Development of Au-Nanoprobes Combined with Loop-Mediated Isothermal Amplification for Detection of Isoniazid Resistance in Mycobacterium tuberculosis

Abstract

Multidrug resistant tuberculosis (MDR-TB) is Mycobacterium tuberculosis that does not respond to isoniazid and rifampicin, so the condition worsens continuously and creates difficulties for treatment by public health control programmes, especially in developing countries. The real time polymerase chain reaction (PCR) combined with agarose gel electrophoresis or strip tests is useful molecular tools for diagnosis of MDR-TB. Novel loop-mediated isothermal amplification (LAMP) can also detect drug resistance, which is a one-point mutation, by designing inner primers of 5′ end specific with the mutant. Au-nanoprobes on hybridisation with LAMP products containing target-specific sequences remain red, whereas test samples without specific sequences in the probe turn purple due to salt-induced aggregation of the Au-nanoprobes. In this study, a strategy was designed based on the LAMP of a DNA sample coupled to specific Au-nanoprobes, which showed the potential to provide a rapid and sensitive method for detecting isoniazid resistance at katG gene position 315 (G→C). 46 clinical samples were tested and showed 100% specificity and sensitivity compared with Genotype® MDR-TB Plus. This method was advantageous because it is rapid, cheap, specific, and sensitive. Further, it does not require thermal cycles for MDR-TB detection.