In a single tube, simultaneous detection of multiple pathogens

The diagnosis of many diseases has been made possible by the amplification of viral RNA and DNA from various biological specimens such as sputum, blood, urine, feces and tissues. In addition, viral RNA and DNA amplification has also enhanced our ability to perform personalized diagnoses, discover drug resistance and find rare genetic information. Polymerase chain reaction (PCR) based gene detection technology in particular has played an important role in the identification of pertinent genetic information when the amount of specimen available is small. The extent to which sample collection can be repeated in the case of tissue specimens is limited; this is because it usually involves an invasive method. This is why it is necessary to be able to analyze a small amount of sample to obtain essential genetic information. Different gene techniques have been developed including those that allow the simultaneous analysis of multiple genes using a single test. These techniques are used in modern medicine for the analysis of genetic information and diagnosis of diseases in individuals so as to provide personalized medical treatment.

The use of real-time PCR for simultaneous detection of multiple genes is more cost effective than next-generation sequencing (NGS). In addition, validating results can be performed easily in a relatively short period of time, a factor that is very important in modern gene diagnosis technology.  Simultaneous detection of multiple genes using real-time PCR has been reported using two methods namely a fluorescence melting curve analysis (FMCA) probe-based method and a probe-free high-resolution melting (HRM) method. For the probe-based FMCA method, one probe and a pair of primers are required for the detection of one gene. To analyze viruses with several variants, multiple probes have to be designed which makes the development of diagnostic kits difficult. In addition, an overlap of melting peaks with the full width at half maximum of the adjacent T_m peaks is observed in co-infected targets. In contrast, the HRM method is limited to being used for multiple detection because it reveals the T_m of the amplified whole amplicon.

To address these limitations, Yong-Tae Kim and led by Professor In Seok Hong from the Department of Chemistry at Kongju National University in Republic of Korea together with Junhye Moon from Sejong Medical Co. Ltd. developed a new method of PCR-based real-time simultaneous multigene detection. In their new method they can simultaneously detect several genes in four targets at one fluorescence channel in a single tube. Their study is published in the Journal of Combining Chemistry and Biology (ChemBioChem).

The research team developed a new method by measuring the melting peak of individual genes without using a probe, but by tagging to a primer. The principle of this innovation was based on a molecular beacon which consisted of a stem sequence and a loop sequence. The stem sequence produced a FRET-based fluorescence signal and the loop sequence hybridized with a particular gene. The authors were able to build this system by using just one fluorophore and controlling the T_m of the external stem sequence notwithstanding the target gene. In addition, the amplification efficiency of this system is 100% when primers of the same amount are used. They plan to conduct further studies to develop a system that will control the fluorescence and quencher of the stem parts and be used to monitor the signal-on method amplification in FMCA and in real-time PCR.