Lack of specificity associated with using molecular beacons in loop mediated amplification assays

Hardinge, Patrick ORCID: https://orcid.org/0000-0002-6292-5186 and Murray, James A. H. ORCID: https://orcid.org/0000-0002-2282-3839 2019. Lack of specificity associated with using molecular beacons in loop mediated amplification assays. BMC Biotechnology 19 , 55. 10.1186/s12896-019-0549-z

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Abstract

Background Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. However the complexity of primer design and the number of primers involved can lead to false positives from non-specific primer interactions. Standard methods of LAMP detection utilise the increasing concentrations of DNA or inorganic pyrophosphate and therefore lack specificity for identifying the desired LAMP amplification. Molecular beacons used in PCR reactions are target specific and may enhance specificity with LAMP. Results We present a potential molecular beacon approach to LAMP detection targeting the single stranded region between loops, and test this for LAMP molecular beacons targeting the 35S promoter and NOS terminator sequences commonly used in GM crops. From these studies we show that molecular beacons used in LAMP, despite providing a change in fluorescent intensity with amplification, appear not to anneal to specific target sequences and therefore target specificity is not a benefit of this method. However, molecular beacons demonstrate a change in fluorescence which is indicative of LAMP amplification products. We identify the LAMP loop structure as likely to be responsible for this change in signal. Conclusions Molecular beacons can be used to detect LAMP amplification but do not provide sequence specificity. The method can be used to determine effectively LAMP amplification from other primer-driven events, but does not discriminate between different LAMP amplicons. It is therefore unsuitable for multiplex LAMP reactions due to non-specific detection of LAMP amplification.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Biosciences
Publisher:	BioMed Central
ISSN:	1472-6750
Funders:	Biotechnology and Biological Sciences Research Council
Date of First Compliant Deposit:	18 July 2019
Date of Acceptance:	19 July 2019
Last Modified:	05 May 2023 02:21
URI:	https://orca.cardiff.ac.uk/id/eprint/124297

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