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Simultaneous multiple target detection in real-time loop-mediated isothermal amplification
 
Nathan A. Tanner, Yinhua Zhang, and Thomas C. Evans
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Supplementary Material

An essential consideration for amplification techniques or any diagnostic methodology is proper identification of positive reactions from false positive or negative reactions, either from nontemplate (amplification of primers without template DNA) or nonspecific amplification (amplification of a nontarget DNA present in the sample), although the latter is unlikely in LAMP due to the use of six sequence-specific primers per target. LAMP and other techniques can result in nontemplate amplification due to several factors, including the nature of LAMP primers and the reaction conditions, which include high concentrations of primer (4.4 μM total) and magnesium (8 mM). The degree of this nontemplate amplification is determined by the specific primer sequences (27), and we demonstrate this in DARQ in Figure 5, which shows a high degree of nontemplate amplification for one primer set (E. coli dnaE), an intermediate level for another (bacteriophage λ), and none for two sets (C. elegans lec-10 and human BRCA1). Importantly for diagnostic testing and multiplexing, the reactions containing many different templates maintained their level of specificity, i.e., the positive and nontemplate (nonspecific) signal was identical for a primer set whether no template or several nonspecific genomic DNAs were present. Figure 5B shows this specificity, with each primer set amplifying target identically in the presence of either a single genomic DNA (dashed lines) or four genomic DNAs (solid lines). Each negative control reaction (dotted lines) was carried out with three nonspecific genomic DNAs, and no effect was seen on nontemplate amplification, e.g., C. elegans lec-10 primers gave no signal without any genomic DNA (Figure 5A) or in the presence of bacteriophage λ, human, and E. coli DNA (Figure 5B). This allows accurate detection and quantification of a target (e.g., human BRCA1) in the presence of high levels of other genomic DNAs (e.g., 100 ng bacteriophage λ, 82.5 ng C. elegans, and 100 ng E. coli). Although we did not test clinical samples with DARQ, LAMP has been widely used for specimen diagnostics, afforded by an increased tolerance to typical contaminants and inhibitors (29). There is no obvious reason that DARQ detection would not effectively translate into diagnostics using clinical samples, however this needs to be experimentally verified.




Figure 5.  Nontemplate, negative control signals in DARQ LAMP reactions. (Click to enlarge)


DARQ detection provides a method for multiplex detection of LAMP amplification with no need for additional primer optimization or probe design, requiring only the use of a 5′ modified LAMP primer (FIP) with complementary detection oligonucleotide (Fd). This detection methodology may be extendable to other nucleic acid amplification techniques by the addition of a duplex “tail” to a primer in the reaction. Upon extension from the opposite direction, the duplex would be displaced with detection upon release of quenching. Our detection method is a simple extension of LAMP to accommodate robust, target-specific, and multiplex detection. As molecular diagnostics become more prominent and accepted in healthcare, the ability to detect multiple targets and use of internal controls will further the utility and flexibility of LAMP.

Acknowledgments

The authors thank Dr. Gregory Lohman for helpful discussion, Drs. Andrew Gardner, Gregory Lohman, and Jennifer Ong for critical reading of the manuscript, and New England Biolabs, Inc. for financial and material support.

Competing interests

The authors are employed by New England Biolabs, Inc., manufacturer of the described enzymes.

Correspondence
Address correspondence to Thomas C. Evans, Jr., New England Biolabs, Inc., 240 County Rd., Ipswich, MA, USA. e-mail: [email protected]

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