Fluorescent LAMP Primer Design

Fluorescently labelled quenched primers^{13 (link)} were synthesised by Sigma with the fluorophore attached to the 5’ end of the oligonucleotide without further adaptation. JOE (6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein) selected for detection on the yellow channel of a Qiagen (Hilden, Germany) RotorGene thermal cycler. Labelled FIP LAMP primers for 35Sp and NOSt purified to the HPLC level (Table 3).Table 3

LAMP quenched fluorescent primers Modified 35S promoter and NOS terminator FIP primers with JOE (6-carboxy-4’,5’-dichloro-2’,7’-dimethoxyfluorescein) attached to the 5’ end^{13 (link)}.

Target, Type, Notation	Lgth	Primer Sequence (5’ to 3’)
35Sp, LAMP, JOE-FIP	45	[JOE]CCACGTCTTCAAAGCAAGTGG-TTTT-GGATAGTGGGATTGTGCGTC
NOSt, LAMP, JOE-FIP	46	[JOE]GCATGACGTTATTTATGAGA-TTTT-TCGCGCTATATTTTGTTTTCTA

The quenched fluorescent LAMP primer was synthesised and HPLC purified by Sigma Aldrich (Poole, UK).

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Hardinge P, & Murray J.A. (2020). Full Dynamic Range Quantification using Loop-mediated Amplification (LAMP) by Combining Analysis of Amplification Timing and Variance between Replicates at Low Copy Number. Scientific Reports, 10, 916.

Publication 2020

RotorGene thermal cycler

Adaptation Hplc Oligonucleotide Primer

Corresponding Organization : Cardiff University

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Variable analysis

independent variables

Fluorescently labelled quenched primers

dependent variables

Detection on the yellow channel of a Qiagen (Hilden, Germany) RotorGene thermal cycler

control variables

The fluorophore (JOE) attached to the 5' end of the oligonucleotide without further adaptation
Labelled FIP LAMP primers for 35Sp and NOSt purified to the HPLC level

controls

Positive control: None specified
Negative control: None specified

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