Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Netrapal Singh,1,2 Bhawna Dahiya,1 Venkatraman Srinivasan Radhakrishnan,3 Tulika Prasad,3 Promod K Mehta1,4 1Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India; 2Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology, Chinese Academy of Scie...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Singh N, Dahiya B, Radhakrishnan VS, Prasad T, Mehta PK
Formato:	article
Lenguaje:	EN
Publicado:	Dove Medical Press 2018
Materias:	MB-GNP-I-PCR Functionalized GNPs ELISA LOD Medicine (General) R5-920
Acceso en línea:	https://doaj.org/article/12519051a88141aaab0444e15fc9be66
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:12519051a88141aaab0444e15fc9be66
record_format	dspace
spelling	oai:doaj.org-article:12519051a88141aaab0444e15fc9be662021-12-02T07:56:25ZDetection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay1178-2013https://doaj.org/article/12519051a88141aaab0444e15fc9be662018-12-01T00:00:00Zhttps://www.dovepress.com/detection-of-mycobacterium-tuberculosis-purified-esat-6-rv3875-by-magn-peer-reviewed-article-IJNhttps://doaj.org/toc/1178-2013Netrapal Singh,1,2 Bhawna Dahiya,1 Venkatraman Srinivasan Radhakrishnan,3 Tulika Prasad,3 Promod K Mehta1,4 1Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India; 2Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China; 3Advanced Instrumentation Research & Facility (AIRF) and Special Centre for Nanoscience (SCNS), Jawaharlal Nehru University (JNU), New Delhi, Delhi, India; 4Microbiology Department, Central University of Haryana, Mahendergarh, India Purpose: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. Methods: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. Results: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. Conclusion: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis. Keywords: MB-GNP-I-PCR, functionalized GNPs, ELISA, LODSingh NDahiya BRadhakrishnan VSPrasad TMehta PKDove Medical PressarticleMB-GNP-I-PCRFunctionalized GNPsELISALODMedicine (General)R5-920ENInternational Journal of Nanomedicine, Vol Volume 13, Pp 8523-8535 (2018)
institution	DOAJ
collection	DOAJ
language	EN
topic	MB-GNP-I-PCR Functionalized GNPs ELISA LOD Medicine (General) R5-920
spellingShingle	MB-GNP-I-PCR Functionalized GNPs ELISA LOD Medicine (General) R5-920 Singh N Dahiya B Radhakrishnan VS Prasad T Mehta PK Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
description	Netrapal Singh,1,2 Bhawna Dahiya,1 Venkatraman Srinivasan Radhakrishnan,3 Tulika Prasad,3 Promod K Mehta1,4 1Centre for Biotechnology, Maharshi Dayanand University, Rohtak, Haryana, India; 2Institute of Synthetic Biology (iSynBio), Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China; 3Advanced Instrumentation Research & Facility (AIRF) and Special Centre for Nanoscience (SCNS), Jawaharlal Nehru University (JNU), New Delhi, Delhi, India; 4Microbiology Department, Central University of Haryana, Mahendergarh, India Purpose: Immuno-PCR (I-PCR), an ultrasensitive method, combines the versatility of ELISA with the exponential amplification capacity of PCR. Coupling of detection antibodies with the reporter DNA is a critical step of I-PCR. Gold nanoparticles (GNPs) and magnetic beads (MBs) are relatively easy to attach with the antibodies and DNA. Therefore, we designed MB-coupled GNP-based I-PCR (MB-GNP-I-PCR) assay for the detection of Mycobacterium tuberculosis antigen. Methods: GNPs were synthesized by chemical reduction and seed-mediated synthesis. Functionalized GNPs were prepared by coupling GNPs with the detection antibodies and reporter DNA and were characterized. Detection limit of M. tuberculosis-specific purified early secreted antigenic target-6 (ESAT-6) (Rv3875) was determined by MB-GNP-I-PCR. Results: Transmission electron microscopy revealed spherical and slightly polydispersed GNPs of ~20 and ~60 nm size. Coupling of antibodies to GNPs was indicated by a shift in absorption maxima from 524 to 534 nm, which was confirmed by transmission electron microscopy. A color reaction with ELISA and the presence of 76 bp product by PCR further validated the coupling of detection antibodies and signal DNA to the functionalized GNPs. Also, attachment of capture antibodies with MBs was confirmed by magneto-ELISA. Detection limit of purified ESAT-6 by MB-GNP-I-PCR was determined to be 10 fg/mL, 105-fold lower than analogous ELISA. Notably, no sample matrix effect was observed in the saliva samples of healthy individuals spiked with the purified ESAT-6. Conclusion: Unlike conventional I-PCR (solid format), MB-GNP-I-PCR (liquid format) is relatively simple with the reduced background signals, which can be further exploited for the clinical diagnosis of tuberculosis. Keywords: MB-GNP-I-PCR, functionalized GNPs, ELISA, LOD
format	article
author	Singh N Dahiya B Radhakrishnan VS Prasad T Mehta PK
author_facet	Singh N Dahiya B Radhakrishnan VS Prasad T Mehta PK
author_sort	Singh N
title	Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
title_short	Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
title_full	Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
title_fullStr	Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
title_full_unstemmed	Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
title_sort	detection of mycobacterium tuberculosis purified esat-6 (rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-pcr assay
publisher	Dove Medical Press
publishDate	2018
url	https://doaj.org/article/12519051a88141aaab0444e15fc9be66
work_keys_str_mv	AT singhn detectionofmycobacteriumtuberculosispurifiedesat6rv3875bymagneticbeadcoupledgoldnanoparticlebasedimmunopcrassay AT dahiyab detectionofmycobacteriumtuberculosispurifiedesat6rv3875bymagneticbeadcoupledgoldnanoparticlebasedimmunopcrassay AT radhakrishnanvs detectionofmycobacteriumtuberculosispurifiedesat6rv3875bymagneticbeadcoupledgoldnanoparticlebasedimmunopcrassay AT prasadt detectionofmycobacteriumtuberculosispurifiedesat6rv3875bymagneticbeadcoupledgoldnanoparticlebasedimmunopcrassay AT mehtapk detectionofmycobacteriumtuberculosispurifiedesat6rv3875bymagneticbeadcoupledgoldnanoparticlebasedimmunopcrassay
_version_	1718399084806537216

Detection of Mycobacterium tuberculosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay

Ejemplares similares