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New PCR Method Claims Single-Tube 96-Plex Assays Using Standard Equipment


NEW YORK – A new PCR method using endogenous barcodes and molecular beacons has been shown to detect dozens of targets in a single reaction using standard lab equipment. The method, called MeltArray, was used to detect as many as 62 targets simultaneously in a recent study.

Qingge Li, a molecular diagnostics researcher at Xiamen University, is senior author on the proof-of-concept study which was published last month in PNAS. In an email, Li said that his team is continuing to perfect the MeltArray method and soon expects to report analysis of a 96-target single-tube assay.

At this level of multiplexing, MeltArray could fill the gap between real-time PCR and sequencing, Li said.

The method "enables immediate application to current nucleic acid testing labs equipped with real-time PCR machines," Li said, noting that most nucleic acid testing labs have this equipment.

The MeltArray method employs the 5'-flap endonuclease activity of Taq DNA polymerase to cleave target-specific "mediator probes" into "mediator primers," which serve as endogenous barcodes during PCR.

In the study, Li and his colleagues developed a 20-plex assay to detect human Y chromosome microdeletions, a 24-plex assay to simultaneously identify and quantify respiratory pathogens, a 62-plex assay to determine E. coli serovars, and a 10-target "minisequencing" SNP genotyping assay for KRAS mutations.

Most of the assays used an SLAN 96S real-time PCR thermal cyclers from Hongshi Medical Technology, while the 62-plex used a six-color fluorometric thermal cycler that was able to carry out thermal melting analyses after amplification.

The assays were then validated with clinical samples. For example, the KRAS mutation test was validated in 107 freshly frozen and 60 formalin-fixed paraffin-embedded samples from colon cancer patients.

Fred Kramer, a former colleague of Li's and a molecular geneticist at the Public Health Research Institute of the New Jersey Medical School, said in an email that the method marks "a major advance in the design of practical, highly multiplex[ed] PCR assays."

The use of standard spectrofluorometric thermal cyclers can also make the MeltArray method broadly applicable, he said.

How it works

MeltArray uses a universal set of mediator sequences that are attached to target-specific probes, Kramer said. After amplification, mediator sequences hybridize to hairpin-shaped universal molecular beacon reporters, which then un-quenches an attached fluorophore and results in a signal.

"The key inventive step is that for a given [hairpin-shaped] molecular beacon reporter, different mediator sequences can bind to different positions in the loop ... so that when those bound mediator sequences serve as primers, different length double-stranded segments are holding the molecular beacon reporter open," Kramer said.

The MeltArray readout is a combination of fluorescence detection and melt temperature analysis.

Specifically, the binding between mediator and reporter is temperature sensitive, such that slowly raising the temperature results in the shortest extended mediator dissociating from the molecular beacon reporter and re-quenching the fluorescent signal, followed by the next longest mediator, and so on.

"The combination of the color of the molecular beacon reporter and the temperature at which its signal is dimmed identifies the target sequence from which the mediator sequence was cleaved from its probe," Kramer said.

The hairpin reporters can be labeled with different fluorophores, and each can bind many mediator sequences that dissociate at different temperatures, yielding a highly multiplexed PCR assay in a single tube.

Kramer emphasized that these molecular beacon reporters and mediator sequences are the same no matter the targets of interest, which enables "the design of a wide variety of multiplex diagnostic PCR assays."

The method still has some limitations that need further improvement, the PNAS study authors noted.

For example, as an endpoint detection methodology, its quantitative ability is restricted, making it currently more suited for screening rather than quantitative detection. The authors propose using fluorescent thermal cyclers with more optical channels, instruments that can analyze the full spectrum as some flow cytometer do, or perhaps the use of digital PCR thermal cyclers equipped to carry out melt curve analyses. The 30-minute hybridization step for the mediator primers also lengthens the overall turnaround time, but could potentially be offset by using rapid PCR technologies.

Multiplexing using standard equipment is reminiscent of the approach taken by ChromaCode, or another method that uses data analytics.

However, Li noted that MeltArray differs from these in that it is a new chemistry that works with existing data processing software, whereas the latter approaches use conventional chemistries but add novel data processing algorithms. And, he added, "MeltArray can detect many more targets in one reaction than the two data-driven approaches directly on a current real-time PCR machine."

Li previously developed multi-color melt curve-based assays for drug-resistant tuberculosis as well as for the deletional and non-deletional mutations that can cause a blood disorder known as alpha-thalassemia. These were licensed by Xiamen, China-based molecular diagnostics firm Zeesan Biotech. However, the MeltArray technology has not yet been licensed to any company, he said, and the team is now seeking partners.