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Nucleotide polymorphism-based single-tube test for robust molecular identification of all currently described Brucella species.

ABSTRACT: Among the numerous molecular methods described during the last 20 years to identify Brucella, multiplexed amplification methods offer the cheapest and simplest technical solution for molecular identification. However, one disadvantage of such methods is their need to undergo technical revalidation each time a new marker is added to the system. Moreover, polymorphic markers cannot be assessed at the single-nucleotide level in these assays. Since new Brucella species are continuously being described, open methodologies able to accommodate new markers while preserving all other system parameters have an obvious advantage. We present a ligase chain reaction (LCR)-based method that simultaneously assesses multiple genetic markers at the single-nucleotide level. Most of the selected markers originate from a multilocus sequence typing (MLST) database that has been extensively validated on hundreds of different Brucella strains. When assayed on both reference and field strains, the method yields characteristic capillary electrophoresis profiles for each of the 10 Brucella species described to date and displays discriminatory potential below the species level for some. Since the LCR methodology is insensitive to interference resulting from the use of multiple oligonucleotides in a single mixture, the way is open for smooth future updates of the proposed system. Such updates are inevitable, given the pending description of new Brucella species.

SUBMITTER: Wattiau P

PROVIDER: S-EPMC3187137 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Nucleotide polymorphism-based single-tube test for robust molecular identification of all currently described Brucella species.

Wattiau Pierre P Whatmore Adrian M AM Van Hessche Mieke M Godfroid Jacques J Fretin David D

Applied and environmental microbiology 20110729 18

Among the numerous molecular methods described during the last 20 years to identify Brucella, multiplexed amplification methods offer the cheapest and simplest technical solution for molecular identification. However, one disadvantage of such methods is their need to undergo technical revalidation each time a new marker is added to the system. Moreover, polymorphic markers cannot be assessed at the single-nucleotide level in these assays. Since new Brucella species are continuously being describ ...[more]

PMID: 21803907

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Project description:Summary In this work, we developed a simple and robust assay to rapidly detect SNPs in nucleic acid samples. Our approach combines loop-mediated isothermal amplification (LAMP)-based target amplification with fluorescent probes to detect SNPs with high specificity. A competitive “sink” strand preferentially binds to non-SNP amplicons and shifts the free energy landscape to favor specific activation by SNP products. We demonstrated the broad utility and reliability of our SNP-LAMP method by detecting three distinct SNPs across the human genome. We also designed an assay to rapidly detect highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants from crude biological samples. This work demonstrates that competitive SNP-LAMP is a powerful and universal method that could be applied in point-of-care settings to detect any target SNP with high specificity and sensitivity. We additionally developed a publicly available web application for researchers to design SNP-LAMP probes for any target sequence of interest. Graphical abstract Highlights • A simple and rapid assay to detect specific SNPs in complex nucleic acid samples• Approach leverages competitive probes to specifically detect SNP over non-SNP sequences• Demonstrated utility across diverse targets and sample types• Web application for researchers to design SNP-LAMP probes for any target of interest Motivation Single-nucleotide polymorphisms (SNPs) are the most common source of genetic variation between individuals and have implications in human disease, pathogen drug resistance, and agriculture. SNPs are often detected using DNA sequencing or PCR, both of which can be difficult to deploy for on-site testing or in low-resource settings. There is a need for new SNP detection methods that can be performed rapidly without advanced laboratory equipment or a cold supply chain. Detecting single-nucleotide polymorphisms (SNPs) is important for understanding human disease, the emergence of pathogen variants, and the application of genetic agricultural breeding programs. Hyman et al. develop a simple and rapid assay to detect SNPs in complex nucleic acid samples. A web-based tool allows researchers to design custom SNP probes.

Dataset Information

Nucleotide polymorphism-based single-tube test for robust molecular identification of all currently described Brucella species.

Publications

Nucleotide polymorphism-based single-tube test for robust molecular identification of all currently described Brucella species.

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