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 05 969 484 190 5 969 484 190 05969484190 5969484190 05969484190 KIT s201 T-SCRN MPX v2.0 96T US-IVD cobas TaqScreen MPX Test, version 2.0 00875197004045 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 for HIV-1 Group M RNA, HCV RNA, and HBV DNA in plasma specimens from individual human donors, including donors of whole blood, blood components, source plasma and other living donors. The test is also intended for use in testing plasma specimens to screen organ and tissue donors for HIV-1 Group M RNA, HCV RNA, and HBV DNA when specimens are obtained while the donor’s heart is still beating, and in testing blood specimens from cadaveric (non-heart beating) donors. This test is not intended for use on samples of cord blood. 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 not more than 6 individual specimens. For donors of hematopoietic stem/progenitor cells (HPCs) sourced from bone marrow, peripheral blood or cord blood, and for donors of donor lymphocytes for infusion (DLI), plasma may be tested in pools comprised of not more than 6 individual specimens. For donations of source plasma, sample may be tested in pools comprised of not more than 96 individual specimens.Whereas this test can detect HIV-1 Group O RNA and HIV-2 RNA, detection of HIV-1 Group O RNA or HIV-2 RNA in donor specimens negative for anti-HIV-1 Group O antibodies or anti-HIV-2 antibodies, respectively, has not been demonstrated in clinical studies.This test is intended to be used in conjunction with licensed serology tests for HIV, HCV, and HBV.For an individual specimen, results are simultaneously detected and discriminated for HIV, HCV, and HBV.This test is not intended for use as an aid in diagnosis of infection with HIV, HCV, or HBV.The cobas® TaqScreen MPX Test, v2.0 can be considered a supplemental test that confirms HIV infection for specimens that arerepeatedly reactive on a licensed donor screening test for antibodies to HIV and reactive for HIV 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 arerepeatedly reactive on a licensed donor screening test for antibodies to HCV and reactive for HCV 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 arerepeatedly reactive on a licensed donor screening test for Hepatitis B surface antigen, and reactive for HBV on the cobas® TaqScreen MPX Test, v2.0. en 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, 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, HCV RNA and HBV DNA in plasma specimens from individual human donors, including donors of whole blood, blood components, source plasma and other living donors.This test is also intended for use in testing plasma specimens to screen individual organ and tissue donors when specimens are obtained while the donor’s heart is still beating, and in testing blood serum and plasma from cadaveric (non-heart-beating) donors. This test is not intended for use on samples of cord blood.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 not more than 6 individual specimens. For donors of hematopoietic stem/progenitor cells (HPCs) sourced from bone marrow, peripheral blood or cord blood, and for donors of donor lymphocytes for infusion (DLI), plasma may be tested in pools comprised of not more than 6 individual specimens. For donations of source plasma, sample may be tested in pools comprised of not more than 96 individual specimens.Whereas this test can detect HIV -1 Group 0 RNA and HIV -2 RNA, detection of HIV -1 Group 0 RNA and HIV -2 RNA in donor specimens negative for anti-HIV-1 Group 0 antibodies or anti-HIV-2 antibodies, respectively, has not been demonstrated in clinical studiesThis test is intended to be used in conjunction with licensed 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 Hepatitis B Virus, Hepatitis C Virus, and Human Immunodeficiency Virus (Types 1 + 2) Nucleic Acid Test Kit (PCR-Fluorescence Assay) (cobas® TaqScreen MPX Test, version 2.0), 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, Human Immunodeficiency Virus Type 1 (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 screening of samples from blood donors, including HIV-1 Group M RNA, HIV-1 Group O RNA, HIV-2 RNA, HCV RNA and HBV DNA in pooled or individual samples. Donors may be donors of whole blood, donors of various blood components (red blood cells, platelets and plasma) and other types of donors.All plasma to be tested may be screened as individual specimens. For donations of whole blood and blood components, plasma specimens may be tested individually or in pools after mixing equal volumes of specimens in conjunction with serology tests for HIV, HCV and HBV.For specimens from blood donors, the reagent tests a pool of 6 specimens. If the result of the pooled sample is positive, resolution testing of the individual specimens is conducted. The result of an individual resolution test serves as the final test result for that specimen. For source plasma specimens, the reagent can at most test a pool of 96 specimens. If the result of the pooled sample is positive, resolution testing is conducted. The result of an individual resolution test serves as the final test result for that specimen. The final resolution testing of an individual specimen can discriminate between reactivity of HIV, HCV and HBV viruses.For specimens repeatedly reactive on screening tests for HIV antibodies, HCV antibodies and Hepatitis B surface antigen, and also reactive on the cobas® TaqScreen MPX Test v2.0, the cobas® TaqScreen MPX Test, v2.0 may be considered a supplemental test that confirms the infection of these specimens with HIV, HCV, and HBV. By itself, the test cannot serve 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 Hamilton MICROLAB® STAR/STARlet IVD Pipettor (optional)Automated 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 Pipettor The 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 Instrument Nucleic 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® Analyzer After 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 RNA, 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 RNAs, 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 Amplification Reverse 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 Amplification Selective 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 nonamplifiable. 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® Analyzer During 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 Pooling and Data Management (PDM) SoftwareRoche PDM software allows the user to review and report results. The Roche PDM software assigns test results for all tests as nonreactive, 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 a Laboratory Information System (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 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 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 The cobas® TaqScreen MPX Test, v2.0 used on the cobas s 201 system is based on 4 major steps as follows:1. Automated Specimen Pooling/Pipetting and Control Pipetting using the Hamilton MICROLAB® STAR/STARlet Pooling Instrument; this step may also be completed manuallyAutomated Extraction of Sample Nucleic Acid and Preparation of the2. PCR Reaction System using the COBAS®AmpliPrep Instrument3. Automated Amplification of Nucleic Acid and Real Time Automated Detection and Discrimination of PCR Products using the COBAS® TaqMan® Analyzer4. Fully Automated Data Management using the Pooling and Data Management (PDM) SoftwareOptionally, Automated Specimen Pooling/Pipetting and Control Pipetting using the Hamilton Microlab® STAR Pooling InstrumentThe Hamilton MICROLAB®STAR/STARlet Pooling Instrument automates addition of samples to individual specimens, pooling, and pipetting of test controls. Source collection tubes containing plasma and negative or positive controls tested in the same batch are simultaneously loaded onto the Pooling Instrument, which automatically pipettes specimens from the collection tubes and carries out pooling according to the specific instruction, such as individual testing, pooled testing or retesting. Negative and positive controls are also pipetted to obtain a group of samples and controls constituting a batch. When the pooling of a batch and pipetting of test controls is completed on the Pooling Instrument, the batch is transferred to the COBAS®AmpliPrep Instrument for the next process phase.The COBAS®AmpliPrep Instrument automatically uncaps, pipettes, incubates and elutes extracted nucleic acids, preparing the reaction solution for PCR amplification. Batches which have completed all processes on the COBAS®AmpliPrep Instrument are automatically transferred to the COBAS®TaqMan®Analyzer for amplification and detection. After completion of testing, data is sent to the PDM, and the operator carries out appropriate treatment of data on the PDM platform.During the pooling process on the HAMILTON Microlab®STAR/STARlet, transferring samples to deep-well plates (for temporary storage) before removing the samples from the deep-well plates for pooling is an option. Another option is not to use the HAMILTON Microlab®STAR/STARlet Pooling Instrument, instead selecting manual sample loading in the software operating system, and then directly loading the manually prepared test batch, including a set of samples and controls, into the COBAS®AmpliPrep Instrument.Automated Specimen Preparation using the COBAS®AmpliPrep InstrumentViral nucleic acids in the specimens and added RNA Internal Control (IC) molecules (which serve as the internal control for the specimen extraction, amplification and detection processes) are simultaneously processed in this step. Thecobas® TaqScreen MPX Test, v2.0 contains all reagents necessary for accomplishing 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 decomposition by nucleases. The released nucleic acids bind to the silica surface of the 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 high salt concentration and high detergent conditions of the lysis reaction. The 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 surface of the Magnetic Glass Particles at an elevated temperature in Elution Buffer.Automated Amplification of Nucleic Acid using the COBAS® TaqMan® AnalyzerAfter isolation of the purified nucleic acids from plasma using COBAS® AmpliPrep, the Master Mix (MPX2 MMX) in the cobas® TaqScreen MPX Test, v2.0 performs the amplification, detection and discrimination of HIV (HIV-1 Group M, HIV-1 Group O and HIV-2), HCV RNA, HBV DNA and IC RNA. Once activated by the addition of manganese acetate, the Amplification Reagent in the cobas® TaqScreen MPX Test, v2.0 permits reverse transcription (for RNA targets), followed by specific amplification of highly conserved regions of HIV-1 Group M RNA, HIV-1 Group O RNA, HIV-2 RNA, HCV RNA, HBV DNA and IC RNA using specific primers. In addition, concurrent detection is accomplished by collecting fluorescent signals released from 5'-nucleolytic degradation of the HIV-1 (Groups M and O), HIV-2, HCV, HBV and IC probes, which are also present in the Amplification Reagent. 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 simultaneously 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 the target nucleic acid from the specimen is achieved in thecobas® TaqScreen MPX Test, v2.0 by the use of the AmpErase (uracil-N-glycosylase) enzyme and deoxyuridine triphosphate (dUTP). The AmpErase enzyme recognizes and lyses DNA strands containing deoxyuridine24 but has no effect on RNA containing deoxythymidine or ribouridine.25, 26 Deoxyuridine is not usually present in DNA, but as deoxyuridine triphosphate is used as one of the dNTPs in the Amplification Reagent, only the amplicon contains deoxyuridine. The deoxyuridine-containing amplicon is destroyed by the AmpErase enzyme prior to amplification of the target DNA. Also, any nonspecific product formed after activation of thecobas® TaqScreen MPX Test, v2.0 Amplification Reagent by manganese is destroyed by the AmpErase enzyme. The AmpErase enzyme, which is included in the Amplification 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 55°C 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 to form single stranded DNA. The specific oligonucleotide probes hybridize with the single stranded form of the amplified DNA. Amplification, Hybridization and Detection occur simultaneously.Detection of PCR Products 27, 28The cobas® TaqScreen MPX Test, v2.0 Amplification Reagent contains detection probes which are specific for HIV-1 (Groups M and O), HIV-2, HCV, HBV and IC nucleic acids. Each detection probe is labeled with 1) one of four fluorescent dyes which act as a reporter and 2) another dye which acts as a fluorescence quencher. Three special 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 must be 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 with 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 may be expressed. 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 reporter dyes representing the viral targets and IC.Automated Data Management using the Pooling and Data Management (PDM) SoftwareThe Roche 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 results and 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

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