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CDC Scientists Develop 'PET-PCR' Fluorogenic Primers to Enable Inexpensive Field-Based Malaria MDx

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This article has been updated from a previous version to correct the name and title of an interview subject.

Scientists from the US Centers for Disease Control and Prevention have developed a new primer technology that, when used in real-time PCR assays, obviates the need for internal probes or intercalating dyes and makes them easier and cheaper to use than existing real-time PCR diagnostic chemistries.

The researchers, primarily from CDC's Division of Parasitic Diseases and Malaria, have also used the primers in a real-time PCR assay to specifically detect Plasmodium falciparum, the most common malaria-causing parasite; and generally detect a number of other Plasmodium species with 100 percent sensitivity and specificity compared to a gold standard of nested PCR.

In addition, having successfully tested the assay in the CDC's laboratories in Atlanta, the researchers said this week that they have begun field testing the diagnostic in Haiti as part of that country's national malaria surveillance program.

The CDC researchers developed the new chemistry, called self-quenching photo-induced electron transfer, or PET, fluorogenic primers, because of shortcomings they perceived in existing real-time PCR-based diagnostic tools for use in resource-poor areas of the world, where malaria prevalence is high.

"There are several [molecular testing] options out there, and a lot of these, at least the real-time [PCR] platforms, are in some of our field stations in developing countries where malaria [is problematic]," CDC's Naomi Lucchi, microbiologist and associate fellow at CDC, and one of the technology's developers, told PCR Insider this week.

Of these existing technologies, TaqMan-based real-time PCR is most commonly used, but the CDC researchers experienced "a lot of challenges" using the technology in resource-poor settings or in the field, Lucchi said.

"One challenge is just the probes themselves," she said. "The probes are pretty expensive; the cold chain involved with them — you have to keep them at minus-20 [degrees Celsius] — and just the whole design of the TaqMan probes was a little challenging for us, even in labs that were sufficiently capable to do this."

Kumar Udhayakumar, microbiologist, chief of the genetics and immunology laboratory of the Laboratory Research and Development Unit in CDC’s malaria branch, and a co-developer of the technology, noted that "anybody who has worked in a laboratory condition in the developing world … can tell you that with the conditions in … even a well-equipped laboratory … the challenge is always getting the reagents."

Udhayakumar added that the impetus to develop the new primers "grew out of our own frustration with experiments in the field, even in a highly sophisticated lab … in Kenya. When we started doing these experiments, we needed to send the reagents from [Atlanta]. It's a huge logistical challenge … so we decided to focus on ways to make reagents that would work in these conditions. The focus in our laboratory is developing the kinds of technology that are more relevant for the field."

The PET-PCR primers, which Lucchi, Udhayakumar, and colleagues describe in a paper published last month in PLOS One, use a modified method for labeling existing real-time PCR primers.

The method involves modifying the 5' end of one of the primers with a 17-base oligonucleotide tail itself labeled with a fluorophore, either 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 … PET mechanism; hence the name PET-PCR," the researchers wrote.

When nucleic acid amplification occurs, however, the stem loop structure of the PET primer opens up, and fluorescence increases due to a dequenching effect of the two guanosine residues located in the overhang positions and also due to the formation of the complementary DNA strand.

This self-quenching feature obviates the need for probe designs involving internal quenching, which in turn reduces assay cost because probes from commercial vendors are not necessary, the researchers claim. Further, single labeled PET-PCR primers do not require high-performance liquid chromatography purification, resulting in a high yield of product and further reducing costs compared to commonly used dual-labeled probes.

Lucchi said that a typical reaction using TaqMan probes costs in the vicinity of $2.50, whereas a PET-PCR reaction costs about $1.80 — a difference that adds up quickly when multiple tests are run.

Furthermore, the PET-PCR primers can be either stored in the refrigerator without loss of activity for up to a month or be lyophilized and reconstituted when ready for use, according to the researchers.

"We found the half life of the [TaqMan] probes to be very difficult," Lucchi said, "whereas you can pretty much keep the [PET-PCR primers] at 4 degrees Celsius without loss of activity. We are also toying with the idea of making this even simpler by lyophilizing everything, so you'd … basically be sending to the malaria-endemic areas something in a pellet form … so the end user just adds their water, their sample, and they're ready to go."

In their PLOS One study, the researchers compared a PET-PCR assay for Plasmodium falciparum and Plasmodium species using previously described viral targets, to a nested PCR assay so chosen as the gold standard because of its superior sensitivity over microscopy and other traditional testing methods.

The group tested DNA from various Plasmodium species from 50 clinical samples from the CDC molecular diagnostic parasitology reference laboratory; as well as 69 Plasmodium falciparum-positive samples from a study in Kenya.

All of the Plasmodium falciparum samples tested were shown to be positive by the Plasmodium falciparum-specific and the Plasmodium-specific PET-PCR primer sets. The genus-specific primers detected all other non-falciparum samples tested. Compared to the nested PCR assay, both sets of PET-PCR primers had 100 percent sensitivity and specificity.

The group also tested multiplexed assays combining the Plasmodium falciparum-specific primers and the genus-specific primers. The latter were able to accurately detect all the Plasmodium samples, but the P. falciparum-specific primer set failed to detect two P. falciparum samples.

The researchers calculated limits of detection for P. falciparum of 3.2 parasites/µl using both Plasmodium genus and P. falciparum-specific primer; and 5.8 parasites/µl for P. ovale, 3.5 parasites/µl for P. malariae, and 5 parasites/µl for P. vivax using the genus-specific primer set.

Lucchi said that the researchers are "happy with the sensitivity and specificity," but that the group does need to refine the assay a bit, particularly in terms of developing primers specific for other species besides P. falciparum.

The researchers validated the assays on real-time PCR instruments from several manufacturers. "You should be able to use this on anything that can detect fluorescence," Lucchi said. In addition, the group could theoretically multiplex more, at least to the current limits of real-time PCR, which is in the range of four to six targets.

"In the case of malaria, the fact that you have the genus there takes care of all malaria," Lucchi said. "You could differentiate [species] … but the more variables you put in your reaction, you start losing sensitivity. It is a trade-off when you start using three or four colors. We think with two colors you should be OK, because in most malaria endemic areas it will be P. falciparum and then you can use your genus[-specific primers]."

However, additional multiplexing could come in handy if the primer technology is used to detect other infectious diseases, which Lucchi said is plausible.

Next steps for the researchers include a more rigorous evaluation of the primers in the field. Udhayakumar said that the group has already conducted a preliminary evaluation in Haiti, where the primers "have performed very well."

"We have actually trained someone there and transferred this technology there for them to use for their national malaria surveillance this year," he said. "We would like to see how it performs in their hands. In the same way we are talking with colleagues in other parts of the world."

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