IVDT_In Vitro Diagnostics Technology

IVD Technology, Fall 2013

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INSTRUMENTATION Performance Comparison of Three Molecular Diagnostic Test Systems Te extraction efciency of manual, semi-automated, and fully automated systems is analyzed. BY IRA ASHMAN P rior to the advent of polymerase chain reaction (PCR) chemistry in 1983, detection of infectious agents in biological specimens relied heavily on labor-intensive and often fawed culture-based methods. PCR provided researchers and clinical lab professionals a quicker and easier option to detect infectious agents, as results became available in a matter of hours. Yet original PCR formats proved less than ideal for most clinical lab applications, notably for workfow complexity and multiple instrument requirements together with high potential for errors resulting from cross-contamination of samples with amplicons. Te 1990s saw a major leap forward with the development of real-time PCR, which employed fuorescence detection technology and simplifed several aspects of the workfow. Varying degrees of automation evolved that not only provided workfow advantages and permitted use of closed systems to essentially eliminate cross-contamination errors but still required multiple instruments and workstations to perform testing. Several target and label amplifcation-based technology alternatives came into play over the years, yet real-time PCR emerged as the dominant clinical lab modality for infectious disease detection in terms of both commercial kits and user-developed protocols. Several systems that ofer varying levels of automation and modularity are currently available on the market, including the frst fully automated system capable of performing manufacturer-developed tests and user-defned protocols (UDPs). Te efciency and consistency with which a particular approach can extract nucleic acids from microorganisms in biological samples is a critically important contributor to assay sensitivity, the maximization of which is, in turn, critical to the medical utility of assays. Tis article presents results from internal feasibility studies that compare the extraction efciencies of three systems with the following features: • manual extraction and purifcation • automated extraction and purifcation • full walk-away automation for extraction, purifcation, amplifcation, and detection. Te manual extraction and purifcation method for DNA uses either fast-spin columns or vacuum procedures and for this study, purifed nucleic acids were analyzed using a stand-alone PCR instrument. Te manual protocol starts with enzymatic lysis of bacteria and capture of nucleic acids on silica gel membranes. Bufers and enzymes are optimized to lyse samples, stabilize nucleic acids, and enhance selective adsorption to the membranes. Lysed samples are then treated with alcohol and loaded onto the spin columns. Two wash steps remove trace quantities of any remaining potential PCR inhibitors, and the purifed DNA is eluted either with water or a low-salt bufer. Te automated nucleic acid isolation and purifcation system is capable of processing up to 32 samples per batch. Purifed nucleic acid samples can be transferred to a separate PCR instrument for amplifcation and detection. A combination of lysis bufer and proteinase K provide for cell disruption and protein digestion. Nucleic acids bind to the surface of magnetic glass particles, which are sequestered and washed to remove cellular debris, and nucleic acids are eluted from the beads at high temperature. Te instrument transfers reagents from container strips into a processing cartridge, after which lysates are transferred from the sample cartridge into reagent wells. Te reaction mixture is then moved to a second row of wells, which contain additional reagents. Next, the instrument sequesters beads in order to remove supernatants, and transfers the beads to a cartridge with wells that contain an elution bufer. Finally, purifed nucleic acid samples are transferred to a storage cartridge for subsequent analysis. Te BD MAX system integrates sample lysis, extraction, purifcation, amplifcation, and detection in a I VD T EC H N O L O G Y | F A LL 2 0 1 3 3 1 magenta cyan black ES320966_IV1309_031.pgs 09.19.2013 05:46 UBM

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