Loop-Mediated Isothermal Amplification Label-Based Gold Nanoparticles Lateral Flow Biosensor for Detection of Enterococcus faecalis and Staphylococcus aureus.

Yi Wang,Changyun Ye,Yan Wang,Jianguo Xu,Hui Li,Lu Zhang

doi:10.3389/fmicb.2017.00192

Abstract

The report describes a simple, rapid and sensitive assay for visual and multiplex detection of Enterococcus faecalis and Staphylococcus aureus based on multiple loop-mediated isothermal amplification (mLAMP) and lateral flow biosensor (LFB). Detection and differentiation of the Ef0027 gene (E. faecalis-specific gene) and nuc gene (S. aureus-specific gene) were determined using fluorescein (FITC)-and digoxin-modified primers in the mLAMP process. In the presence of biotin- and FITC-/digoxin-modified primers, the mLAMP yielded numerous biotin- and FITC-/digoxin-attached duplex products, which were detected by LFB through biotin/streptavidin interaction (biotin on the duplex and streptavidin on the gold nanoparticle) and immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the LFB test line). The accumulation of gold nanoparticles generated a characteristic red line, enabling visual and multiplex detection of target pathogens without instrumentation. The limit of detection (LoD), analytical specificity and feasibility of LAMP-LFB technique were successfully examined in pure culture and blood samples. The entire procedure, including specimen (blood samples) processing (30 min), isothermal reaction (40 min) and result reporting (within 2 min), could be completed within 75 min. Thus, this assay offers a simple, rapid, sensitive and specific test for multiplex detection of E. faecalis and S. aureus strains. Furthermore, the LAMP-LFB strategy is a universal technique, which can be extended to detect various target sequences by re-designing the specific LAMP primers.

Highlights

Gram-positive bacteria, especially Enterococcus faecalis (E. faecalis) and Staphylococcus aureus (S. aureus), are among leading causes of nosocomial infection, such as bloodstream, surgical site and urinary tract infections (Filetoth, 2008)
Loop-mediated isothermal amplification (LAMP)-lateral flow biosensor (LFB) for Detection of Enterococcus faecalis and Staphylococcus aureus related with skin, throat and nose, is one of the most important pathogens in hospital- and community-acquired infections associated with high mortality (Foster et al, 2014)
A double-labeled detectable product (BIP∗/LB∗), which is similar to the detectable backward inner primer (BIP)∗/LF∗ product, can be produced when the BIP primer is labeled with biotin at the 5′ end and LB primer for hapten

Summary

Introduction

Gram-positive bacteria, especially Enterococcus faecalis (E. faecalis) and Staphylococcus aureus (S. aureus), are among leading causes of nosocomial infection, such as bloodstream, surgical site and urinary tract infections (Filetoth, 2008). LAMP-LFB for Detection of Enterococcus faecalis and Staphylococcus aureus related with skin, throat and nose, is one of the most important pathogens in hospital- and community-acquired infections associated with high mortality (Foster et al, 2014). Reliable detection of E. faecalis and S. aureus is important for accurate diagnosis and instituting adequate antimicrobial therapy. PCR and PCR-based detection of target pathogens directly in clinical samples has been established and was used as a fast alternative to the traditional culture techniques (Peters et al, 2007; Wang et al, 2016b). A simple and specific assay for rapid diagnosis of the two pathogens is extremely important to institute adequate antimicrobial therapy, facilitate clinical care, infection control, and epidemiologic investigations

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Conclusion