Development of a multiple cross displacement amplification combined with nanoparticles-based biosensor assay to detect Neisseria meningitidis

Abstract

Development of a multiple cross displacement amplification combined with nanoparticles-based biosensor assay to detect Neisseria meningitidis Shijun Li*,1, Chunting Liu*,1, Ying Liu,1 Qing Ma,1 Yue Wang,1 Yi Wang2,31Laboratory of Bacterial Infectious Disease of Experimental Center, Guizhou Provincial Center for Disease Control and Prevention, Guiyang, Guizhou, 550004, People’s Republic of China; 2Key Laboratory of Major Diseases in Children, Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, 10045, People’s Republic of China; 3Ministry of Education, National Key Discipline of Pediatrics (Capital Medial University), Beijing Pediatric Research Institute, Beijing Children’s Hospital, Capital Medical University, National Center for Children’s Health, Beijing, 10045, People’s Republic of China*These authors contributed equally to this workBackground: Neisseria meningitidis is a leading pathogen of meningococcal disease in humans worldwide. Multiple cross displacement mplification (MCDA) combined with nanoparticles-based lateral flow biosensor (MCDA-LFB) has been reported for the rapid detection of several bacterial pathogens in recent years. Here, therefore we developed an MCDA-LFB assay for the rapid detection of N. meningitis.Methods: A set of 10 primers specifically to recognize 10 different regions of the ctrA gene of N. meningitidis were designed. MCDA was developed and combined with a LFB to detect the ctrA gene of N. meningitidis. The reaction time and temperature condition for the MCDA-LFB were optimized and then the MCDA-LFB was applied to detect the DNA from clinical samples.Results: MCDA-LFB assay was successfully established for the detection of N. meningitidis based on the ctrA gene. The MCDA assay was optimized at 64°C for only 35 mins and the products of amplification were directly sensed by LFB. The whole operation, including DNA template preparation (∼20 mins), MCDA reaction (35 mins) and results interpretation (∼2 mins) could be finished in no more than 60 mins. The detection limit was as low as 10 fg/reaction (around 3 CFUs/reaction) of pure N. meningitidis DNA, with no cross-reaction with other bacterial DNA.Conclusion: The MCDA-LFB techniques developed in the present study are an effective tool for the rapid detection of N. meningitidis, especially in resource-poor countries in meningococcal disease epidemic period.Keywords: Neisseria meningitidis, multiple cross displacement amplification, lateral flow biosensor, MCDA-LFB, limit of detection