Immunocapture Magnetic Beads Enhanced the LAMP-CRISPR/Cas12a Method for the Sensitive, Specific, and Visual Detection of Campylobacter jejuni

Abstract

Campylobacter jejuni is one of the most important causes of food-borne infectious disease, and poses challenges to food safety and public health. Establishing a rapid, accurate, sensitive, and simple detection method for C. jejuni enables early diagnosis, early intervention, and prevention of pathogen transmission. In this study, an immunocapture magnetic bead (ICB)-enhanced loop-mediated isothermal amplification (LAMP) CRISPR/Cas12a method (ICB-LAMP-CRISPR/Cas12a) was developed for the rapid and visual detection of C. jejuni. Using the ICB-LAMP-CRISPR/Cas12a method, C. jejuni was first captured by ICB, and the bacterial genomic DNA was then released by heating and used in the LAMP reaction. After the LAMP reaction, LAMP products were mixed and detected by the CRISPR/Cas12a cleavage mixture. This ICB-LAMP-CRISPR/Cas12a method could detect a minimum of 8 CFU/mL of C. jejuni within 70 min. Additionally, the method was performed in a closed tube in addition to ICB capture, which eliminates the need to separate preamplification and transfer of amplified products to avoid aerosol pollution. The ICB-LAMP-CRISPR/Cas12a method was further validated by testing 31 C. jejuni-positive fecal samples from different layer farms. This method is an all-in-one, simple, rapid, ultrasensitive, ultraspecific, visual detection method for instrument-free diagnosis of C. jejuni, and has wide application potential in future work.

Keywords: Campylobacter jejuni; ICB-LAMP-CRISPR/Cas12a; food-borne pathogens; point of care; visual detection.

Figure 1

Design and working principle of the ICB-LAMP-CRISPR/Cas12a method. (A) Schematic of the ICB-LAMP-CRISPR/Cas12a method. Campylobacter jejuni was captured by the prepared ICB and separated magnetically. Five microliters of template DNA of C. jejuni was added to the LAMP mixture, which was placed at the bottom of the tube and sealed with 20 μL of mineral oil. The CRISPR/Cas12a reaction reagents are added inside the lid. After 30 min of LAMP amplification at 65 °C, the tube was shaken to mix with Cas12a reagents for cleavage. Once the Cas12a nuclease is activated by recognizing the DNA target, it splits the quenched fluorescent ssDNA-FQ probe indiscriminately, generating a fluorescence signal visible to the naked eye under blue light. (B) Enhanced sensitivity of the ICB-LAMP-CRISPR/Cas12a method. The sensitivity was enhanced in three parts: the enrichment of ICB, the high efficiency of LAMP amplification, and the indiscriminate cleavage of the fluorescent ssDNA-FQ probe. (C) Enhanced specificity of the ICB-LAMP-CRISPR/Cas12a method. The specificity was enhanced from three parts: the specific antibodies of C. jejuni coated in the magnetic beads, the LAMP primers designed based on the conserved hipO gene, and the cleavage activity of Cas12a guide by the specific sgRNA. (D) Work conditions of the nearly instrument-free POC diagnostics. Equipment and consumables needed for running the ICB-LAMP-CRISPR/Cas12a method include a heat block, pipettes, pipette tips, sample tubes, and T-green transilluminator.

Figure 2

Construction of the ICB-LAMP reaction system. (A) Capture time evaluation of ICB. One milliliter of C. jejuni (8 × 10³ CFU/mL) was mixed with 10 μL of ICB, and the optical density (OD) in the supernatant was measured every 10 min. No significant difference was observed after 20 min of incubation. (B) LAMP primer selection. Five groups of LAMP primers based on the hipO gene were obtained and used in the LAMP reaction, and primer 2 had the best amplification effect. (C) LAMP primers and sgRNA binding sites.

Figure 3

sgRNA screening. (A) Endpoint CRISPR/Cas12a for sgRNA screening. Six sgRNAs were designed based on the target DNA and used in CRISPR/Cas12a at 37 °C for 30 min, and sgRNA 4 worked well in the standard program, with strong fluorescent light under LED blue light. (B) Real-time CRISPR/Cas12a for sgRNA screening. The real-time monitoring was conducted in a CFX96 Touch Real-Time PCR Detection System for 30 min, and the endpoint fluorescence intensity and time with a fluorescence threshold of 1000 were monitored. Each experiment was repeated three times with similar results.

Figure 4

Evaluation of eight CRISPR/Cas12a reactions (R) with various components through endpoint imaging after 30 min of incubation and real-time fluorescence detection. The LAMP products, Cas12a, sgRNA, and the ssDNA-FQ reporter were tested. After incubation at 37 °C for 30 min, only reaction 4, containing the target nucleic acid sequence, sgRNA, Cas12a, and the ssDNA-FQ reporter, produced a superbright fluorescence signal under LED blue light. The real-time monitoring was conducted in a CFX96 Touch Real-Time PCR Detection System for 30 min, and the endpoint fluorescence intensity and time with a fluorescence threshold of 1000 were monitored.

Figure 5

Specificity evaluation ICB-LAMP-CRISPR/Cas12a method. The specificity of ICB-LAMP-CRISPR/Cas12a was compared with PCR, qPCR, and LAMP. C. jejuni NCTC 11168 and 6 non-C. jejuni (C. coli, E. coli, S. flexneri, K. pneumoniae, P. mirabilis, and S. enteritidis) were used as the tested sample. The specificity evaluation results of PCR and LAMP were shown by agarose gel electrophoresis. The specificity evaluation results of qPCR and ICB-LAMP-CRISPR/Cas12a are shown by the real-time amplification curve and end-point fluorescence. (A) The specificity evaluation of PCR. (B) The specificity evaluation of qPCR. (C) The specificity evaluation of LAMP. (D) The specificity evaluation of the ICB-LAMP-CRISPR/Cas12a method.

Figure 6

Sensitivity evaluation ICB-LAMP-CRISPR/Cas12a. The sensitivity of ICB-LAMP-CRISPR/Cas12a was compared with- that of PCR, qPCR, LAMP, and ICB-LAMP. Serial tenfold dilutions of C. jejuni (8 × 10⁰–8 × 10¹⁰ CFU/mL) were used for the sensitivity evaluation. The sensitivity evaluation results of PCR, LAMP, and ICB-LAMP were shown by agarose gel electrophoresis. The sensitivity evaluation results of qPCR and ICB-LAMP-CRISPR/Cas12a are shown by the real-time amplification curve and end-point fluorescence. (A) The sensitivity evaluation of PCR. (B) The sensitivity evaluation of qPCR. (C) The sensitivity evaluation of LAMP. (D) The sensitivity evaluation of ICB-LAMP. (E) The sensitivity evaluation of ICB-LAMP-CRISPR/Cas12a.

Figure 7

The time evaluation ICB-LAMP-CRISPR/Cas12a. A total of 1 mL 8 × 10³ CFU/mL C. jejuni was detected by the ICB-LAMP-CRISPR/Cas12a method, and monitored by a CFX96 Touch Real-Time PCR Detection System and real-time photograph detection for 30 min.

Figure 8

Evaluation of ICB-LAMP-CRISPR/Cas12a in C. jejuni isolates. Thirty-one C. jejuni-positive fecal samples (n = 31) were used in the actual sample evaluation of ICB-LAMP-CRISPR/Cas12a.