Visual Detection of Brucella spp. in Spiked Bovine Semen Using Loop-Mediated Isothermal Amplification (LAMP) Assay

Abstract

Several pathogens including Brucella spp. are shed in semen of infected bulls and can be transmitted to cows through contaminated semen during artificial insemination. The present study reports omp2a and bcsp31 gene based loop-mediated isothermal amplification (LAMP) assays for detection of Brucella genomic DNA in semen from infected bulls. The positive results could be interpreted visually by change in colour of reaction mixture containing hydroxyl naphthol blue (HNB) dye from violet to sky blue. LAMP assays based on omp2a and bcsp31 could detect as little as 10 and 100 fg of B. abortus S19 genomic DNA, respectively. Sensitivity of omp2a and bcsp31 LAMP assays for direct detection of organisms in bovine semen was 2.28 9 10 1 CFU and 2.28 9 10 2 CFU of B. abortus S19 in spiked bovine semen, respectively. The omp2a LAMP assay was found equally sensitive to TaqMan probe based real-time PCR and 100 times more sensitive than conventional PCR in identifying Brucella in spiked semen. The diagnostic applicability of the omp2a LAMP assay was evaluated with seventy-nine bovine semen samples and results were re-evaluated through TaqMan probe based real-time PCR and conventional PCR. Taken together, the omp2a LAMP assay is easy to perform, rapid and sensitive in diagnosis of Brucella spp. in bovine semen.

Closed tube loop-mediated isothermal amplification assay for rapid detection of hepatitis B virus in human blood

“…To surmount such obstacles of an opened tube LAMP system, closed tube LAMP assays using a fluorometer (Genie II/III systems) and commercially available master mix (OptiGene, UK) were developed and validated for rapid detection of HBV in DNA and blood samples. This approach has been demonstrated by few previous studies; the visualization of LAMP reaction via closed tube system decreased cross contamination ( Tao et al., 2011 ; Karthik et al., 2014 ; Prusty et al., 2016 ). Furthermore, the LAMP master mix can be used for Genie II/III systems (OptiGene, UK) and also for other mobile/molecular devices such as Smart-DART (Diagenetix, USA), PCR, real-time PCR, and even colorimetric assays ( Reddy et al., 2011 ; Davoodian and Sadeghifard, 2011 ).…”

Rapid Veterinary Diagnosis of Bovine Reproductive Infectious Diseases from Semen Using Paper-Origami DNA Microfluidics

“…To evaluate analytical sensitivity in real samples in the field, different concentrations of cultured organisms were spiked into healthy semen samples collected from a germplasm centre managed by IVRI in India. Fifty colony forming units (CFU) of Brucella, Figure 3 (a) (quantified data normalized in Figure 3 (b), (c) and (d)), were detected which is a 4-fold improvement in sensitivity compared with assays using the visual detection of the colour change from hydroxynaphthol blue (HNB) for LAMP assay 18 . The device also enabled the detection of Leptospira as low as 50 organisms, a performance which compares favourably with published results 38 .…”

“…As an alternative that does not require complex temperature cycling, decreasing the requirements for well-equipped facilities, loop-mediated isothermal amplification, LAMP, has emerged as a powerful isothermal means of performing NATs yielding high specificity and sensitivity 16,17 . LAMP has recently been used to rapidly detect Brucella 18 , Leptospira 19 and BoHV-1 3 as single assays performed under laboratory conditions. LAMP has also been carried out within microfluidic chips 20 and capillary platforms 21,22 demonstrating the potential of the technique for point-of-care diagnosis.…”

Establishment and Evaluation of a Loop-Mediated Isothermal Amplification Assay for Detection of Raccoon Dog in Meat Mixtures

“…A successful and suitable LAMP assay result is not only determined by DNA amplification efficiency; the method selected for monitoring the reaction product is equally important, and many detection methods have been developed [23,24]. The final reaction product can be directly observed by the reaction of magnesium pyrophosphate precipitation [25], the color change which is caused by DNAbinding dyes [26] and indirect colorimetric indicators [27], or the special band at the test line on the strip using lateral Journal of Food Quality 7 flow dipstick (LFD) assay [28], and the LAMP products are also analyzed by gel electrophoresis at the endpoint [29]. The real-time LAMP reaction can be monitored by a turbidity meter or a real-time fluorescence detector [30].…”