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Simultaneous multiple target detection in real-time loop-mediated isothermal amplification
Nathan A. Tanner, Yinhua Zhang, and Thomas C. Evans
New England Biolabs, Ipswich, MA, USA
BioTechniques, Vol. 53, No. 2, August 2012, pp. 81–89
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Supplementary Material

Loop-mediated isothermal amplification (LAMP) is a rapid and reliable sequence-specific isothermal nucleic acid amplification technique. To date, all reported real-time detection methods for LAMP have been restricted to single targets, limiting the utility of this technique. Here, we adapted standard LAMP primers to contain a quencher-fluorophore duplex region that upon strand separation results in a gain of fluorescent signal. This approach permitted the real-time detection of 1–4 target sequences in a single LAMP reaction tube utilizing a standard real-time fluorimeter. The methodology was highly reproducible and sensitive, detecting below 100 copies of human genomic DNA. It was also robust, with a 7-order of magnitude dynamic range of detectable targets. Furthermore, using a new strand-displacing DNA polymerase or its warm-start version, Bst 2.0 or Bst 2.0 WarmStart DNA polymerases, resulted in 50% faster amplification signals than wild-type Bst DNA polymerase, large fragment in this new multiplex LAMP procedure. The coupling of this new multiplex technique with next generation isothermal DNA polymerases should increase the utility of the LAMP method for molecular diagnostics.

Sequence-specific isothermal nucleic acid amplification techniques represent a growing sector of molecular diagnostics, offering rapid, sensitive detection without the need for thermal cycling equipment as required for the PCR. Furthermore, isothermal techniques typically provide comparable or better detection limits compared with PCR but in a fraction of the reaction time (1, 2). These methods have particular interest to field or point-of-care molecular diagnostics due to advantages in efficiency, cost, and instrumentation (3). In contrast to PCR, which denatures double-stranded DNA (dsDNA) with heat, isothermal amplification techniques typically use enzymatic activity to provide strand separation of dsDNA. Sequence specificity is provided by oligonucleotide primers that anneal to the target sequence and are extended by a strand displacing DNA polymerase. Several techniques require multiple enzymes to work in concert [e.g., strand displacement amplification (SDA), helicase dependent amplification (HDA), and isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN)], but loop-mediated isothermal amplification (LAMP) provides sequence-specific amplification using only a strand-displacing DNA polymerase (techniques reviewed in References 1 and 2). In addition to the DNA polymerase, LAMP uses four core primers (FIP, BIP, F3, and B3) recognizing six distinct sequence regions on the target (Figure 1), with two primers containing complementary sequence in order to create loop structures that facilitate exponential amplification (4). The use of multiple target sequence regions confers a high degree of specificity to the reaction. Two additional primers, termed loop primers, can be added to increase reaction speed, resulting in six total primers used per target sequence (5). The LAMP reaction rapidly (completed in as little as 5 min) generates amplification products as multimers of the target region in various sizes and is substantial in total DNA synthesis (>10 μg, >50× PCR yield) (4-6).

Figure 1.  DARQ in LAMP. (Click to enlarge)

Measurement of the LAMP product is typically performed by fluorescence detection of dsDNA with an intercalating or magnesium-sensitive fluorophore (4, 7), bioluminescence through pyrophosphate conversion (8), turbidity detection of precipitated magnesium pyrophosphate (9, 10), or even visual examination through precipitated Mg2P2O7 or fluorescence (11, 12). These methods are robust and familiar, and visual methods are ideal for use in field diagnostics, but detect total DNA amplification in a reaction and are thus limited to detection of a single target. As isothermal techniques are further adopted as diagnostic tools, the ability to detect multiple targets in a single sample will be important. Currently, quantitative PCR (qPCR) enables probe-specific multiplex detection and the ability to perform tests with an internal standard for definitive negative results. However, qPCR probes require extensive design and optimization for use and may not effectively translate to the LAMP reaction (13-16). Previous studies have demonstrated methods of multiplex LAMP detection, although they have been limited in capability. A variety of methods have used end point analysis, through agarose gel (17, 18) or pyrosequencing (19) methods, but these do not allow real-time detection and require further processing and instrumentation. In addition, the sensitivity of LAMP reactions to carryover contamination is so great that manufacturer recommendations (Eiken Chemical, Tokyo, Japan) suggest not opening LAMP reaction vessels, or doing so in separate facilities with separate equipment, further decreasing the desirability of post-LAMP manipulation. Previous real-time methods use nonspecific quenching, either through loss-of-signal guanine quenching (20) or gain-of-signal fluorescence using labeled primers and an intercalating dye (21). These methods can be less sensitive, and nonspecific quenching limits the selection of fluorophores available for multiplexing. Our approach was to develop a gain-of-signal, target-specific LAMP detection that is easily implemented and not limited in design or sensitivity.

The LAMP forward and back interior primers (FIP and BIP) contain 5′ flaps (Figure 1; F1c sequence) that, upon synthesis and displacement, will anneal to a complementary downstream region (F1). We chose this region for development of detection probes, as it is inherent to LAMP and contains sequence that is specific to each target, precluding any need for probe sequence optimization. LAMP also requires a strand displacing DNA polymerase (typically Bst DNA polymerase, large fragment), a component we utilize for detection through strand displacement. Using previously designed LAMP primers as a basis, we synthesized the FIP modified at the 5′ end with either a dark quencher or fluorophore. For probe creation, we annealed oligonucleotides complementary to the flap region (F1c) with a 3′ dark quencher or fluorophore spectrally overlapping with the fluorophore or dark quencher of the FIP (Figure 1A). This duplex primer maintains its function as a LAMP primer, but upon synthesis from the reverse direction the flap duplex is separated, resulting in detection of amplification by release of quenching (Figure 1B). A similar method using a short nested probe rather than amplification primer was applied to qPCR (22). Our method, which we termed detection of amplification by release of quenching or DARQ, is adaptable for any LAMP reaction and requires no additional probe design or testing, merely synthesis of a 5′-modified FIP and a 3′-modified complementary probe (termed Fd). Similar quencher:fluorophore duplex primers have been previously used in isothermal amplification, including LAMP with suggested use for mulitplexing, but to date no demonstration of real-time, multiplex LAMP has been achieved in this fashion (23-25). Here, we demonstrate the use of this method for LAMP detection in single and multiplex reactions, detecting up to four distinct LAMP targets in a single reaction.

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