RPA-CRISPR/Cas12a-Based Detection of Haemophilus parasuis

Author:

Zhang Kunli1ORCID,Sun Zeyi12,Shi Keda1,Yang Dongxia1,Bian Zhibiao1,Li Yan1,Gou Hongchao1,Jiang Zhiyong1,Yang Nanling1,Chu Pinpin1,Zhai Shaolun1ORCID,Wei Zhanyong2,Li Chunling1

Affiliation:

1. Institute of Animal Health, Guangdong Academy of Agricultural Sciences, Key Laboratory of Livestock Disease Prevention of Guangdong Province, Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province, Ministry of Agriculture and Rural Affairs, Guangzhou 510640, China

2. College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, China

Abstract

Haemophilus parasuis (H. parasuis, HPS) is a prominent pathogenic bacterium in pig production. Its infection leads to widespread fibrinous inflammation in various pig tissues and organs, often in conjunction with various respiratory virus infections, and leads to substantial economic losses in the pig industry. Therefore, the rapid diagnosis of this pathogen is of utmost importance. In this study, we used recombinase polymerase amplification (RPA) and clustered regularly interspaced short palindromic repeats (CRISPR) technology to establish a convenient detection and analysis system for H. parasuis that is fast to detect, easy to implement, and accurate to analyze, known as RPA-CRISPR/Cas12a analysis. The process from sample to results can be completed within 1 h with high sensitivity (0.163 pg/μL of DNA template, p < 0.05), which is 104 -fold higher than the common PCR method. The specificity test results show that the RPA-CRISPR/Cas12a analysis of H. parasuis did not react with other common pig pathogens, including Streptococcus suis type II and IX, Actinobacillus pleuropneumoniae, Escherichia coli, Salmonella, Streptococcus suis, and Staphylococcus aureus (p < 0.0001). The RPA-CRISPR/Cas12a assay was applied to 15 serotypes of H. parasuis clinical samples through crude extraction of nucleic acid by boiling method, and all of the samples were successfully identified. It greatly reduces the time and cost of nucleic acid extraction. Moreover, the method allows results to be visualized with blue light. The accurate and convenient detection method could be incorporated into a portable format as point-of-care (POC) diagnostics detection for H. parasuis at the field level.

Funder

National Natural Science Foundation of China

Guangdong Academy of Agricultural Sciences

Guangzhou Science and Technology Bureau

Department of Agriculture and Rural Affairs of Guangdong Province

Special Fund for Scientific Innovation Strategy—Construction of High-Level Academy of Agriculture Science

Publisher

MDPI AG

Subject

General Veterinary,Animal Science and Zoology

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