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.
Highlights
Campylobacter jejuni (C. jejuni) is a foodborne bacterial pathogen that causes gastroenteritis in humans [1]
We report a method for the detection of C. jejuni that integrates immunocapture magnetic beads (ICB), loopmediated isothermal amplification (LAMP), and CRISPR/Cas12a (ICB-LAMP-CRISPR/Cas12a) to enhance the sensitivity and specificity of this method
Because the NHS-magnetic beads were coated with C. jejuni polyclonal antibody (Figure 1A), C. jejuni was captured by ICB and resuspended in 5 μL of ddH2 O, and the template
Summary
Campylobacter jejuni (C. jejuni) is a foodborne bacterial pathogen that causes gastroenteritis in humans [1]. C. jejuni is widely distributed in food animal species, especially chickens, and it is transmitted to humans primarily through the foodborne route [2,3]. C. jejuni infection generally causes self-limited diarrhea in humans; it may induce severe or systemic infections in immunocompromised or young and elderly patients [3]. In the U.S, it is estimated that Campylobacter is responsible for more than 1.3 million cases of illnesses each year [4]. In England, the total cost of Campylobacter species infection was estimated to be 116 million dollars each year [5]. Rapid and accurate detection of foodborne pathogens, such as C. jejuni, could help to improve public health and increase food safety
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