Infection and Drug Resistance (Apr 2020)
Nanoparticles-Based Biosensor Coupled with Multiplex Loop-Mediated Isothermal Amplification for Detection of Staphylococcus aureus and Identification of Methicillin-Resistant S. aureus
Abstract
Luxi Jiang, Xiaomeng Li, Rumeng Gu, Deguang Mu Department of Respiratory Medicine, Zhejiang Provincial People’s Hospital, People’s Hospital of Hangzhou Medical College, Hangzhou, Zhejiang, People’s Republic of ChinaCorrespondence: Deguang Mu Tel +86-571-85893509Email [email protected]: Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (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 S. aureus, and differentiate MRSA from methicillin-susceptible S. aureus (MSSA).Materials and Methods: We designed a set of six primers targeting the nuc gene of S. aureus, and a set of five primers targeting the mecA gene of MRSA. The lateral flow biosensor visually reported the S. aureus-LAMP results within 2 mins. S. aureus species and non-S. aureus 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 S. aureus stains, and distinguish MRSA from MSSA, and can be used as a new molecular method for detection of S. aureus in laboratories.Keywords: S. aureus, MRSA, MSSA, limit of detection, lateral flow biosensor, loop-mediated isothermal amplification, mLAMP
