Synthetic long oligonucleotides to generate artificial templates for use as positive controls in molecular assays: drug resistance mutations in influenza virus as an exampleReportar como inadecuado




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Virology Journal

, 8:405

First Online: 16 August 2011Received: 19 January 2011Accepted: 16 August 2011

Abstract

BackgroundPositive controls are an integral component of any sensitive molecular diagnostic tool, but this can be affected, if several mutations are being screened in a scenario of a pandemic or newly emerging disease where it can be difficult to acquire all the necessary positive controls from the host. This work describes the development of a synthetic oligo-cassette for positive controls for accurate and highly sensitive diagnosis of several mutations relevant to influenza virus drug resistance.

ResultsUsing influenza antiviral drug resistance mutations as an example by employing the utility of synthetic paired long oligonucleotides containing complementary sequences at their 3- ends and utilizing the formation of oligonucleotide dimers and DNA polymerization, we generated ~170bp dsDNA containing several known specific neuraminidase inhibitor NAI resistance mutations. These templates were further cloned and successfully applied as positive controls in downstream assays.

ConclusionThis approach significantly improved the development of diagnosis of resistance mutations in terms of time, accuracy, efficiency and sensitivity, which are paramount to monitoring the emergence and spread of antiviral drug resistant influenza strains. Thus, this may have a significantly broader application in molecular diagnostics along with its application in rapid molecular testing of all relevant mutations in an event of pandemic.

List of abbreviationsNAINeuraminidase inhibitor

PCRpolymerase chain reaction

RLBreverse line blot hybridization

LCRLigase chain reaction

RCARolling Circle Amplification.

Electronic supplementary materialThe online version of this article doi:10.1186-1743-422X-8-405 contains supplementary material, which is available to authorized users.

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Autor: Bin Wang - Megan C Steain - Dominic E Dwyer - Anthony L Cunningham - Nitin K Saksena

Fuente: https://link.springer.com/



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