Molecular Design Real Time Loop-Mediated Isothermal Amplification Method for Rapid Detection of Neisseria Meningitidis; [Метод Молекулярного Конструирования Петлевой Изотермической Амплификации В Реальном Времени Для Быстрого Обнаружения Neisseria Meningitidis] Publisher Pubmed



Soleimani M¹ ; Morovvati A² ; Majidzadeha K³
Source: Folia Medica Published:2021

Abstract

Introduction: Detecting Neisseria meningitides using conventional methods is time consuming and laborious. Development of a reliable, 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. Loop-mediated isothermal amplification (LAMP) method is a simple gene amplification method which can be performed at a single temperature without the need for thermal cycling. Aim: 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. Materials and methods: LAMP reaction was set up and optimized by four primers. Amplification results were assessed by obtaining real time turbidity graphs from each LAMP reaction tube using real time turbidimeter apparatus. A standard curve was generated from turbidity graphs corresponding to ten-fold serial dilution of crgA gene containing recombinant plasmid. Results: LAMP assay could isolate only N. meningitides, whereas no amplification was obtained with negative control isolates, which indicates 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 correlation coefficient equal to 0.92. Conclusions: The REAL LAMP assay is a rapid, simple, cost-effective, sensitive, specific method for detection of N. meningitides. It has the potential of finding application in epidemiological studies and biodefense situations. © Folia Medica.