Real-Time Fluorescence Loop Mediated Isothermal Amplification for the Diagnosis of Malaria

Abstract

BackgroundMolecular diagnostic methods can complement existing tools to improve the diagnosis of malaria. However, they require good laboratory infrastructure thereby restricting their use to reference laboratories and research studies. Therefore, adopting molecular tools for routine use in malaria endemic countries will require simpler molecular platforms. The recently developed loop-mediated isothermal amplification (LAMP) method is relatively simple and can be improved for better use in endemic countries. In this study, we attempted to improve this method for malaria diagnosis by using a simple and portable device capable of performing both the amplification and detection (by fluorescence) of LAMP in one platform. We refer to this as the RealAmp method.Methodology and Significant FindingsPublished genus-specific primers were used to test the utility of this method. DNA derived from different species of malaria parasites was used for the initial characterization. Clinical samples of P. falciparum were used to determine the sensitivity and specificity of this system compared to microscopy and a nested PCR method. Additionally, directly boiled parasite preparations were compared with a conventional DNA isolation method. The RealAmp method was found to be simple and allowed real-time detection of DNA amplification. The time to amplification varied but was generally less than 60 minutes. All human-infecting Plasmodium species were detected. The sensitivity and specificity of RealAmp in detecting P. falciparum was 96.7% and 91.7% respectively, compared to microscopy and 98.9% and 100% respectively, compared to a standard nested PCR method. In addition, this method consistently detected P. falciparum from directly boiled blood samples.ConclusionThis RealAmp method has great potential as a field usable molecular tool for diagnosis of malaria. This tool can provide an alternative to conventional PCR based diagnostic methods for field use in clinical and operational programs.

A Novel, Sensitive Assay for High-Throughput Molecular Detection of Plasmodia for Active Screening of Malaria for Elimination

“…Although real-time PCR and LAMP were recently reported to be able to detect plasmodia directly from whole blood (17,31), the sensitivities were compromised compared with those obtained using purified DNA. For example, the direct qPCR missed 5 positive samples out of 74 compared with conventional qPCR (31), and the detection limit of LAMP, when used directly on heated blood instead of DNA, changed from 1 to 10 parasites/l to 40 parasites/l (17), a sensitivity not significantly better than that of microscopy or RDT.…”

“…In a recent malaria survey in an elimination setting, RDT missed the majority of Plasmodium vivax infections while giving a number of false-positive results for P. falciparum infection (4), indicating that RDT is not appropriate for general malaria screening. Molecular methods, such as PCR (13), quantitative PCR (qPCR) (14), reverse transcription-PCR (15), nucleic acid sequence-based amplification (NASBA) (16), and loop-mediated isothermal amplification (LAMP) (17), are more sensitive than microscopy and RDT, yet their dependence on DNA/RNA extraction can strongly influence the performance of the diagnosis (14,16,17), and in surveillance settings, DNA/RNA extraction can be labor-intensive to perform on large numbers of samples. Importantly, when performed in parallel on 96-well plates, the target amplification nature of these methods, even those with closed systems such as real-time PCR, requires a very high standard of laboratory practice to avoid contamination that leads to falsepositive results, restricting their application to well-equipped laboratories with specially trained technicians (18).…”

Real-time DNA Amplification and Detection System Based on a CMOS Image Sensor

“…2,3 Even though PCR is an efficient gene-amplification system, the expensive device, requirement of a trained person, long turnaround times, non portability, and difficulties in the quantification of end products are considered to be limitations of the real-time diagnosis field. 4 As a result, real-time PCR and quantitative PCR systems have been developed to both amplify and detect a DNA simultaneously. 5 Therein, the real-time gene-amplification system has transformed the life-science research and molecular diagnostics fields to the next level, performed as a rapid detection, minimal amount quantification with real-time amplification.…”

Centrifugal LabTube platform for fully automated DNA purification and LAMP amplification based on an integrated, low-cost heating system

“…nanotechnology-based sensors (Zhang et al 2005) or microfluidic systems, such as droplet -based , Chiou et al 2013, centrifugal (Cho et al 2007), paper (Martinez et al 2009, Fu et al 2010, Zuo et al 2013, capillary (T akayama et al 1999, Yager et al 2008, Gervais and Delamarche 2009, pneumatic (Legendre et al 2006, Mohan et al 2013 devices and lateral flow assays (Yager et al 2008, Chen et al 2010, You et al 2011). For the past decade, this has led to numerous publications about promising, early-stage technologies (Baeumner et al 2003, Leonard et al 2003, Kost 2006, Lazcka et al 2007, Lucchi et al 2010, Asiello and Baeumner 2011, Duarte et al 2011, Karlsson et al 2011, Schudel et al 2011, Matlock-Colangelo and Baeumner 2012, Lounsbury et al 2013, Mohan et al 2013. Despite some commercially available systems (e.g.…”