IVDT_In Vitro Diagnostics Technology

IVD Technology, November/December 2012

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MOLECULAR DIAGNOSTICS dependant amplifi cation (HDA), loop- mediated amplifi cation (LAMP), or recombinase/polymerase amplifi cation (RPA) have any capability to prevent primer-primer interactions that lead to amplifi ed primer artifacts, which limit sensitivity and multiplexing. To overcome this problem we developed a novel method called blocked-primer helicase-dependant amplifi cation (bpHDA), which uses the isothermal nucleic acid amplifi ca- tion method HDA to exponentially amplify target DNA sequences coupled with a blocked primer/RNase H2-mediated target-specifi c "hot start." In bpHDA, modifi ed blocked primers are constructed with a single ribonucleotide linkage 3 to 5 bases upstream of a 3'-end block, to prevent primer extension. Once blocked, prim- ers hybridize to complementary target sequences, and thermostable RNase H2 derived from Pyrococcus abyssi is activated, cleaving the ribonucleotide linkage in the primer present in duplex DNA. Th e short segment of the primer 3' of the ribonucleotide dissoci- ates, liberating the block and creating a free 3'-hydroxyl, which is now capable of primer extension. RNase H2 used here has very little activity at tempera- tures below 400ºC and is highly active at 650ºC, the temperature required for HDA to amplify target sequences optimally. Because primer-primer hybrids are unstable at elevated tem- peratures, primer artifact is not ampli- fi ed. Unblocked primers can partici- pate in DNA amplifi cation reactions using HDA, which uses the enzyme DNA helicase to unwind double- stranded amplifi ed DNA unlike heat in PCR. Exponential amplifi cation occurs identically to PCR with primers being extended by DNA polymerase (Figure 1). Because primer artifact is mitigated in bpHDA, we are able to use higher primer concentrations allowing for faster reactions. For example, the genome of Mycobacterium tuberculosis is very GC-rich, making amplifi ca- tion of target sequences diffi cult due to artifact formation. In an applica- r 65ºC, Cleavage X Extension tional benefi t of bpHDA is improved consistency in amplifi cation perfor- mance compared with standard HDA protocols. In a duplex designed to co- amplify toxigenic Clostridium diffi cile and a sample process control organ- ism, Staphylococcus aureus, we observed robust and consistent amplifi cation with bpHDA, whereas standard HDA yielded inconsistent amplifi cation due to the presence of competing primer artifact.2 genomic DNA in 17 minutes by real- time amplifi cation reactions.2 Figure 1: BpHDA. At lower temper- atures, blocked primer (black) bound to the target sequence (white) can- not be extended by DNA polymerase due to the presence of a 3' block- ing group (X = 3-carbon spacer). At elevated temperatures, RNase H2 is activated, and a ribonucleotide (r) near the 3'-end of the primer is cleaved, liberating a 3'-hydroxyl group, allowing for primer extension by DNA polymerase (circle). For other bacterial genomes with more typical GC content, rates of amplifi cation are very high. For example, amplifi cation of the tcdB gene within C. diffi cile genome occurs with a doubling time measured to be 28 seconds, allowing for detection of the amplifi cation of a single copy of tion directed at detecting mutations in the rpoB gene of M. tuberculosis, we compared the performance of bpHDA to HDA with unmodifi ed primers. In standard HDA, we were only able to use up to 50 nM primer concentra- tions. Under these conditions ampli- fi cation reactions were slow, requiring one hour to generate a detectable signal from 30,000 copies of genomic DNA with a limit of detection of 100 copies even after 90 minutes of ampli- fi cation time. For bpHDA, similar results were observed at 50 nM primer amounts; at higher primer concentra- tions, a single copy of genomic DNA was detectable after 40 minutes of amplifi cation time, whereas with stan- dard HDA, only primer artifact was observed.1 18 IVD TECHNOLOGY | NOVEMBER/DECEMBER 2012 Th is is particularly signifi cant at lower-input organism amounts. Detection Technology Array technology currently is domi- nated by fl uorescence or electrochemi- cal signaling methods. However, these technologies require either expensive imaging approaches or more com- plex surface preparation. Th e Portrait PA5000 system utilizes inexpensive silicon chips modifi ed such that inter- molecular interactions that occur on the surface of the chip are amplifi ed and can transduce intensity changes in the surface color that are apparent to the naked eye, permitting visual detec- tion of attomole quantities of nucleic acid hybrids. Because of this, low cost CMOS or CCD cameras can be used to capture chip images for subsequent image analysis, lowering device cost. Epitaxial silicon wafers are coated and processed using semiconductor processing equipment, allowing for highly scalable, low-cost produc- tion. Th e wafer coating consists of hydrophobic polymeric siloxane that is conformal to the molecularly fl at polished silicon surface. Th e molecular fl atness and low chemical reactivity of the modifi ed silicon surface combine to exhibit very low nonspecifi c bind- ing properties. To facilitate probe attachment, two additional chemical coatings are applied to create a surface with functional aldehyde groups. We then apply DNA probes modifi ed with terminal hydrazide groups, creating a stable hydrazone linkage allowing for one-step chemical attachment. Th e chemistry is very effi cient, allowing ivdtechnology.com An addi- X OH

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