Four different Plasmodium
species with different clinical implications infect humans in different combinations around the world, with recent studies demonstrating that P. knowlesi
is also capable of causing human malaria due to zoonotic transmission 
. Approximately 225 million cases of malaria and 781,000 deaths due to malaria occurred in 2009 
. Due to the apparent decline in the number of malaria cases and deaths in the last few years, there is a concerted global effort to control and eliminate malaria with the support of many public and private initiatives (reviewed in 
). Therefore, there is a clear need for diagnostic tools that are robust enough to accurately detect the species of infecting parasite(s), to identify the transmission foci of malaria reservoirs (often which may be submicroscopic and asymptomatic) and to monitor the success of malaria control and elimination programs.
The existing tools for malaria diagnosis include microscopy, parasite antigen/enzyme detection kits [commonly referred to as rapid diagnostic tests (RDTs)] and molecular tools (nucleic acid based tools). Microscopy remains the gold standard for the diagnosis of malaria in many malaria endemic countries. Although microscopy is relatively inexpensive and can be used to differentiate parasite species as well as provide quantitative data on the level of parasitemia, several limitations including poor sensitivity 
begs for novel methods in the elimination era. RDTs have become an alternative tool for malaria diagnosis. While some RDTs are Plasmodium
-specific, (pan) detecting the genus-specific aldolase and lactate dehydrogenase enzymes, the majority of the RDTs are specific for the P. falciparum
histidine-rich protein –2 (Pf HRP-2). The recent discovery that some P. falciparum
parasites in parts of South America and Africa have deleted the hrp-2
has brought into question the use of the HRP-2 based RDT tests due to potential false negatives. Another drawback of RDTs is the fact that they cannot be used to determine parasite densities and such quantification is increasingly essential with respect to both, antimalarial drug resistance surveillance and malaria control programs. With limits of detection at ~100 parasites/µl (reviewed in 
), RDTs can only be useful for routine diagnosis and treatment. With the limitations of both microscopy and RDTs, there is a clear realization that more sensitive diagnostic tools are needed for the detection of subclinical but transmissible infections to guide and monitor malaria elimination programs 
Molecular tools are far more sensitive in detecting low level infections and accurately detecting species of malaria parasite (reviewed in 
). There are many versions of molecular tools for malaria diagnosis ranging from conventional PCR-based assays, real-time PCR assays and more recently, isothermal amplification assays (reviewed in 
). When considering the development of tools for large scale field application, it is desirable to consider cost, robustness, and ease of use. The real-time PCR platform has some advantages for large scale screening as it requires no major post-PCR steps to acquire the results and a large number of samples can be analyzed at one time.
Real-time PCR assays depend on the use of fluorophores or DNA intercalating fluorescent dyes. As in the cases of TaqMan probes, molecular beacons and scorpion, sequence-specific oligonucleotide probes are dual labeled with a fluorescent dye and quencher. Although these molecular methods are robust and simpler than conventional PCR methods for large scale screening, the reagents are expensive and require special handling, which then creates practical challenges for field use in malaria endemic countries. A less complicated real-time PCR technique is the use of non-specific DNA intercalating dyes such as SYBR Green, SYTO-9, and calcein, which emit fluorescence signals when bound to double stranded DNA. Another drawback for these non-specific intercalating dyes is that they do not allow for multiplexing. Other alternatives for the detection of real-time PCR assays have been described including the direct labeling of one of the primers (forward or reverse) with a single fluorophore in a manner that facilitates self-quenching without the need of a quencher. These self-quenching primers facilitate the use of real-time PCR without the need for internal dual-labeled sequence specific probes. An example of this is the Light Upon Extension (LUX) primer that is labeled with an internal single fluorophore near the 3′ end of the primer in a hairpin structure which allows for the quenching before amplification 
. These self-quenching, single labeled primers are less expensive compared to the dual labeled probe. However, the design of LUX primers requires the specific software, LUX Designer.
Here we describe a novel method for labeling real-time PCR primers based on the principle that was recently described in a patent filed by Jothikumar et al. (patent # PCT/US2008/084347). In this simplified technique, the 5′ end of one of the primers is modified by the addition of a 17-bases oligonucleotide tail that is labeled with a fluorophore (HEX or FAM) on its 5′ end, (). In the absence of amplification, this tail forms a loop and remains in a closed conformation resulting in effective quenching of fluorescence due to close proximity of four G bases (i.e., the two overhang GG and two complementary GG residues in the hairpin formation) via photo-induced electron transfer (PET) mechanism; hence the name PET-PCR (not to be confused with the PET fluorescence dye from ABI). When participating in nucleic acid amplification, the stem loop structure of the PET primer opens up. The subsequent fluorescence increase is due to the de-quenching effect of the two guanosine residues located in the overhang positions and also due to the formation of the complementary DNA strand.
To evaluate this concept, we designed PET-PCR primers for the specific amplification of the Plasmodium genus and P. falciparum species. We demonstrate the utility of this assay using 119 clinical samples of different malaria species infections, including mixed infections, and using well quantified Plasmodium species to estimate the limits of detection of this assay.