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A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods

View Article: PubMed Central - PubMed

ABSTRACT

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

No MeSH data available.


Comparative analysis of qPCR and qLAMP assay amplification success. Plots displaying qPCR [(a) & (b)] and qLAMP [(c) & (d)] successful amplification data from three separate reactions per 3 experiments (n = 9).
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fig0015: Comparative analysis of qPCR and qLAMP assay amplification success. Plots displaying qPCR [(a) & (b)] and qLAMP [(c) & (d)] successful amplification data from three separate reactions per 3 experiments (n = 9).

Mentions: Standard curve-based analyses of the C. trachomatis and M. genitalium qPCR assays (Fig. 2a and b, Table S2a) show that the assays achieved good linearity and precision (mean R2 measurements of 1.00), displayed good levels of PCR sensitivity (Fig. 3a and b) and are capable of repeatedly detecting ∼5 copies of target material under experimental conditions.


A novel approach for evaluating the performance of real time quantitative loop-mediated isothermal amplification-based methods
Comparative analysis of qPCR and qLAMP assay amplification success. Plots displaying qPCR [(a) & (b)] and qLAMP [(c) & (d)] successful amplification data from three separate reactions per 3 experiments (n = 9).
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121211&req=5

fig0015: Comparative analysis of qPCR and qLAMP assay amplification success. Plots displaying qPCR [(a) & (b)] and qLAMP [(c) & (d)] successful amplification data from three separate reactions per 3 experiments (n = 9).
Mentions: Standard curve-based analyses of the C. trachomatis and M. genitalium qPCR assays (Fig. 2a and b, Table S2a) show that the assays achieved good linearity and precision (mean R2 measurements of 1.00), displayed good levels of PCR sensitivity (Fig. 3a and b) and are capable of repeatedly detecting ∼5 copies of target material under experimental conditions.

View Article: PubMed Central - PubMed

ABSTRACT

Molecular diagnostic measurements are currently underpinned by the polymerase chain reaction (PCR). There are also a number of alternative nucleic acid amplification technologies, which unlike PCR, work at a single temperature. These ‘isothermal’ methods, reportedly offer potential advantages over PCR such as simplicity, speed and resistance to inhibitors and could also be used for quantitative molecular analysis. However there are currently limited mechanisms to evaluate their quantitative performance, which would assist assay development and study comparisons. This study uses a sexually transmitted infection diagnostic model in combination with an adapted metric termed isothermal doubling time (IDT), akin to PCR efficiency, to compare quantitative PCR and quantitative loop-mediated isothermal amplification (qLAMP) assays, and to quantify the impact of matrix interference. The performance metric described here facilitates the comparison of qLAMP assays that could assist assay development and validation activities.

No MeSH data available.