Optimal Oligonucleotide Sequence and Condition for Detecting Tuberculosis by Colorimetric Gold Nanoparticles

Purpose: Tuberculosis (TB) is a multi-systemic and lethal disease. It is one of the most common causes of death from a single infectious agent worldwide. All of current diagnostic methods, including Mantoux test, acid-fast bacilli (AFB) smear and culture, AFB staining, and nucleic acid amplification, have their own shortcoming and are difficult to perform in low-income countries where TB patients are most common. Therefore, the purpose of our study was to determine the optimal complementary oligonucleotide sequence to hybridize to the target fragment of IS6110 (a target sequence extracted from TB patients) and optimal the condition, in order to provide a more rapid, cost-effective, convenient, and reliable diagnostic tool for tuberculosis via the colorimetric gold nanoparticles (AuNPs) and microfluidic paper-based analytical devices (μPAD) device to detect Mycobacterium tuberculosis. Methods: To determine the optimal condition for ssDNA oligonucleotide sequence for hybridization, same combination of oligonucleotide sequences under identical annealing temperature and duration, with 1:1 ratio of ssDNA and dsDNA, were tested under different denaturation temperatures and durations. Seven different oligonucleotide sequences were then compared to determine the ideal sequence for hybridization. The band intensity on gel electrophoresis was analyzed by UV-light transilluminator. Results: The gel electrophoresis results revealed that oligonucleotide sequence 5'-CTC GTC CAG CGC CGC-3' and 5'-TAG GCG TCG GTG ACA-3' yielded the highest hybridization rate with the 123 bp target sequence of IS6110. Conclusion: The colorimetric sensing method of gold nanoparticles possesses the potential of being a highly accurate, user-friendly, rapid, and equipment-free diagnostic tool for detecting Mycobacterium tuberculosis.