RPA-CRISPR/Cas12a-Based Detection of Haemophilus parasuis

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.

Keywords: CRISPR/Cas12a; Haemophilus parasuis; RPA; detection.

Figure 1

An RPA-CRISPR-Cas12a platform for the detection of H. parasuis. (A) Schematic illustration of the RPA. (B) Incubation of the amplified products (A) and Cas12 proteins to form a DNA-Cas12 complex under 25 °C for 10 min. Protospacer adjacent motif (PAM) sites guide the CRISPR/Cas12a-crRNA complex to recognize target sites to activate Cas12a effectors. (C) The crRNA recognized and bound the target sequences to activate the Cas12a protein. The activated Cas12a protein nonspecifically cleaves single-stranded DNA reporter molecules by trans-cleavage. The positive sample produces fluorescence and is detected by blue light.

Figure 2

Optimization results of primer pairs and crRNA in the reaction system. (A) HPS ompP2 genome map showing primers and crRNAs. (B) Fluorescent results of 1# crRNA combined with 4 primer pairs (F1R1, F2R1, F3R3, and F3R4). (C) Fluorescent results of 2# crRNA combined with 3 primer pairs (F3R3, F3R4, and F4R4). Mean ± SD for 3 technical replicates for (B,C).

Figure 3

Optimization results of concentrations of ssDNA reporter, Cas12a and crRNA in the reaction system. (A) The concentrations of the ssDNA reporter molecule were optimized at 450 nmol/L, 400 nmol/L, 350 nmol/L, 300 nmol/L, 250 nmol/L, and 200 nmol/L in the CRISPR-Cas12a reaction mixture containing 1 μL Cas12a (1 μmol/L), 1 μL crRNA (1 μmol/L), and 3 μL 10 × NEB buffer 2.1. The results were observed under blue light. (B) The optimal shear efficiency concentration was determined by an orthogonal test with the different concentrations of Cas12a (100 nmol/L, 50 nmol/L, 30 nmol/L, and 15 nmol/L) and crRNA (100 nmol/L, 50 nmol/L, and 30 nmol/L), respectively. The concentration of Cas12a and crRNA was optimized with the optimal ssDNA concentration as the test concentration. The optimal reaction conditions were selected by observing the fluorescence intensity.

Figure 4

Specificity analysis. (A) Specificity assessment of the RPA-CRISPR/Cas12a-assay for the ompP2 gene of H. parasuis. Only the DNA from H. parasuis produced fluorescence signals, whereas DNAs from other pathogens and the negative control did not elicit discernible fluorescence signals. Seven swine bacterial pathogens, including Streptococcus suis type II and IX (SS-2, SS-9), Actinobacillus pleuropneumoniae (APP), Escherichia coli (E. coli), Salmonella, Staphylococcus aureus (S. aureus), and Erysipelothrix rhusiopathiae (E. rhusiopathiae), were evaluated. (B) Fluorescent results of different swine bacterial pathogens detected by the RPA-Cas12a assay were collected at 30 min for the Cas12a reaction. Three replicates were conducted for each test. Fluorescence intensity values are shown in the graph as mean ± SD. (N: negative control, ddH₂O; **** p < 0.0001).

Figure 5

Sensitivity analysis. (A) Serial dilutions of HPS5 DNA for RPA-Cas12a limit of detection determination. The RPA product (3 μL) was added to 30 μL of the CRISPR-Cas12a reaction mixture for cleavage assays. The reaction results were observed in the fluorescence intensity under blue light. (B) The fluorescence intensity of each sample was collected at 30 min for the Cas12a reaction. Bar graphs represent fluorescent signals for the Cas12a reaction from A. Three replicates were conducted for each test. Fluorescence intensity values are shown in the graph as mean ± SD. (N: negative control, ddH₂O; * p < 0.05, *** p < 0.001, **** p < 0.0001). (C) The serial dilutions of HPS5 DNA were amplified with specific primer sets in a classical PCR reaction. The amplification results were detected by agarose gel electrophoresis.

Figure 6

Detection of HPS clinical isolation strain. (A) The serotypes 1/2/3/4/5/6/7/8/9/10/11/12/13/14/15 of H. parasuis (HPS) were detected by the RPA-CRISPR/Cas12a assay. (B) Bar graphs representing fluorescent signals detected by the RPA-Cas12a assay were collected at 30 min for the Cas12a reaction. Fluorescence intensity values are shown in the graph as mean ± SD for 3 technical replicates.