Specificity and processing rate enhancement of Mycobacterium tuberculosis diagnostic procedure using antibody-coupled magnetic nanoparticles

Abstract

Mycobacterium tuberculosis (MTB) is the immediate cause of one of the most dangerous lung diseases that threaten developing countries, where the number of patients diagnosed with multi–drug resistant (MDR) strains is increasing significantly. In this study, we have developed a new diagnostic procedure using magnetic nanoparticles functionalised with amino group (NP–NH2) to couple with anti–MTB antibody for a more specific PCR–based detection method. The results show that the NP–NH2–antibody complex generated through 1–ethyl–3–[3–dimethylaminopropyl] carbodiimide (EDC) coupling allows rapid and specific detection of Mycobacterium bovis from the BCG vaccine which possesses the signature IS6110 sequence widely used in MTB diagnosis. Furthermore, this complex is stable and provides reproducible detection for at least 4 weeks when stored in phosphate–buffered saline solution with Tween, bovine serum albumin (BSA) and sodium azide (PBS–TBN) at 4ºC. As a result, the required time for the whole procedure to prepare samples for PCR could be completed within an hour owing to the elimination of the DNA purification step. We have tested this method on sputum samples collected from Vietnam Hospital 103 thus proving its validity. Further experiments are underway to employ the magnetic feature of the NP–NH2–antibody complex to develop an automated kit facilitating high throughput screening and reducing contamination risk.