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Updated: Jul 31, 2026
Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1
Published on: March 13, 2018
Molecular design real time loop-mediated isothermal amplification method for rapid detection of Neisseria
Mohammad Soleimani1, Abbas Morovvati2, Keyvan Majidzadeh-A1
1AJA University of Medical Sciences, Tehran, Iran.
Introduction:
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.
Objective:
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.
Material And Methods:
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.
Results:
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.
Insights
A new real-time LAMP assay rapidly and accurately detects Neisseria meningitides. This method offers a specific and sensitive alternative for diagnosing meningococcal disease, improving patient outcomes.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Disease Detection
Background:
- Conventional Neisseria meningitides detection is slow and labor-intensive.
- Rapid diagnostics are crucial for controlling meningococcal disease outbreaks.
- Existing PCR methods require specialized equipment, limiting accessibility.
Purpose of the Study:
- Develop a quantitative real-time LAMP assay for Neisseria meningitides detection.
- Accurately quantify bacterial load in patients with meningococcal disease.
Main Methods:
- Optimized LAMP assay with four specific primers.
- Assessed amplification using real-time turbidity graphs and a turbidimeter.
- Generated a standard curve from serial dilutions of the crgA gene.
Main Results:
- The LAMP assay demonstrated 100% specificity, detecting only N. meningitides.
- Achieved a limit of detection of approximately 5 copies of the crgA gene per reaction.
- Showed excellent linear correlation (R²=0.92) between gene copy number and time threshold.
Conclusions:
- The developed real-time LAMP assay is a specific and sensitive tool for N. meningitides detection.
- This method allows for accurate quantification of bacterial load.
- LAMP offers a rapid, cost-effective alternative to conventional and PCR-based methods for meningococcal disease diagnosis.
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