Bhawna Dahiya1, Suman Sharma1, Anish Khan1, Ekta Kamra1, Preeti Mor1, Abhishek Sheoran2, Vishnubhatla Sreenivas3, Mandira Varma-Basil4, Krishna B Gupta5, Mahesh C Gupta6, Dhruva Chaudhary7, Promod K Mehta1. 1. Centre for Biotechnology, Maharshi Dayanand University (MDU), Rohtak 124001, Haryana, India. 2. Department of Statistics, Ramanujan College, University of Delhi, New Delhi 110019, India. 3. Department of Biostatistics, All India Institute of Medical Sciences, New Delhi 110029, India. 4. Department of Microbiology, Vallabhbhai Patel (VP) Chest Institute, University of Delhi, Delhi 110007, India. 5. Department of TB & Respiratory Medicine, University of Health Sciences (UHS), Rohtak 124001, Haryana, India. 6. Department of Pharmacology, UHS, Rohtak 124001, Haryana, India. 7. Pulmonary & Critical Care Medicine, UHS, Rohtak 124001, Haryana, India.
Abstract
Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts were made to improve the technology and diagnostic potential of real-time immuno-PCR (RT-I-PCR). Methods: We designed gold nanoparticle (GNP)-based RT-I-PCR (GNP-RT-I-PCR) assay for the detection of Mycobacterium tuberculosis CFP-10 (Rv3874) protein in clinical samples of TB patients. Results: A wide quantitative detection range of CFP-10 was found to be 0.5-5 × 104 pg/ml in bodily fluids of TB patients, which can evaluate the progression of disease. Moreover, sensitivities of 83.7 and 76.2% were observed in pulmonary (n = 49) and extrapulmonary TB (n = 42) patients, respectively, with specificities of 93.5-93.8% (n = 63). Conclusion: Conjugation of detection antibodies and oligonucleotides to functionalized GNPs of GNP-RT-I-PCR is relatively easier, compared with streptavidin-biotin/succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate system employed in RT-I-PCR. Our assay also showed better diagnostic performance than RT-I-PCR, which may provide a viable platform for the development of an efficient TB diagnostic test.
Aim: Timely and reliable diagnostic test for tuberculosis (TB) is immediately required. Attempts were made to improve the technology and diagnostic potential of real-time immuno-PCR (RT-I-PCR). Methods: We designed gold nanoparticle (GNP)-based RT-I-PCR (GNP-RT-I-PCR) assay for the detection of Mycobacterium tuberculosis CFP-10 (Rv3874) protein in clinical samples of TB patients. Results: A wide quantitative detection range of CFP-10 was found to be 0.5-5 × 104 pg/ml in bodily fluids of TB patients, which can evaluate the progression of disease. Moreover, sensitivities of 83.7 and 76.2% were observed in pulmonary (n = 49) and extrapulmonary TB (n = 42) patients, respectively, with specificities of 93.5-93.8% (n = 63). Conclusion: Conjugation of detection antibodies and oligonucleotides to functionalized GNPs of GNP-RT-I-PCR is relatively easier, compared with streptavidin-biotin/succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate system employed in RT-I-PCR. Our assay also showed better diagnostic performance than RT-I-PCR, which may provide a viable platform for the development of an efficient TB diagnostic test.