Fluorescent Detection of Methicillin Resistant Staphylococcus aureus by  Loop-mediated Isothermal Amplification Assisted with Streptavidin-coated Quantum  Dots

Aily  Aliasgharian; Pooria  Gill; Mohammad  Ahanjan; Adele  Rafati

doi:10.18502/ajmb.v14i1.8546

Aily Aliasgharian Research Center for Thalassemia, Mazandaran University of Medical Sciences, Sari, Iran
Pooria Gill Immunogenetics Research Center, Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran
Mohammad Ahanjan Department of Medical Microbiology, Faculty of Medicine, Mazandaran University of Medical Sciences, Sari, Iran
Adele Rafati Immunogenetics Research Center, Department of Medical Nanotechnology, Faculty of Advanced Technologies in Medicine, Mazandaran University of Medical Sciences, Sari, Iran

DOI: https://doi.org/10.18502/ajmb.v14i1.8546

Keywords: : LAMP, MRSA, MSSA, Quantum dots, mecA gene

Abstract

Background: Methicillin Resistance Staphylococcus aureus (MRSA) could be considered as a major concern in medicine can cause nosocomial infection and bacteremia, especially in patients using catheter and household medical devices.

Methods: Using molecular diagnostic methods are important for identification of MRSA from the Methicillin Sensitive Staphylococcus aureus (MSSA). Here we described a fluorescent assay using biotin-labelling Loop-mediated isothermal amplification (LAMP) method assisted with streptavidin-coated Quantum Dots (QDs) for detection of MRSA. For comparison, another fluorescent assay using LAMP assisted with Green Viewer (GV; a fluorescent dye) was applied for detection of MRSA. The mecA gene was selected as the target for amplification by LAMP and for biotin-labeling of the LAMP amplicons, biotin-11-dUTP was mixed with the dNTPs (deoxy Nucleotide Phosphates) in LAMP reaction. For determining the clinical performance of the developed assay, 30 blood samples with MRSA positive results were tested with QD-LAMP, the conventional LAMP, GV-LAMP, and Polymerase Chain Reaction (PCR).

Results: Obtained results indicated that % sensitivity of QD-LAMP was 86.66% for detection of mecA positive MRSA samples; however, the Limit of Detection (LoD) of QD-LAMP was 1.5×10⁴ Colony Forming Unit (CFU).

Conclusion: The results suggested that the QD-LAMP assay was easy to operate and could be used for detection of MRSA in parallel to the blood culture with less sensitivity for detection of bacteremia and pediatric septicemia with low counts of MRSA.