For in vitro diagnostic use. Others cobas TaqScreen MPX Test version 2.0 IVD cobas® TaqScreen MPX Test, version 2.0 RMD-s201-MPX-001 05969492190 KIT s201 T-SCRN MPX v2.0 96T CE-IVD cobas TaqScreen MPX Test, version 2.0 00875197004052 Reagents, kits 1 kit 96 tests true The cobas® TaqScreen MPX Test, version 2.0 (v2.0) for use with the cobas s 201 system, is a qualitative in vitro test for the direct detection of Human Immunodeficiency Virus Type 1 (HIV-1) Group M RNA, HIV-1 Group O RNA, Human Immunodeficiency Virus Type 2 (HIV-2) RNA, Hepatitis C Virus (HCV) RNA and Hepatitis B Virus (HBV) DNA in human plasma.This test is intended for use to screen donor samples for HIV-1 Group M RNA, HIV-1 Group O RNA, HIV-2 RNA, HCV RNA and HBV DNA in plasma specimens from individual human donors, including donors of whole blood, blood components (red cells, platelets and plasma) and other living donors. This test is also intended for use to screen organ and tissue donors when specimens are obtained while the donor’s heart is still beating and in blood specimens from cadaveric (non-heart-beating) donors.Plasma from all donors may be screened as individual specimens. For donations of whole blood and blood components, plasma specimens may be tested individually or in pools comprised of aliquots of individual specimens in conjunction with serology tests for HIV, HCV and HBV.For an individual specimen, results are simultaneously detected and discriminated for HIV, HCV and HBV.The cobas® TaqScreen MPX Test, v2.0 can be considered a supplemental test that confirms HIV infection for specimens that are repeatedly reactive on a licensed donor screening test for antibodies to HIV and reactive on the cobas® TaqScreen MPX Test, v2.0.The cobas® TaqScreen MPX Test, v2.0 can be considered a supplemental test that confirms HCV infection for specimens that are repeatedly reactive on a licensed donor screening test for antibodies to HCV and reactive on the cobas® TaqScreen MPX Test, v2.0.The cobas® TaqScreen MPX Test, v2.0 can be considered a supplemental test that confirms HBV infection for specimens that are repeatedly reactive on a licensed donor screening test for hepatitis B surface antigen, and reactive on the cobas® TaqScreen MPX Test, v2.0.This test is not intended for use as an aid in diagnosis of infection with HIV, HCV or HBV. en The cobas® TaqScreen MPX Test, v2.0 used on the cobas s 201 system is based on 4 major processes:Automated Specimen Pooling/Pipetting and Control Pipetting using the optional Hamilton MICROLAB® STAR/STARlet IVD PipettorAutomated Specimen Preparation using the COBAS® AmpliPrep InstrumentAutomated Amplification of Nucleic Acid and Real Time Automated Detection and discrimination of PCR products using the COBAS® TaqMan® AnalyzerAutomated Data Management using the Pooling and Data Management (PDM) SoftwareAutomated Specimen Pooling and Pipetting using the Hamilton MICROLAB STAR/STARlet IVD PipettorThe optional Hamilton MICROLAB STAR/STARlet IVD Pipettor automates pipetting of pools and individual donor specimens, transfer of aliquots to Deep Well Plates (optional) and pipetting of Test Controls as part of the cobas s 201 system. The cobas s 201 system is used for testing donor pools and resolution testing of reactive pools to identify the reactive individual donor specimens. The cobas s 201 system is designed to process specimens in batches. A batch is defined as a collection of specimens and controls that are pipetted, extracted, amplified and detected together. When the pipetting of a batch is completed on the Hamilton MICROLAB STAR/STARlet IVD Pipettor, the batch is transferred into the COBAS® AmpliPrep Instrument for the next phase of the process. Manually pipetted specimens may be loaded directly onto the COBAS® AmpliPrep Instrument without prior use of the Hamilton MICROLAB STAR/STARlet IVD Pipettor.Note: For testing of cadaveric specimens, the specimen should first be manually diluted 1:5 in cobas® TaqScreen Cadaveric Specimen Diluent (CADV SPEC DIL) prior to pipetting using the Hamilton MICROLAB STAR/STARlet IVD Pipettor.Automated Specimen Preparation using the COBAS® AmpliPrep InstrumentNucleic acids from the targets and added Armored RNA Internal Control (IC) molecules (which serve as the specimen preparation and amplification/detection process control) are simultaneously processed. The cobas® TaqScreen MPX Test, v2.0 contains reagents that accomplish five sequential steps on the COBAS® AmpliPrep Instrument. The Proteinase Solution digests proteins to promote lysis, inactivate nucleases and facilitate the release of RNA and DNA from viral particles. Addition of Lysis Reagent to the specimen results in viral lysis and nuclease inactivation by denaturation of proteins. RNA and DNA are released and simultaneously protected from nucleases. The released nucleic acids bind to the silica surface of the added Magnetic Glass Particles. This is mainly due to the net positive charge on the glass particle surface and net negative charge of the nucleic acids under the chaotropic salt concentration and ionic strength of the Lysis reaction. Wash Reagent removes unbound substances and impurities such as denatured proteins, cellular debris and potential PCR inhibitors (such as hemoglobin, etc.), and reduces the salt concentration. Purified nucleic acids are released from the Magnetic Glass Particles at an elevated temperature with Elution Buffer.Automated Amplification of Nucleic Acid using the COBAS® TaqMan® AnalyzerAfter isolation of the purified nucleic acids from human plasma during automated specimen preparation, the cobas® TaqScreen MPX Test, v2.0 Master Mix (MPX2 MMX) is used for the amplification, detection and discrimination of HIV (HIV-1 Group M, HIV-1 Group O and HIV-2) and HCV RNA, HBV DNA and IC RNA. Once activated by the addition of manganese acetate, the cobas® TaqScreen MPX Test, v2.0 Master Mix permits reverse transcription (for RNA targets), followed by PCR amplification of highly conserved regions of HIV-1 Group M, HIV-1 Group O, HIV-2 and HCV RNA, HBV DNA and IC RNA using specific primers. Concurrent detection of the amplified nucleic acid is accomplished by the generation of fluorescent signals from 5'-nucleolytic degradation of the HIV-1 (Groups M and O), HIV-2, HCV, HBV and IC probes, also present in the Master Mix. Four unique fluorescent dyes are used: one dye labels the IC probe, and other three dyes label the HIV, HCV and HBV probes, permitting independent identification of the HIV, HCV and HBV targets, and the IC. All three HIV targets are identified using the same dye and thus are not discriminated from each other.Reverse Transcription and PCR AmplificationReverse transcription and amplification reactions are performed with a thermostable recombinant enzyme, Z05D DNA Polymerase. In the presence of manganese (Mn2+), Z05D DNA Polymerase has reverse transcriptase and DNA polymerase activities. This allows both reverse transcription and PCR amplification to occur in the same reaction mixture.PCR amplification is accomplished using the Z05D DNA Polymerase, which extends the annealed primers along the target templates to produce a double-stranded DNA (amplicon). This process is repeated for multiple cycles, with each cycle doubling the amount of amplicon DNA. Amplification occurs only in the region of the target genomes between the primers; the entire genomes are not amplified.Selective AmplificationSelective amplification of target nucleic acid from the specimen is achieved in the cobas® TaqScreen MPX Test, v2.0 by the use of AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes and catalyzes the destruction of DNA strands containing deoxyuridine24, but not DNA containing deoxythymidine or RNA containing ribouridine.25,26 Deoxyuridine is not present in naturally occurring DNA, but is always present in amplicon because of the use of deoxyuridine triphosphate as one of the dNTPs in the MPX2 Master Mix reagent; therefore, only amplicon contains deoxyuridine. Deoxyuridine renders contaminating amplicon susceptible to destruction by the AmpErase enzyme prior to amplification of the target DNA. Also, any nonspecific product formed after initial activation of the cobas® TaqScreen MPX Test, v2.0 Master Mix by manganese is destroyed by the AmpErase enzyme. The AmpErase enzyme, which is included in the MPX2 Master Mix reagent, catalyzes the cleavage of deoxyuridine-containing DNA at the deoxyuridine residues by opening the deoxyribose chain at the C1-position. When heated in the first thermal cycling step, the amplicon DNA chain breaks at the position of the deoxyuridine, thereby rendering the DNA non-amplifiable. The AmpErase enzyme remains inactive for a prolonged period of time once exposed to temperatures above 55ºC and therefore does not destroy target amplicon formed after PCR.Real time Automated Detection of PCR Products using the COBAS® TaqMan® AnalyzerDuring PCR Amplification, the intermittent high temperature during the cycling denatures the Target and IC amplicon to form single stranded DNA. The specific detection oligonucleotide probes hybridize to the single stranded form of the amplified DNA. Amplification, Hybridization and Detection occur simultaneously.Detection of PCR Products27,28The cobas® TaqScreen MPX Test, v2.0 Master Mix contains detection probes which are specific for HIV-1 (Groups M and O), HIV-2, HCV, HBV or IC nucleic acid. The HIV, HCV, HBV and IC detection probes are each labeled with 1) one of four fluorescent dyes which act as a reporter and 2) another dye which acts as a quencher. Three unique reporter dyes are associated with the HIV, HCV or HBV specific probes and are measured at defined wavelengths. A fourth reporter dye is associated with the IC specific probe and is measured at a different wavelength. A single type of quencher dye is used in all probes. This system permits simultaneous detection and discrimination of the amplified HIV, HCV and HBV targets, and the IC, using four wavelengths.Before PCR amplification begins, the probes are intact and the reporter dye fluorescence is suppressed by the quencher dye due to Förster-type energy transfer. During PCR amplification, the probes hybridize to specific single stranded DNA sequences and are cleaved by the 5' to 3' nuclease activity of the Z05D DNA Polymerase at the same time that amplification is occurring. Once the reporter and quencher dyes are separated by this cleavage, the fluorescent activity of the reporter dye is unmasked. With each PCR cycle, increasing amounts of cleaved probes are generated and the cumulative signal of the reporter dye is concomitantly increased.Real time detection and discrimination of PCR products is accomplished by measuring the fluorescence of the released reporter dyes representing the viral targets and IC.Automated Data Management using the PDM SoftwareRoche PDM software allows the user to review and report results. The Roche PDM software assigns test results for all tests as non-reactive, reactive or invalid. In addition to retrieving and examining PCR results, the Roche PDM software allows the operator to print reports, search for results, accept donor results and optionally transmit results to an LIS.24. Longo MC, Berninger MS, Hartley JL. Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions. Gene. 1990; 93:125-128.25. Savva R, McAuley-Hecht K, Brown T, Pearl L. The structural basis of specific base-excision repair by uracil-DNA glycosylase. Nature. 1995; 373:487-493.26. Mol CD, Arvai AS, Slupphaug G, et al. Crystal structure and mutational analysis of human uracil-DNA glycosylase: structural basis for specificity and catalysis. Cell. 1995; 80:869-878.27. Higuchi R, Dollinger G, Walsh PS, Griffith R. Simultaneous amplification and detection of specific DNA sequences. Biotechnology (NY). 1992; 10:413-417.28. Heid CA, Stevens J, Livak JK, Williams PM. Real time quantitative PCR. Genome Res. 1996; 6:986-994. en