Tuberculosis (TB) is one of the leading diseases in different parts of the world; the causative agent of this disease is Mycobacterium tuberculosis (Mtb). The disease is ranked as the 5th top cause of death in low/ middle-income countries and the 9th top cause of death worldwide. The increase in the prevalence and mortality rates of TB across the globe has been attributed to improper medication, as well as the evolution and emergence of drug-resistant bacterial strains. According to a report released by WHO, there were nearly 600,000 new cases with resistance to rifampicin (RIF) antibiotic, which was the most effective first-line drug in 2017, of which around 490,000 had MDR-TB globally. Thus, the prevention and early diagnosis of TB has become a major concern for global medical care. The conventional methods used to detect and assess the antibiotic resistance of Mtb include mycobacterial culture, smear microscopy, and drug-susceptibility testing. Over the years, researchers have explored the use of efficient real-time PCR assays and FDA endorsed Xpert MTB/RIF assay for the detection and differentiation of drug-resistant Mtb from drug-susceptible Mtb. However, the cost efficiency, high degree of sophistication, and requirement of trained personnel are major limitations to the use of these assays in field diagnostic tests. Thus, this study aimed to design a diagnostic system that simultaneously detects DNA fragments and single nucleotide substitution (SNS)-encoded segments associated with drug resistance.
Recently, Shih-Ying Huang and Min-Chieh Chuang at Tunghai University in collaboration with Jia-Ru Chang, Horng-Yunn Dou and Yu-Chieh Liao at the National Health Research Institutes in Taiwan demonstrated a diagnostic system that uses a molecular beacon probe and an assistant strand as the core to simultaneously interact with an Mtb-specific fragment (in IS6110) and a SNS-encoded segment (in rpoB) with a three-tiered (High, Medium, and Low) output for the diagnosis of TB. Their findings are reported in Analytica Chimica Acta.
The research team observed that the fluorescence emitted by the matched (TS2) and mismatched (TT2) four-way junction (4WJ) increased with time. The TT2+TR combinatorial target produced a fluorescence, which was half of that produced by TS2+TR targets. However, the fluorescence produced from other combinations was low and different compared to these aforementioned variables. The authors also observed that the length of the TT2 and TS2 binding arm changed the tiered output relationship and influenced the formation of a four-way DNA junction (4WJ). The 15 bp-binding arm allowed the formation of 4WJ in matched and mismatched forms while the 11 bp-binding arm only allowed the formation of 4WJ in matched forms. Moreover, the ΔG12 of the matched form was found to be more negative compared to the ΔG12 of the mismatched form.
The authors observed that mismatched 4WJ began to dissociate at a temperature of 35.8oC while matched 4WJ began to dissociate at a temperature of 41.9oC. They also noticed that the activation of enzymatic amplification caused an increase in fluorescence at a temperature of 32oC in the presence of enzymes without targets. However, an increase in fluorescence was observed below 20oC in the presence of enzymes with targets while a decrease in fluorescence was observed above 60oC in the presence of enzymes with targets. The greatest difference between mismatched and matched 4WJ forms occurred at 38oC.
Furthermore, the use of KF and Nt.AlwI increased the stability of the three-tiered format and enhanced the fluorescence signal by 64-fold. The authors also observed that the system exhibited three-tiered output format at a target concentration range of 0.3 to 120 nM. In addition, the system accurately detected Mtb in a bacterial load that ranged from 4×100 to 4×103 CFU per reaction.
The study demonstrated successfully the simultaneous detection of Mtb-encoding genes and RIF-resistance with a three-tiered output system for the diagnosis of TB. The findings will advance further studies on the use of a three-tiered output system for the rapid detection of Mtb.
Huang, S.Y., Chang, J.R., Liao, Y.C., Dou, H.Y., and Chuang, M.C. Simultaneous detections of genetic fragment and single nucleotide mutation with a three-tiered output for tuberculosis diagnosis, Analytica Chimica Acta (2018) 1007, 1-9.