Isothermal amplification of nucleic acids for rapid and multiplexed detection of antibiotic-resistance in Campylobacter jejuni

Campylobacter jejuni is a food-borne pathogen commonly found in poultry industry, and has been the main cause for gastroenteritis, incurring high medical cost and childhood morbidity worldwide. Due to the heavy use of antibiotics in the past, antibiotic-resistance in Campylobacter jejuni has been developed in many countries such as Spain, United States, Egypt, Hong Kong etc. In 2017, WHO has categorised antibiotic-resistant Campylobacter as high priority pathogens list. It is important to detect the emergence of antibiotic-resistance in countries where the resistance have yet developed. In addition, this would help better prescription by medical practitioner prior to treatment. In this study, recombinase polymerase amplification was developed to detect C. jejuni, and to identify if the pathogen is resistant to antibiotics. Particular interest was initially placed in the amplification of tetracycline-resistant (TetO) and ciprofloxacin-resistant (gyrA) genes in C. jejuni clinical isolates obtained in Spain. The results show that the amplification can be carried out in 10 min under isothermal condition below 40 °C, and optimisation of multiplex amplification, sensitivity and specificity test against other bacterial strains such as E. coli and L. monocytogenes were carried out. Future work includes the transfer of the assay onto a paper-based analytical device that results in a portable device that is suitable for low resource settings.