Roche Molecular Biochemicals Application Note No. HP 1/1999 Nucleic Acid Purification High Pure Viral Nucleic Acid Kit High Pure 16 System Viral Nucleic Acid Kit Efficiency of Hepatitis C Virus sample preparation Thomas Walter*, Christel Adem, Brigitte Miedl, Sabine Philippi-Schulz** Roche Molecular Biochemicals, Nonnenwald 2, 82377 Penzberg, Germany * Roche Molecular System, Bahnhofstraße 9 15, 82327 Tutzing, Germany **Roche Diagnostics GmbH, Sandhofer Str. 116, 68305 Mannheim, Germany Hepatitis C Virus Pooled plasma COBAS AMPLICOR Introduction Hepatitis C Virus (HCV) is considered to be the principle etiologic agent responsible for 90-95% of the cases of posttransfusional non-a and non-b hepatitis (1,2). The singlestranded, positive sense RNA virus with a genome of approximately 10,000 nucleotides belongs to the family of flaviviridae. HCV can be transmitted by blood and blood products. Thus, the Paul Ehrlich Institut (PEI), the Federal office for serum and vaccines in Langen, Germany, has set guidelines for the risk reduction of HCV contamination in blood products and published requirements, which have been in effect since April 1999, for the validation of routine testing by HCV-nucleic acid amplification technology in blood banks (3). We have investigated the compliance of the High Pure Viral Nucleic Acid Kits in combination with the automated COBAS AMPLICOR PCR amplification / detection system with these requirements. Method HCV WHO International Standard (96/790) was obtained from the National Institute for Biological Standard and Control (NIBSC) and has a concentration of 50,000 IU/mL after reconstitution. The WHO International Standard was serially diluted with human EDTA plasma pools (each pool containing 48 plasma samples). The diluted samples contained 512, 260, 130, 65, 33, 16, 8, 4 and 2 IU/mL of HCV RNA (for the High Pure 16 System Viral Nucleic Acid Kit) or 260, 130, 65, 33, 16 and 8 IU/mL of HCV RNA (for the High Pure Viral Nucleic Acid Kit)*. For both kits, eight replicates of each pool at each dilution were tested to give a total of 24 (for the High Pure 16 System Viral Nucleic Acid Kit) or 16 (for High Pure Viral Nucleic Acid Kit) test results for each dilution. A negative and a positive control from the AMPLICOR HCV specimen preparation kit Version 2.0 was tested with each set of replicates as single determination. Experiments were performed with two different lots of reagents by two different operators for each purification kit. The purification protocol from the package insert was modified to accommodate the HCV internal control (IC) to give optimal compatibility with the COBAS AMPLICOR HCV V2.0, qualitative test. The complete protocol for the High Pure 16 System Kit is shown. A test result was considered positive if both the HCV and IC OD at 660 nm were 0.15. *(see tables 2-1 and 2-2 for relevant dilutions)
Materials and Protocol Materials High Pure 16 System Viral Nucleic Acid Kit, 6 x 16 purifications High Pure 16 System Heating Block High Pure Viral Nucleic Acid Kit COBAS AMPLICOR HCV Control Kit V2.0 8 Sets COBAS AMPLICOR HCV Specimen Preparation Kit V2.0 96 determinations COBAS AMPLICOR HCV Amplification Kit, V2.0, 96 determinations COBAS AMPLICOR HCV Detection Kit, V2.0, 96 determinations COBAS AMPLICOR Internal Control Detection Kit, V2.0 100 determinations Absolute ethanol, absolute iso-propanol Cat. No. 2 011 816 (Roche Molecular Biochemicals) Cat. No. 2 011 867 (Roche Molecular Biochemicals) Cat. No. 1 858 874 (Roche Diagnostics) ART: 1 111 167 (Roche Diagnostics) ART: 1 111 205 (Roche Diagnostics) ART: 1 111 000 (Roche Diagnostics) ART: 1 111 140 (Roche Diagnostics) ART: 0 757 608 (Roche Diagnostics) Table 1: Materials for Sample Preparation of Plasma or Serum Samples Protocol Preparation of the master-mix: Pipette 100 µl HCV Mn 2+ V2.0 into MasterMix vial (from COBAS AMPLICOR HCV, V2.0, ART: 1 111 000) to prepare Working MasterMix sufficient for 12 samples/controls. Invert Working MasterMix 15 x. For each sample, pipette 50 µl Working MasterMix in A-Tube, leave cover open and store at 4 8 C in a sealed plastic bag. (1) Preparation of reagents and samples: When opening a new High Pure Viral Nucleic Acid Kit dissolve and aliquot the poly(a) carrier RNA: Pipette 0.5 ml Elution Buffer (vial 5, colorless lid) to the poly(a) carrier RNA (vial 2, white lid) and close with rubber lid. Invert 15 x until the poly(a) carrier RNA has completely dissolved. Aliquot 80 µl in sterile, nuclease-free tubes and store at 20 C. Dissolve and aliquot the proteinase K: Pipette 5 ml Elution Buffer (vial 5, colorless lid) to the proteinase K (vial 3, white lid) and close with rubber lid. Invert 15 x until the proteinase K has completely dissolved. Aliquot 800 µl in sterile, nuclease-free tubes and store at 20 C. Wash Buffer, working solution: Wash Buffer (vial 4, blue lid) add 40 ml absolute ethanol, respectively and mix. Inhibitor Removal Buffer, working solution: Inhibitor Removal Buffer (vial 4a, black lid) add 15 ml absolute ethanol, respectively and mix. Before starting a series of samples: Preparation of the working-lysis reagent for 16 samples: Thaw one tube each of aliquoted poly(a) carrier RNA and proteinase K and mix with 4 ml Lysis/Binding Buffer (vial 1, green lid). Add 120 µl HCV IC V2.0 (IC= Internal Control from COBAS AMPLICOR HCV Specimen Preparation Kit, ART: 1 111 205) and mix thoroughly. Prepare lysis rack (colorless rack). Heat incubation block to 50 C. Heat Specimen Diluent to 50 C. Thaw samples and vortex for at least 5 sec. each. Do not vortex MasterMix! Comments Store activated MasterMix not longer than 4 h at 2 8 C. Label with content and date. The solution is stable for 12 months at 20 C. Label with content and date. The solution is stable for 12 months at 20 C. Ethanol should be of highest quality and not be stored in metal container (Inhibitors!) Mark addition of ethanol and date on the vial. Stable until the expiration date. Ethanol should be of highest quality and not be stored in metal container (Inhibitors!). Mark addition of ethanol and date on the vial. Stable until the expiration date. Always vortex HCV IC before addition to the lysis buffer. Working/Lysis Reagent is not stable and has to be prepared freshly for each series of samples. Use immediately.
Protocol (2) Label one well of the lysis rack as positive control, one well tube as negative control and one well for each patient sample. Pipette 250 µl working-lysis reagent into each well. (3) For each control, pipette 200 µl Normal-Human-Plasma (NHP) in the respective wells and vortex. Subsequently pipette 200 µl patient serum in the respective wells. Finally add 20 µl negative- or 20 µl positive AMPLICOR controls in the respective wells, close wells and vortex gently. (4) Incubate lysis rack for 10 min at 50 C in the heating block. Comments Conical tubes from Sarstedt with screw lid, Cat.-No. 72.692.005 1.5 ml possibly with special stand Cat.-No. 93.848.100 are recommended. AMPLICOR Controls: Always vortex the controls for 10 15 seconds before use. In any case pipette NHP, patient samples and controls in the order given, to inactivate RNases in the NHP! Use the High Pure 16 System Heating Block, Cat. No. 2 011 867 (5) Centrifuge lysis rack briefly (30 sec) in a table top microtiterplate centrifuge. (6) Add 100 µl iso-propanol to every well and vortex. Centrifuge lysis rack briefly (30 sec) in a table top microtiterplate centrifuge. (7) Transfer samples completely to the upper reservoir of the prepared High Pure filter tube rack (yellow rack). Close the lids and centrifuge for 2 min at 2,000 x g in a microtiterplate centrifuge. Iso-propanol should be of highest quality and not be stored in metal container (Inhibitors!) The protocol is standardized for 200 µl sample volume. For different sample volumes see instructions in the package insert of the High Pure 16 System Viral Nucleic Acid Kit (8) Add 400 µl Inhibitor Removal Buffer working solution (black lid) to the upper reservoir of the filter tubes. Close the lids and centrifuge for 2 min at 2,000 x g in a microtiterplate centrifuge. (9) Add 450 µl Wash Buffer working solution (blue lid) to the upper reservoir of the filter tubes. Close the lids and centrifuge for 2 min at 2,000 x g in a microtiterplate centrifuge. (10) Add 450 µl Wash Buffer working solution (blue lid) to the upper reservoir of the filter tubes. Close the lids and centrifuge for 3 min at 2,200 x g in a microtiterplate centrifuge to remove wash buffer completely (11) Dispose waste container and insert filter tube rack in the elution rack (blue rack). For elution of viral nucleic acids pipette 200 µl of the 50 C preheated Specimen Diluent (from COBAS AMPLICOR HCV Specimen Preparation Kit ART: 1 111 205) to the High Pure Filter Tubes. Close the lids and centrifuge for 3 min at 2200 x g in a microtiterplate centrifuge (12) Discard the High Pure Filter Tube rack and cover the Elution rack with the elution rack cover (blue). (13) Add 50 µl of the purified control- and patient samples to the Working MasterMix (each 50 µl) in the A-ring. Prepared Samples and controls can be stored at room temperature for 3 h before addition to the MasterMix. If amplification is not possible within this time, prepared samples can be stored at 20 C to 80 C up to 1 month. After addition of the prepared samples to the Working Master- Mix in the A-tubes, the amplification has to start within 45 min.
Results The results from this study are presented in Table 2-1 for the High Pure 16 System Viral Nucleic Acid Kit and in Table 2-2 for the High Pure Viral Nucleic Acid Kit. For both purification procedures the COBAS AMPLICOR HCV qualitative test V2.0 detected the WHO International Standard at 260, 130, 65 and 33 IU/mL 100% of the time. At 16 IU/mL, the COBAS AMPLICOR HCV qualitative test V2.0 detected 20 of the 24 replicates for High Pure 16 System Viral Nucleic Acid Kit and 9 of 16 replicates for the High Pure Viral Nucleic Acid Kit resulting in a 83.3% and 56.25 % positive rate, respectively. A Probit analysis of the experiments indicating the minimal HCV concentration that is detected positive with 95% probability was performed. It was found that the High Pure 16 System Viral Nucleic Acid Kit and the High Pure Viral Nucleic Acid Kit have a comparable detection limit of 30 IU/ml and 33 IU/mL, respectively (Table 3 and Diagram). There was no lot to lot variation for both kits and identical results have been obtained by two different operators. Thus, the PEI specification for detection of HCV in plasma pools corresponding to a concentration of 5000 IU/mL in an individual donation can be fulfilled with a proposed pool size of 48.
Results (continued) OD at 660 nm HCV RNA Target and Internal Control Replicate 8 IU/mL 16 IU/mL 33 IU/mL 65 IU/mL 130 IU/mL 260 IU/mL No. HCV IC HCV IC HCV IC HCV IC HCV IC HCV IC 1 0.003 > 4.000 1.567 > 4.000 1.447 > 4.000 > 4.000 3.603 > 4.000 > 4.000 > 4.000 > 4.000 2 0.004 3.768 0.167 > 4.000 3.824 > 4.000 3.260 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 3 0.389 > 4.000 0.003 2.639 3.456 > 4.000 3.699 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 4 0.004 > 4.000 0.003 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 5 3.111 3.888 1.315 9.999 > 4.000 > 4.000 3.707 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 6 0.703 3.888 2.713 3.888 3.407 > 4.000 3.532 > 4.000 > 4.000 > 4.000 > 4.000 3.710 7 1.849 3.888 0.894 3.888 3.018 3.134 > 4.000 3.947 > 4.000 3.947 3.947 > 4.000 8 1.027 3.736 1.045 3.611 3.771 > 4.000 3.294 > 4.000 3.947 2.771 > 4.000 3.947 9 0.009* 0.013* 1.175 3.614 3.123 > 4.000 2.676 3.996 3.998 > 4.000 > 4.000 > 4.000 10 0.007 9.999 1.101 3.314 1.422 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 11 0.645 9.999 1.813 9.999 3.453 3.519 > 4.000 3.519 > 4.000 > 4.000 > 4.000 > 4.000 12 1.805 3.313 1.977 2.565 2.120 > 4.000 2.861 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 13 0.736 3.301 0.007 3.602 1.835 > 4.000 3.705 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 14 0.360 3.163 0.655 2.179 1.743 > 4.000 3.051 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 15 0.008 3.727 0.685 2.636 3.246 3.768 3.100 > 4.000 3.945 3.166 > 4.000 3.342 16 0.311 3.360 0.650 3.302 3.546 > 4.000 2.192 3.944 > 4.000 3.643 3.945 > 4.000 17 0.005 9.999 0.786 3.912 1.951 > 4.000 3.824 3.824 > 4.000 3.824 > 4.000 > 4.000 18 0.820 9.999 1.603 9.999 2.864 3.533 3.222 > 4.000 3.824 3.699 > 4.000 > 4.000 19 1.834 9.999 0.006 3.913 2.124 > 4.000 3.709 3.312 > 4.000 > 4.000 > 4.000 > 4.000 20 0.010 3.847 2.113 2.154 2.631 3.399 3.290 3.203 > 4.000 3.767 > 4.000 3.943 21 0.009 3.723 1.016 3.547 1.838 3.377 2.310 1.853 3.767 3.642 3.943 > 4.000 22 0.008 0.308 1.763 2.519 1.323 3.143 2.230 3.620 3.642 3.745 > 4.000 3.745 23 0.010 3.179 1.009 3.046 2.407 > 4.000 3.633 > 4.000 > 4.000 3.855 3.730 > 4.000 24 0.005* 0.002* 2.314 > 4.000 3.633 3.855 3.855 > 4.000 > 4.000 > 4.000 > 4.000 3.332 No. Positive 12/22 20/24 24/24 24/24 24/24 24/24 % Positive 54.5% 83.3% 100% 100% 100% 100% * Invalid result due to failure to recover Internal Control Table 2-1. Results of High Pure 16 System Viral Nucleic Acid Kit (results are shown only for 260 8 IU/mL dilutions)
Results (continued) OD at 660 nm HCV RNA Target and Internal Control Replicate 8 IU/mL 16 IU/mL 33 IU/mL 65 IU/mL 130 IU/mL 260 IU/mL No. HCV IC HCV IC HCV IC HCV IC HCV IC HCV IC 1 0.004 > 4.000 2.031 > 4.000 2.758 > 4.000 3.603 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 2 0.003 > 4.000 0.003 > 4.000 3.779 > 4.000 2.121 > 4.000 2.966 2.701 > 4.000 > 4.000 3 0.817 > 4.000 0.621 > 4.000 1.820 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 > 4.000 4 0.004 3.495 0.724 > 4.000 3.836 > 4.000 2.104 3.298 3.261 > 4.000 > 4.000 3.738 5 0.007 > 4.000 0.006 > 4.000 3.738 > 4.000 3.437 > 4.000 > 4.000 > 4.000 > 4.000 3.738 6 0.006 > 4.000 1.836 > 4.000 2.925 > 4.000 2.942 3.562 > 4.000 > 4.000 > 4.000 2.303 7 0.360 3.951 2.789 3.774 3.406 1.145 2.598 > 4.000 3.774 3.348 > 4.000 3.075 8 0.669 2.930 1.020 3.553 2.561 3.553 2.746 2.910 3.774 3.252 > 4.000 3.775 9 0.006 3.165 2.110 > 4.000 0.912 3.943 3.243 1.162 3.202 3.943 3.545 3.943 10 0.848 > 4.000 0.005 > 4.000 1.956 > 4.000 2.031 3.918 > 4.000 > 4.000 > 4.000 > 4.000 11 1.032 3.617 0.004 2.804 3.316 > 4.000 > 4.000 > 4.000 > 4.000 3.919 > 4.000 3.743 12 0.005 > 4.000 0.004 > 4.000 3.315 > 4.000 > 4.000 3.742 > 4.000 3.441 > 4.000 > 4.000 13 1.012 > 4.000 1.628 > 4.000 2.458 > 4.000 2.840 > 4.000 > 4.000 3.770 > 4.000 > 4.000 14 0.004 > 4.000 0.003 > 4.000 2.184 > 4.000 > 4.000 1.332 > 4.000 > 4.000 > 4.000 > 4.000 15 0.003 > 4.000 0.003 3.594 1.489 3.373 3.770 2.925 > 4.000 3.168 > 4.000 > 4.000 16 1.832 > 4.000 1.017 > 4.000 > 4.000 3.854 2.775 > 4.000 > 4.000 > 4.000 > 4.000 3.009 No. Positive 7/16 9/16 16/16 16/16 16/16 16/16 % Positive 43.75% 56.25% 100% 100% 100% 100% Table 2-2: Results of High Pure Viral Nucleic Acid Kit (results are shown only for 260 8 IU/mL dilutions) Method Lower 95% conf. limit Concentration [IU/mL] upper 95% conf. limit High Pure 16 System Viral Nucleic Acid Kit 18 30 51 High Pure Viral Nucleic Acid Kit 20 33 56 Table 3: Results of Probit analysis
% PCR Positive Results Sensitivity: HCV Detections Limit 100,0% 80,0% 60,0% High Pure 16 System High Pure Viral NA 40,0% 20,0% 0,0% 8 16 33 65 130 260 HCV [IU/mL] Diagram: Sensitivity, HCV Detection Limit Discussion Sample preparation is an important part of nucleic acid amplification determining in part the detection limit of the test system. Through the removal of PCR inhibitors, sample preparation is also important in determining the upper sample volume that can be applied to the reaction. This study proved the successful purification of HCV Viral RNA from plasma pools and the compatibility of the High Pure Viral Nucleic Acid Kits with COBAS AMPLICOR HCV amplification. References (1) Alter, H. 1991 Descartes before the horse: I clone therefore I am: The Hepatitis C virus in current perspective. Annals of Internal Medicine 115:644-649. (2) McHutchinson, J., Person, J., Govindarajan, S. et al. 1992. Improved detection of Hepatitis C antibodies in high-risk populations. Hepatology 15: 19-25. (3) Anforderungen an Validierung bzw. Routinebetrieb der HCV-NAT im Blutspendewesen at http://www.pei.de/massnahm/valid.htm
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