Polymerase Chain Reaction, PCR, was invented in 1985 and awarded the Nobel Prize for chemistry in 1993. To date, almost thirty years after its initial conception, it is still the most sensitive method for detection of specific DNA sequences in biological specimens. An important addition to PCR was the invention of real-time PCR employing specific fluorescent DNA probes to detect the accumulation of amplication products. Real-time PCR is the method of choice for sensitive detection of pathogens in clinical specimens. Therefore, Check-Points has chosen this technology for clinical applications where a very high sensitivity is required.
Check-Points has developed two multiplex real-time PCR tests for detection of multidrug resistance (MDR) in gram-negative bacteria: Check-Direct CPE and Check-Direct ESBL. The challenge in the design of such tests is to combine multiple PCR primer pairs into a single PCR without compromising the detection sensitivity. This is a combination of intelligent design and empirical testing to select the best primer sets. Check-Points has chosen to use molecular beacons for real-time detection of amplification products employing up to 5 different fluorophores. Molecular beacons are highly specific: they only bind to amplification products if the resulting double-stranded structure has a higher stability than the stem-and-loop structure of the beacon probe. This characteristic makes molecular beacons also suitable for SNP detection. This is important as SNPs may alter the MDR spectrum of CPE and ESBL.