Detection of Mycobacterium tuber culosis purified ESAT-6 (Rv3875) by magnetic bead-coupled gold nanoparticle-based immuno-PCR assay
Loading...
Date
2018
Journal Title
Journal ISSN
Volume Title
Publisher
International Journal of Nanomedicine
Abstract
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.
Description
Keywords
MB-GNP-I-PCR, functionalized GNPs, ELISA, LOD