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Genmark pcr2/19/2024 ![]() The GenMark eSensor RVP has been shown to be highly comparable to other multiplex PCR platforms as well as singleplex real-time PCR in terms of diagnostic sensitivity and specificity, which measures the level of correlation between two methods. Viral pathogenic nucleic acid can be detected with confidence when measurements are at or exceed 3 nanoamps (nA) on the GenMark XT-8 instrument. Upon application of a low voltage current, the hybridized molecule bound to this solid phase brings the ferrocene in close proximity to the gold electrode where reversible electron transfer can occur and the resulting current can be measured. This hybridized molecule is then exposed to another sequence-specific probe which is bound to a solid phase, a gold electrode. Once the amplicons are in a single-stranded state, they are hybridized to a complementary virus-specific signal probe tagged with ferrocene, a reducing agent. Nucleic acids from targeted viral pathogens are amplified using a multiplex PCR reaction followed by denaturation of the double stranded molecules into single oligonucleotide strands using exonuclease. is a multiplex PCR panel that detects the amplification of various viral gene fragments electrochemically. The Respiratory Viral Panel (RVP) manufactured by GenMark Diagnostics, Inc. New and improved workflow designs make it possible for laboratories with varied molecular technical ability to implement multiplex PCR platforms. Multiplex PCR methods, those that target more than one pathogen in a single test, benefit diagnostics in a clinical laboratory due to their ability to detect and rule-out many related pathogens in the same amount of time. The relationship between TCID 50/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported. Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). ![]() Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. The GenMark eSensor RVP LODs were obtained by converting the TCID 50/mL concentrations reported in the package insert to copies/μL using qPCR. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens.
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