Development of isothermal TaqMan assays for detection of biothreat organisms.
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.
Tong
Yanhong
Y
BioHelix Corporation, Beverly, MA 01915, USA. tong@biohelix.com
Tang
Wen
W
Kim
Hyun-Jin
HJ
Pan
Xiaojing
X
Ranalli
Tamara
T
Kong
Huimin
H
D001408:Bacillus anthracis / Q000235:genetics*; D001681:Biological Assay / Q000379:methods*; D004247:DNA / Q000235:genetics; D004265:DNA Helicases / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000191:economics / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000235:genetics*; D013696:Temperature; D014734:Vibrio cholerae / Q000235:genetics*
D017931:DNA Primers; D015342:DNA Probes; D004247:DNA; D019914:Taq Polymerase; D004265:DNA Helicases