TaqMan probe (dual-labeled DNA probe)–based real-time detection, one of the most sensitive and specific fluorescent detection methods, has been widely utilized in conjunction with polymerase chain reaction (PCR). Helicase-dependent amplification (HDA) is an isothermal amplification technology that has a similar reaction scheme to PCR, but replaces thermocycling with a helicase capable of unwinding a DNA duplex. Here we describe a novel isothermal real-time detection method (HDA-TaqMan) that combines the advantages of both HDA and a TaqMan assay. In this assay, the reactions of DNA unwinding, primer annealing, polymerization, probe hybridization, and subsequent hydrolysis by the polymerase are coordinated and synchronized to perform at a single temperature. It not only provides a useful tool for real-time detection of HDA, but also provides an isothermal format for the TaqMan system. With this platform, we have successfully developed rapid real-time isothermal assays for biodefense targets that include Vibrio cholerae and Bacillus anthracis.
Nucleic acid amplification platforms can be plagued with non-specific amplification that can lead to false-positive or -negative results in real-time detection. The use of sequence-specific probes rather than non-specific DNA intercalating dyes can ameliorate this false-positive problem. Currently there are several sequence-specific probes for use in real-time amplification and detection, for example, molecular beacons (1), hydrolysis probes [TaqMan, 5′ nuclease assays (2)], and FRET hybridization probes (3). The TaqMan assay is one of the most frequently utilized platforms for both research and molecular diagnostics. Several new technologies have been combined with the TaqMan platform to improve the detection performance (i.e., signal-to-noise ratio). For example, locked nucleic acids (LNA) and minor groove binding protein (MGB) can enhance the melting temperature (Tm) of a shorter length probe (4). A TaqMan probe with the MGB or LNA modification can increase the sensitivity and accuracy of allele determination in real-time PCR or other methods that use differential hybridization to distinguish polymorphisms (5,6,7).
Interestingly, the TaqMan probe has not been applied to the real-time detection of isothermal amplification products. There are several platforms for isothermal amplification, including strand displacement amplification [SDA (8)], transcription-mediated amplification [TMA (9)], rolling cycle amplification [RCA (10)], loop-mediated amplification LAMP [11,]), nucleic acid sequence-based amplification [NASBA (12)], and helicase-dependent amplification [HDA (13,14,15)]. Most of the isothermal amplification technologies rely on a 5′-3′ exonuclease deficient DNA polymerase (SDA, RCA, LAMP) or an RNA polymerase (TMA, NASBA). The limitation of the choices of polymerases and the complicated reaction schemes have been a barrier to the implementation of TaqMan assays in isothermal real-time detection. Instead, isothermal amplification platforms have used a variety of other probe-based detection methods such as molecular beacons with NASBA (16), and fluorescence transfer probes (dual-dye labeled hairpin probe) with SDA (17).
HDA differs from other isothermal amplification technologies because it employs a similar reaction mechanism to PCR, with the exception that HDA uses a helicase enzyme rather than heat to separate double-stranded DNA or RNA. Like PCR, the simple reaction scheme requires a pair of primers, a protein mix (helicase and DNA polymerase), and buffer. The similarity between HDA and PCR and the simplicity of the HDA scheme makes it easier to utilize different real-time PCR detection platforms with HDA. In addition, HDA has the potential to be utilized in conjunction with a portable real-time analyzer, allowing for the creation of a low-cost isothermal quantitative platform. In our previous research, we have successfully developed real-time HDA assays that employ EvaGreen DNA intercalating dye, molecular beacons, and MGB Eclipse probes (Nanogen, Inc., Bothell, WA, USA) (15,18). We also found that real-time signal can be detected from a TaqMan MGB probe used with the commercially available thermostable HDA (tHDA) IsoAmp II kit (BioHelix Corp., Beverly, MA, USA) that contains the exonuclease-deficient DNA polymerase (data not shown). However, because the TaqMan MGB probe had slightly higher background fluorescence than an MGB Eclipse probe, the signal-to-noise ratio was low. In a standard tHDA reaction, while the uncleaved TaqMan probe can bind to the complementary target to generate fluorescent signal, the amplified DNA could also bind to the target as well. This binding competition causes the fluorescent signal to stop increasing at a certain point even under asymmetric amplification conditions. This also reduced the signal-to-noise ratio. All of these factors had prevented the use of a TaqMan probe in conjunction with tHDA. In this article, we have adapted the HDA platform for use with TaqMan probes for real-time detection through the use of a DNA polymerase with 5′ to 3′ exonuclease activity and developed the isothermal HDA-TaqMan method.
Vibrio cholerae and Bacillus anthracis are two highly important biothreat organisms. V. cholerae, a class B biothreat agent, can cause cholera, a severe diarrheal disease. It can be quickly spread through contaminated water and can cause a high degree of mortality. Screening the water supply or other contaminated materials to detect the presence of this bacterium will allow for the prevention of infection as well as earlier treatment, both of which will in turn reduce mortality. B. anthracis, a class A biothreat agent, has been previously utilized in a bioterrorism attack in Washington, DC in 2001. The ability to reliably, sensitively, and cost-effectively detect B. anthracis in environmental samples is critical for the prevention of widespread casualties. In this article, we have applied the HDA platform to the rapid real-time identification of the above organisms.