Sample-ready multiplex qPCR assay for detection of malaria

Abstract

BackgroundMicroscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization.MethodsA recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, “wet” assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance.ResultsThe limit of detection for the MMSR assay was 0.244 parasites/μL for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to “wet” assay which was 0.39 and 3.13 parasites/μL for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the “wet” assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the “wet” assay.ConclusionThe MMSR assay has the same robust performance characteristics as the “wet” assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget.

Use of real-time multiplex PCR, malaria rapid diagnostic test and microscopy to investigate the prevalence of Plasmodium species among febrile hospital patients in Sierra Leone

“…This study explored the utility of a room temperaturestable, pre-aliquoted molecular assay for detection of malaria in a remote, low-resource location in West Africa. The MMSR PCR test was developed to detect multiple Plasmodium species and to identify the two predominant species responsible for human malaria, P. falciparum and P. vivax [1,22]. The assay uses Sample-Ready ™ format in which all necessary components (primers, probes, Taq polymerase and the reaction buffer) are provided as a freeze-dried pellet in a reaction tube compatible with most 0.1 mL real time PCR cycler blocks.…”

“…The MMSR assay used in this study is only one of many NAATs developed specifically for malaria detection but has several unique advantages. While costly (14.50 USD each), it offers wide coverage (detection of all and identification of two most prevalent Plasmodium species in a single reaction) and capability of measurement of the parasite density with room temperature stability and simple setup requiring just one pipetting [21,22]. Most homebrew and commercially available PCR-based assays with comparable features (e.g.…”

“…For PCR-based detection, DNA extracted from venous blood (QIAamp DNA Mini Kit, Qiagen, Germantown, MD, USA) was analysed using MMSR (BioGX, Birmingham, AL, USA), a TaqMan-based real-time PCR assay. Developed and validated by researchers at Walter Reed Army Institute of Research (WRAIR) [21,22], MMSR is designed to detect four targets in a single assay: P. falciparum, P. vivax, Plasmodium spp. (universal malaria target) and RNaseP (sample extraction control).…”

Protection against Plasmodium falciparum malaria by PfSPZ Vaccine

“…AEs and TBSs were assessed at least daily from day 7 to day 18 after CHMI, and then again on days 20, 22, 25, and 28 after initiation of CHMI or until detection of parasitemia as previously described (13). qPCR was done retrospectively (see supplemental materials for specifics) (29,30). Clinical laboratory tests were also monitored.…”

Peer Review #1 of "Genome-Enhanced Detection and Identification (GEDI) of plant pathogens (v0.1)"

“…The conceptual and practical simplicity of qPCR, in combination with its speed and sensitivity, have made it the technology of choice in many diagnostic applications, including microbial quantification (Yu et al, 2005;Narihiro and Sekiguchi, 2011;Thonar et al, 2012) and pathogen detection (Orlofsky et al, 2015). Multiple qPCR assays have become extremely powerful to detect fungi, bacteria and parasites (Kamau et al, 2014;Gosiewski et al, 2014 and we believe that this pipeline should enable development of qPCR assays in crop and tree pathogens. The pipeline described herein could be easily applied to any DNA-based detection methods (Yeo and Wong, 2002), such as loop-mediated isothermal amplification (LAMP), hybridization-based microarray (Huang et al, 2006) or PCR-ELISA (Löffler et al, 1998).…”