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. 2015 Aug;9(8):DC06-9.
doi: 10.7860/JCDR/2015/13962.6315. Epub 2015 Aug 1.

Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp

Sukanya Sudhaharan  1 , Lavanya Vanjari  2 , Neeraja Mamidi  2 , Nagapriyanka Ede  2 , Lakshmi Vemu  3
Affiliations

Evaluation of LAMP Assay Using Phenotypic Tests and Conventional PCR for Detection of nuc and mecA genes Among Clinical Isolates of Staphylococcus spp

Sukanya Sudhaharan et al. J Clin Diagn Res. 2015 Aug.

Abstract

Introduction: The purpose of this study is to develop a nuc and mecA gene specific Loop-mediated isothermal Amplification (LAMP) assay for rapid identification and detection of methicillin resistant Staphylococcus aureus among clinical isolates.

Materials and methods: A total of 100 (70 from pus and 30 from blood), clinical isolates of Staphylococcus spp were screened for the nuc gene to differentiate between S.aureus and Coagulase negative Staphylococci (CONS) by a nuc gene specific LAMP assay. The isolates were also screened for the presence of the mec Agene by the mecA specific LAMP assay. The results were compared with the phenotypic identification and methicillin resistance by Vitek-2 system (bioMérieux, Marcy l'Etoile, France) and conventional PCR.

Results: Among 100 Staphylococcus isolates, there were 82 (82%) Staphylococcus aureus isolates and 18 (18%) coagulase negative Staphylococcus as detected by the Vitek 2, conventional PCR and the LAMP assay using the nuc gene. The mecA gene was detected by the LAMP assay in 56(56%) isolates (44 Methicillin resistant Staphylococcus aureus (MRSA) and 12 Methicillin resistant coagulase negative Staphylococcus (MRCONS), which were also identified by the Vitek 2 and conventional PCR as methicillin resistant. The results of the LAMP assay were available within 90min as compared to the Vitek 2 results (18- 24hours) and conventional PCR (3-4 hours).

Conclusion: The present study proved that LAMP assay can be used for the simultaneous differentiation of Staphylococcal spp and detection of methicillin resistance.

Keywords: Benchside assay; Methicillin resistance; Turnaround time; Vitek 2.

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Figures

[Table/Fig-2]:
[Table/Fig-2]:
nuc and mecA conventional PCR agarose gel image by using LAMP outer (F3 B3) primers
[Table/Fig-3]:
[Table/Fig-3]:
nuc and mecA conventional PCR agarose gel image by using published primers
[Table/Fig-4]:
[Table/Fig-4]:
Visual fluorescence and gel images of LAMP products of MRSA and MSSA
[Table/Fig-5]:
[Table/Fig-5]:
Visual fluorescence and gel images of LAMP products of MRCONS and MSCONS
[Table/Fig-6]:
[Table/Fig-6]:
Comparison of Turnaround time and cost per test for phenotypic, conventional PCR and LAMP Assay
See this image and copyright information in PMC

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