182383
pubmed: 38182383
pii: S0003-2670(23)01322-3
doi: 10.1016/j.aca.2023.342101
pii:
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

342101

Informations de copyright

Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.

Déclaration de conflit d'intérêts

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Auteurs

Jie Hao (J)

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China.

Mengyan Jia (M)

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China.

Yiting Liu (Y)

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China.

Zhenlin Lv (Z)

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China.

Junming Chen (J)

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China.

Wenguang Xiong (W)

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China; National Reference Laboratory of Veterinary Drug Residues, South China Agricultural University, Guangzhou, 510642, China. Electronic address: xiongwg@scau.edu.cn.

Zhenling Zeng (Z)

Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, 510642, China; National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China; National Laboratory of Safety Evaluation (Environmental Assessment) of Veterinary Drugs, South China Agricultural University, Guangzhou, 510642, China; National Reference Laboratory of Veterinary Drug Residues, South China Agricultural University, Guangzhou, 510642, China. Electronic address: zlzeng@scau.edu.cn.

Classifications MeSH