Abstract
The technique of loop-mediated isothermal amplification (LAMP) utilizes four (or six) primers targeting six (or eight) regions within a fairly small segment of a genome for amplification, with concentration higher than that used in traditional PCR methods. The high concentrations of primers used leads to an increased likelihood of non-specific amplification induced by primer dimers. In this study, a set of LAMP primers were designed targeting the prfA gene sequence of Listeria monocytogenes, and dimethyl sulfoxide (DMSO) as well as Touchdown LAMP were employed to increase the sensitivity and specificity of the LAMP reactions. The results indicate that the detection limit of this novel LAMP assay with the newly designed primers and additives was 10 fg per reaction, which is ten-fold more sensitive than a commercial Isothermal Amplification Kit and hundred-fold more sensitive than previously reported LAMP assays. This highly sensitive LAMP assay has been shown to detect 11 strains of Listeria monocytogenes, and does not detect other Listeria species (including Listeria innocua and Listeria invanovii), providing some advantages in specificity over commercial Isothermal Amplification Kits and previously reported LAMP assay.
Highlights
Loop-mediated isothermal amplification, developed and reported by Notomi et al, in 2000 [1], can sensitively and rapidly amplify nucleic acids with two pairs of primers recognizing 6 independent sequences of a target gene under isothermal conditions
The loop-mediated isothermal amplification (LAMP) assay theoretically has the advantage of specificity, selectivity, and rapidity over polymerase chain reaction (PCR) [3,4], nucleic acid sequence based amplification (NASBA) [5,6], strand displacement amplification (SDA) [7], rolling circle amplification (RCA) [8], helicase dependent amplification (HDA) [9], and cross-priming amplification assay (CPA) [10,11]
There were only two primers, non-specific amplification occurred in the isothermal amplification of four primer combinations out of seven combinations, and it was more obvious in the reaction of three primer combinations, as Table 1 shown
Summary
Loop-mediated isothermal amplification, developed and reported by Notomi et al, in 2000 [1], can sensitively and rapidly amplify nucleic acids with two pairs of primers recognizing 6 independent sequences of a target gene under isothermal conditions. The LAMP assay theoretically has the advantage of specificity, selectivity, and rapidity over polymerase chain reaction (PCR) [3,4], nucleic acid sequence based amplification (NASBA) [5,6], strand displacement amplification (SDA) [7], rolling circle amplification (RCA) [8], helicase dependent amplification (HDA) [9], and cross-priming amplification assay (CPA) [10,11]. There is still no report studying non-specific amplification and cause of false positive results in LAMP reactions at present. For practical application of LAMP as well as reduction of the rate of false positive results in LAMP reactions, most researches currently focus on development of closed-tube detection to reduce aerosol pollution and cross pollution, which include the use of turbidity [12], SYBR Green I [13], PicoGreen [14], GelRedTM [15,16], lateral flow dipstick [17], hydroxynaphthol blue dye [18], malachite green [19], microfluidic chips and GMR sensors as well as calcein used by Eiken Chemical Co., Ltd [20,21].
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