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.
journal_name
BMC Biotechnoljournal_title
BMC biotechnologyauthors
Hardinge P,Murray JAHdoi
10.1186/s12896-019-0549-zsubject
Has Abstractpub_date
2019-08-01 00:00:00pages
55issue
1issn
1472-6750pii
10.1186/s12896-019-0549-zjournal_volume
19pub_type
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journal_title:BMC biotechnology
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更新日期:2007-02-15 00:00:00
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abstract:: ...
journal_title:BMC biotechnology
pub_type: 杂志文章
doi:10.1186/s12896-019-0504-z
更新日期:2019-02-18 00:00:00
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