Molecular design real time loop-mediated isothermal amplification method for rapid detection of Neisseria meningitidis.

Abstract

Detection of Neisseria meningitides by using conventional methods is time consuming and laborious. Development of a realiable and rapid method for prompt control and prevention of meningococcal disease is required. Although PCR and real time PCR methods have been developed; they require electrophoresis or expensive Devices. LAMP is a simple gene amplification method which can be performed at a single temperature without the need for thermal cycling. We aimed to develop a quantitative real-time LAMP assay for detection of N. meningitides and accurate quantification of the bacterial load in patients with meningococcal disease. the LAMP reaction was set up and optimized by four primers were designed. Amplification results were assessed by obtained real time turbidity graphs from each LAMP reaction tube using real time turbidimeter apparatus. Standard curve was generated from turbidity graphs corresponding to ten-fold serial dilution of crgA gene containing recombinant plasmid. by LAMP assay just N. meningitides isolated, whereas no amplification was obtained with negative control isolates, and this indicating 100% specificity. The limit of detection (LOD) of our LAMP assay was found to be ~ 5 copies of crgA gene per reaction. REAL LAMP Analysis of the standard curve revealed excellent linear correlation between gene copy number and time threshold. With a coefficient correlation equal to 0.92.