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Developing a real‐time quantitative loop‐mediated isothermal amplification assay as a rapid and accurate method for detection of Brucellosis
Author(s) -
Soleimani M.,
Shams S.,
MajidzadehA K.
Publication year - 2013
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.12290
Subject(s) - loop mediated isothermal amplification , brucellosis , loop (graph theory) , chromatography , brucella abortus , biology , microbiology and biotechnology , virology , chemistry , mathematics , biochemistry , combinatorics , dna
Aim The aim of this study was designing a LAMP method for the rapid detection of B rucella and development of a sensitive quantitative‐ LAMP (Q‐ LAMP ) assay for quantification of brucellosis. Methods and Results In this study for the LAMP detection of the causative agent of brucellosis, we used specifically designed primers to target the omp25 conserved gene of B rucella spp. The sensitivity of the LAMP method was evaluated by preparing serial tenfold dilution of omp25 gene containing plasmid followed by performing the LAMP reaction. To improve the assay as a quantitative test, LAMP products in the serial dilution were evaluated by Loopamp real‐time turbidimeter system and then standard curve was generated by plotting time threshold values against log of copy number. The assay specificity was evaluated using B rucella genomic DNA and a panel containing genomes of 11 gram‐positive and gram‐negative organisms. The LAMP assay was highly specific and no amplification products were observed from the non‐ B rucella organisms. The test sensitivity for visual detection of turbidity or fluorescent colour change and also agarose gel electrophoresis was 560 ng and 5·6 ng, respectively. The lower limit of detection was 17 copies of the gene that could be detected in 50 min. Conclusions The results of this study indicated that the LAMP assay is a simple, rapid, sensitive and specific technique for detection of B rucella spp. that may improve diagnostic potential in clinical laboratories. Significance and Impact of the Study The LAMP assay because of the simplicity and low cost can be preferred to other molecular methods in the diagnosis of infectious diseases.