Abstract
Isothermal nucleic-acid amplification methods such as Loop-Mediated isothermal AMPlification (LAMP) are increasingly appealing alternatives to PCR for use in portable diagnostic system due to the low cost, weight, and power requirements of the instrumentation. As such, interest in developing new probes and other functionality based on the LAMP reaction has been intense. Here, we report on the development of duplexed LAMP assays for pathogen detection using spectrally unique Assimilating Probes. As proof of principle, we used a reaction for Salmonella enterica as a model coupled with a reaction for λ-phage DNA as an internal control, as well as a duplexed assay to sub-type specific quarantine strains of the bacterial wilt pathogen Ralstonia solanacearum. Detection limits for bacterial DNA analyzed in individual reactions was less than 100 genomic equivalents in all cases, and increased by one to two orders of magnitude when reactions were coupled in duplexed formats. Even so, due to the more robust activity of newly available strand-displacing polymerases, the duplexed assays reported here were more powerful than analogous individual reactions reported only a few years ago, and represent a significant advance for incorporation of internal controls to validate assay results in the field.
Highlights
IntroductionIsothermal nucleic acid amplification technologies have a significant advantage over polymerase chain reaction (PCR) as they can be implemented in a single step process at a constant temperature [1]
Isothermal nucleic acid amplification technologies have a significant advantage over polymerase chain reaction (PCR) as they can be implemented in a single step process at a constant temperature [1].Removing the constraint for rapid thermal cycling enables diagnostics to be conducted in small, simple, and low-power instruments in comparatively primitive conditions [2,3,4,5,6]
Detection limits for individual Loop-Mediated isothermal AMPlification (LAMP) reactions conducted under the conditions described here resulted in detection limits of 500 fg of DNA for reactions with Salmonella enterica (Se), Ralstonia solanacearum (Rs), and Rs Race 3 Biovar 2 (R3B2), equivalent to fewer than 100 genome equivalents per reaction in each of these cases (Table 1)
Summary
Isothermal nucleic acid amplification technologies have a significant advantage over polymerase chain reaction (PCR) as they can be implemented in a single step process at a constant temperature [1]. Implementing nucleic acid hybridization with the LFD format, often referred to as nucleic acid lateral-flow (NALF), can be an effective method of leveraging the simplicity of LFD and the power of gene-based diagnostics [22] This approach has been demonstrated for example to detect amplicons from specific LAMP assays [23]. Nucleic acid amplification such as with LAMP involves complex biochemical interactions which may fail due to inhibition or loss of activity of the different reaction components, so that incorporation of proper controls to validate assay performance is critical This is especially true when analyzing complex environmental, food, or clinical samples with unpredictable composition and numerous potential inhibitors. While Rs affects a wide variety of important crops and is extremely persistent in warm, humid tropics, R3B2 strains of Rs are of special concern in the US because they are adapted to cooler climates and could result in serious economic impacts to agricultural production in North America if they become established there [29]
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