IVDT_In Vitro Diagnostics Technology

IVD Technology, Fall 2013

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INSTRUMENTATION Figure 1. Comparison of PCR effciency. Target = Salmonella typhimurium fully-automated walk-away system capable of running 24 samples at a time.1 It uses a combination of lytic enzymes and extraction reagents to generate samples for subsequent amplifcation and detection. Lysis liberates nucleic acids, which are captured on high-afnity magnetic beads. Te instrument employs a magnet to sequester the beads for removal of the supernatant. Te frst reagent solution is added, beads are washed and incubated with a second reagent solution, and the supernatant containing target for analysis is mixed with a third reagent in preparation for PCR analysis. All reagents and consumables required for lysis, nucleic acid extraction, purifcation, amplifcation and detection are loaded into a Unitized Reagent Strip (URS). Strips are loaded into the instrument, which runs through the entire assay process in walk-away mode. In real-time PCR, a useful metric for measuring the relative performance of an assay is percent efciency, which is defned by the following equation: E = (10-1/slope – 1) x 100. Te slope is derived from a graph in which the x-axis contains the log of analyte concentration, and the y-axis contains Ct, the cycle threshold for each data point. Efciency levels between 90% and 110% represent the industry standard for efcacious real-time PCR analysis.2 Results from two sets of feasibility experiments clarify performance diferences among the three aforementioned Figure 2. Comparison of extraction effciency. Target = Salmonella typhimurium systems. A frst set of experiments compares the automated extraction system (which requires a separate instrument for PCR amplifcation/detection) and the fully automated system (which performs all steps on a single instrument) for efciency in extracting DNA from a bacterial target. To this end, it was necessary to frst assess the efciency of the PCR portion of each system. A sample of Salmonella typhimurium spiked to a level of 107 CFU/ml was extracted in both systems. Eluted DNA was subject to fve ten-fold dilutions, and each of the fve diluted samples, which provided from 105 down to 101 copies of DNA per PCR reaction mixture, were amplifed on the respective instruments. Te results (Figure 1) show that for this assay, the PCR portions of both systems are inherently efcient (automated extraction/PCR: E = 97.2%; full automation: E = 93.9%). To compare extraction efciencies of both systems (i.e., automated extraction followed by PCR and the fully automated system), a quantitated S. typhimurium suspension was subject to fve ten-fold dilutions prior to extraction, with each of the fve diluted samples run in triplicate through each system. Results (Figure 2) show that the PCR profle for the fully automated system fts within optimal limits (E = 105.9%), whereas the PCR profle for the automated extraction is outside these limits (E = 70.1%). Extraction on a fully automated system also exhibited higher sensitivity than the automated extraction system, since it was able to detect analyte in the most dilute sample. A second set of experiments sought to compare all three systems (i.e., manual extraction/PCR, automated extraction/PCR, and full automation) in order to determine how extraction-related diferences afect the potential sensitivity of detection. To mimic actual clinical samples, a suspension of S. typhimurium was diluted with bufer containing a stool matrix to the level of 5 x 106 CFU/ml. From that, four additional ten-fold dilutions were made using the same bufer/matrix combination. Te fve resulting solutions were then split three ways. Two sets were run Figure 3. Manual extraction: PCR versus full automation. Target = Salmonella typhimurium 32 IVD TEC HNOLO G Y | FA L L 2013 magenta cyan yellow black ES320823_IV1309_032.pgs 09.19.2013 05:03 UBM

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