Dataset Information

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

ABSTRACT: Brucellosis is a bacterial disease, which, although affecting cattle primarily, has been associated with human infections, making its detection an important challenge. The existing gold standard diagnosis relies on the culture of bacteria which is a lengthy and costly process, taking up to 45 days. New technologies based on molecular diagnosis have been proposed, either through dip-stick, immunological assays, which have limited specificity, or using nucleic acid tests, which enable to identify the pathogen, but are impractical for use in the field, where most of the reservoir cases are located. Here we demonstrate a new test based on hybridization assays with metal nanoparticles, which, upon detection of a specific pathogen-derived DNA sequence, yield a visual colour change. We characterise the components used in the assay with a range of analytical techniques and show sensitivities down to 1000 cfu/ml for the detection of Brucella. Finally, we demonstrate that the assay works in a range of bovine samples including semen, milk and urine, opening up the potential for its use in the field, in low-resource settings.

SUBMITTER: Pal D

PROVIDER: S-EPMC5515423 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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

Pal Dheeraj D Boby Nongthombam N Kumar Satish S Kaur Gurpreet G Ali Syed Atif SA Reboud Julien J Shrivastava Sameer S Gupta Praveen K PK Cooper Jonathan M JM Chaudhuri Pallab P

PloS one 20170718 7

Brucellosis is a bacterial disease, which, although affecting cattle primarily, has been associated with human infections, making its detection an important challenge. The existing gold standard diagnosis relies on the culture of bacteria which is a lengthy and costly process, taking up to 45 days. New technologies based on molecular diagnosis have been proposed, either through dip-stick, immunological assays, which have limited specificity, or using nucleic acid tests, which enable to identify ...[more]

PMID: 28719613

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Project description:Acute hepatopancreatic necrosis disease (AHPND) is a component cause of early mortality syndrome (EMS) of shrimp. In 2013, the causative agent was found to be unique isolates of Vibrio parahaemolyticus (VPAHPND) that contained a 69 kbp plasmid (pAP1) carrying binary Pir-like toxin genes PirvpA and PirvpB. In Thailand, AHPND was first recognized in 2012, prior to knowledge of the causative agent, and it subsequently led to a precipitous drop in shrimp production. After VPAHPND was characterized, a major focus of the AHPND control strategy was to monitor broodstock shrimp and post larvae for freedom from VPAHPND by nucleic acid amplification methods, most of which required use of expensive and sophisticated equipment not readily available in a shrimp farm setting. Here, we describe a simpler but equally sensitive approach for detection of VPAHPND based on loop-mediated isothermal amplification (LAMP) combined with unaided visual reading of positive amplification products using a DNA-functionalized, ssDNA-labled nanogold probe (AuNP). The target for the special set of six LAMP primers used was the VPAHPND PirvpA gene. The LAMP reaction was carried out at 65°C for 45 min followed by addition of the red AuNP solution and further incubation at 65°C for 5 min, allowing any PirvpA gene amplicons present to hybridize with the probe. Hybridization protected the AuNP against aggregation, so that the solution color remained red upon subsequent salt addition (positive test result) while unprotected AuNP aggregated and underwent a color change from red to blue and eventually precipitated (negative result). The total assay time was approximately 50 min. The detection limit (100 CFU) was comparable to that of other commonly-used methods for nested PCR detection of VPAHPND and 100-times more sensitive than 1-step PCR detection methods (104 CFU) that used amplicon detection by electrophoresis or spectrophotometry. There was no cross reaction with DNA templates derived from non-AHPND bacteria commonly found in shrimp ponds (including other Vibrio species). The new method significantly reduced the time, difficulty and cost for molecular detection of VPAHPND in shrimp hatchery and farm settings.

Dataset Information

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

Publications

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

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