Nanoparticles-Based Biosensor Coupled with Multiplex Loop-Mediated Isothermal Amplification for Detection of and Identification of Methicillin-Resistant .

INTRODUCTION

(), including methicillin-resistant (MRSA), is a common human pathogen, which can cause a variety of infections from mild to severe. In this article, a new diagnostic method called multiplex loop-mediated isothermal amplification combined with nanoparticles-based lateral flow biosensor (mLAMP-LFB) has been developed, which was proved to be fast, reliable, and simple for detecting , and differentiate MRSA from methicillin-susceptible (MSSA).

MATERIALS AND METHODS

We designed a set of six primers targeting the gene of , and a set of five primers targeting the gene of MRSA. The lateral flow biosensor visually reported the -LAMP results within 2 mins. species and non- species were used to identify the specificity and sensitivity of the assay.

RESULTS

The best conditions for LAMP were 50 mins at 63°C, and the sensitivity was 100 fg. No cross-reactivity was shown and the specificity of this assay is 100%. This assay requires 20 mins for DNA preparation, 50 mins for isothermal amplification and 2 mins for biosensor detection. The total time is within 75 mins. Among 96 sputum samples, LAMP-LFB and traditional culture method showed the same results, 8 (8.33%) samples were MRSA-positive, and 9 (9.38%) samples were MSSA-positive. Seven (7.29%) samples were MRSA-positive and 7 (7.29%) were MSSA-positive by PCR method. Compared with the culture method, diagnostic accuracy of m-LAMP-LFB assay was 100%. The results showed that the m-LAMP-LFB method has better detection ability than the PCR method.

DISCUSSION

In short, this m-LAMP-LFB assay is a specific and sensitive method that can quickly identify stains, and distinguish MRSA from MSSA, and can be used as a new molecular method for detection of in laboratories.