Abstract
BackgroundLoop 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.ResultsWe 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.ConclusionsMolecular 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.
Highlights
Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics
Specificity of Loop-mediated amplification (LAMP) molecular beacon targeting the forward loop sequence We designed a LAMP molecular beacon targeted to the forward loop sequence of a variant of the 35S promoter from Cauliflower mosaic virus (Fig. 1), with a probe sequence of 14 nucleotides and complementary arms of 5 nucleotides
The use of this primer in a 63 degrees C 35S promoter (35Sp) LAMP assay at a concentration of 320 nanomolar with the omission of the LoopF primer from the reaction mix resulted in a clear difference between the positive and negative samples (Fig. 2: 2A)
Summary
Loop mediated isothermal amplification of nucleic acid templates is a rapid, sensitive and specific method suitable for molecular diagnostics. At the initiation of the amplification hairpin forming forward and backward inner primers (FIP and BIP) strand invade the double stranded DNA target. These primers anneal and are extended by a polymerase with strong displacement activity. The nascent strands are displaced by further primers (F3 and B3) enabling the subsequent formation of double hairpin structures from which amplification can cycle. LAMP amplification has been used for many pathogen detection strategies due to rapid reaction times, high sensitivity, robustness to contaminants and specificity
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