Unmodified Gold Nanoparticles as Diagnostic Agents for Colorimetric Detection of Mycobacterium leprae Infection

Abstract

Purpose: In the past few years, several campaigns have uncovered multiple cases of leprosy that remained undocumented due to lack of awareness and resources in the developing world even after declaring its elimination from several countries. Also, due to lack of clinical expertise in microscopical diagnosis plus close resemblance of Mycobacterium leprae (ML) with M. tuberculosis (MTB), cases of misdiagnosis occur frequently. Attributed to their unique properties and ability to interact with biomolecules on one-to-one basis, gold nanoparticles (GNPs) are widely utilized for diagnostic purposes. Their intense red color, owing to surface plasmon resonance, shifts to blue when interparticle distance vanishes due to aggregation. This could be achieved by binding or wrapping of DNA amplicons onto GNPs to cause aggregation. Methods & Materials: Asymmetric PCR amplification (As-PCR) technique was used to obtain ssDNA amplicons. Amplification was done using primers for RLEP gene as confirmed by gel electrophoresis. MTB and E. coli served as negative controls. Primer ratios were optimized for highest yield of ssDNA amplicons. Column purification was done to remove salts and primers. The purified amplicons were incubated with GNPs (20nm) in equal ratios followed by NaCl addition. The UV-visible spectral analysis of reaction mixture was performed. Results: As-PCR using RLEP primers favourably amplified target sequences. Out of several primer combinations, the ratio 100:1 produced major ssDNA during As-PCR. After incubation with gold colloid and salt addition, the visible change in color from red to blue was observed indicating presence of amplified product that caused GNP aggregation while negative controls retain their color as verified by UV spectra showing a steep wavelength shift. The method showed high specificity with positive samples showing blue colored solution. Conclusion: A simple colorimetric method for detection of M. leprae was demonstrated. Utilizing naked GNPs reduces steps, duration and complexity of the assays providing easy method for visible detection of infected sample without the need of labelling of GNPs. For leprosy being a neglected disease, this method would serve as an important tool to cater to the unmet needs of its rapid, simple and reliable diagnosis.