Sample-ready multiplex qPCR assay for detection of malaria.
BACKGROUND
Microscopy 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.


METHODS
A 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.


RESULTS
The 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.


CONCLUSIONS
The 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.
Walter Reed Army Institute of Research, Military Malaria Research Program, Malaria Vaccine Branch, 503 Robert Grant Ave, Silver Spring, Maryland, USA. edwin.kamau@us.army.mil.
Edwin
E
Kamau
Saba
S
Alemayehu
Karla C
KC
Feghali
Dennis W
DW
Juma
George M
GM
Blackstone
William R
WR
Marion
Peter
P
Obare
Bernhards
B
Ogutu
Christian F
CF
Ockenhouse
D001769:Blood / Q000469:parasitology; D060085:Coinfection / Q000175:diagnosis / Q000469:parasitology; D005612:Freeze Drying / Q000379:methods; D006801:Humans; D008288:Malaria / Q000175:diagnosis* / Q000469:parasitology; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010274:Parasitology / Q000379:methods*; D010961:Plasmodium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013696:Temperature