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Open Access Highly Accessed Methodology article

Rapid and simple detection of methicillin-resistance staphylococcus aureus by orfX loop-mediated isothermal amplification assay

Jianyu Su1, Xiaochen Liu1, Hemiao Cui1, Yanyan Li2, Dingqiang Chen3*, Yanmei Li45* and Guangchao Yu6

Author Affiliations

1 College of Light Industry and Food Sciences, South China University of Technology, Guangzhou 510640, China

2 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China

3 Department of Laboratory Medicine, First Affiliated Hospital of Guangzhou Medical College, Guangzhou 510120, China

4 Guangzhou Women and Children’s Medical Center, Guangzhou 510623, China

5 Department of Microbial Pathogenesis, University of Maryland, Baltimore 21201, USA

6 First Affiliated Hospital of Jinan University, Guangzhou 510620, China

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BMC Biotechnology 2014, 14:8  doi:10.1186/1472-6750-14-8

Published: 24 January 2014

Abstract

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most prevalent pathogens responsible for nosocomial infections throughout the world. As clinical MRSA diagnosis is concerned, current diagnostic methodologies are restricted by significant drawbacks and novel methods are required for MRSA detection. This study aimed at developing a simple loop-mediated isothermal amplification (LAMP) assay targeting on orfX for the rapid detection of methicillin-resistance Staphylococcus aureus (MRSA).

Results

The protocol was designed by targeting orfX, a highly conserved open reading frame in S. aureus. One hundred and sixteen reference strains, including 52 Gram-positive and 64 Gram-negative isolates, were included for evaluation and optimization of the orfX-LAMP assay. This assay had been further performed on 667 Staphylococcus (566 MRSA, 25 MSSA, 53 MRCNS and 23 MSCNS) strains and were comparatively validated by PCR assay using primers F3 and B3, with rapid template DNA processing, simple equipments (water bath) and direct result determination (both naked eye and under UV light) applied. The indispensability of each primer had been confirmed, and the optimal amplification was obtained under 65°C for 45 min. The 25 μl reactant was found to be the most cost-efficient volume, and the detection limit was determined to be 10 DNA copies and 10 CFU/reaction. High specificity was observed when orfX-LAMP assay was subjected to 116 reference strains. For application, 557 (98.4%, 557/566) and 519 (91.7%, 519/566) tested strains had been detected positive by LAMP and PCR assays. The detection rate, positive predictive value (PPV) and negative predictive value (NPV) of orfX-LAMP were 98.4%, 100% and 92.7% respectively.

Conclusions

The established orfX-LAMP assay had been demonstrated to be a valid and rapid detection method on MRSA.

Keywords:
Loop-mediated isothermal amplification (LAMP); MRSA; OrfX