Developing a real-time quantitative loop-mediated isothermal amplification assay as a rapid and accurate method for detection of Brucellosis

Abstract

Aim: The aim of this study was designing a LAMP method for the rapid detection of Brucella 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 Brucella 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 Brucella genomic DNA and a panel containing genomes of 11 gram-positive and gramnegative organisms. The LAMP assay was highly specific and no amplification products were observed from the non-Brucella organisms. The test sensitivity for visual detection of turbidity or fluorescent colour change and also agarose gel electrophoresis was 560 ng and 5Á6 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 Brucella 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.

Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay

“…Currently, this method has been applied to various fields, for example, detection of viral and bacterial diseases in fish, animals, and humans, owing to the high specificity, sensitivity, simplicity and rapidity of this technique [9][10][11][12][13]. A LAMP assay for Brucella was introduced previously, but it only detects Brucella at the genus level using IS711 and the omp 25 gene, which avoids amplification of O. anthropi [9,12,[14][15][16][17]. However, a LAMP assay to detect only B. abortus at the species level has not yet been developed.…”

Development of probe‐based real‐time loop‐mediated isothermal amplification for detection ofBrucella

“…Brucella sp. can cause disease in both humans and animals (Soleimani et al 2013). It leads to tremendous economic losses as well as a potentially debilitating infection in man (Hosein et al 2010).…”

Visual detection of Brucella in bovine biological samples using DNA-activated gold nanoparticles

“…As a consequence Brucella spp have been identified using multiplexed PCR [9] as well as isothermal LAMP assays. [10] Both methods have limited application in the field as they require well-equipped laboratory and skilled personnel, not available in many rural settings in LMICs.…”