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

Abstract

Background: 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.