Exogenous internal positive control

Abstract

The present invention provides an internal positive control for contaminating viruses. The invention provides the use of a second virus as an exogenous internal positive control in methods for verifying the reliability of an assay to detect a first virus, in methods of ensuring the absence of the first virus in a biological sample or pharmaceutical sample and in methods of manufacturing a vaccine free from a first virus.

Claims

1. A method for verifying the reliability of an assay to detect a first virus comprising the steps of: (a) adding an exogenous second virus to a biological sample prior to analysing the viral nucleic acids or polypeptides from the biological sample; and (b) analysing the viral nucleic acids or polypeptides from the biological sample to detect the first and second virus; wherein the first and second virus are the same type of virus and the second virus is an internal positive control; and wherein the first virus is an animal virus and the second virus is a plant virus.

2. A method of confirming that a biological sample is substantially free from a first virus, comprising the steps of: (a) adding an exogenous second virus to a biological sample prior to analysing the nucleic acids from the biological sample; (b) analysing the nucleic acids from the biological sample to detect the first and second virus; and wherein the first and second virus are the same type of virus and the second virus is an internal positive control; and wherein the first virus is an animal virus and the second virus is a plant virus.

3. The method of claim 1, wherein the first and second viruses are both filamentous viruses, icosahedral viruses, or complex viruses.

4. The method of claim 1, wherein the second virus is Alliaria petiolata Tymovirus (ApTV) comprising genomic RNA comprising RNA comprising a poly-ribonucleic acid sequence according to SEQ ID NO: 1-4, where thymidine bases are substituted with uridine bases.

5. The method of claim 1, wherein the first virus is an icosahedral animal virus.

6. The method of claim 1 wherein the biological sample is selected from the group consisting of a vaccine; an intermediate in vaccine production; blood; blood products, serum, plasma, red blood cells, white blood cells, platelets; tissue samples, bone marrow, kidney, liver, heart, lung, and skin.

7. The method of claim 2, wherein the biological sample is an influenza vaccine or an intermediate in the production of an influenza vaccine.

8. The method of claim 7, wherein the influenza vaccine is produced in cell-culture.

9. The method of claim 2 further comprising detecting the presence of the first virus in the absence of detecting the second virus.

10. The method of claim 2 further comprising detecting the presence of the second virus in the absence of the first virus.

11. The method of claim 9 further comprising the step of extracting the nucleic acids from the biological sample after adding the exogenous second virus to the biological sample.

Description

MODES FOR CARRYING OUT THE INVENTION

(1) Biological Samples

(2) Different biological samples were used during the development phase of the MRV RTD-PCR (table 2). Additionally, three potential inhibitory substances were investigated, which could potentially interfere with the internal positive control.

(3) TABLE-US-00002 TABLE 2 Investigated biological samples Batch Influenza Strain Serotype Fermenter harvests (B1): F110711_B1 (A/Solomon H1N1 Islands/3/06) F110714_B1 (A/Wisconsin/67/05) H3N2 F110717_B1 (B/Malaysia/2506/4) Victoria Seed virus: 522SSV0805 (B/Florida/4/06) Yamagata 522SSV0809 (A/Brisbane/59/07) H1N1 522SSV0811 (A/Uruguay/716/07) H3N2

(4) For the investigations of the influence of inhibitory substances on the performance of the RTD-PCR method, three different components were used. MDCK host cell DNA, soluble MDCK host cell proteins of a B1 supernatant and a concentrated influenza virus solution were selected as potential inhibitory substances. The MDCK host cell DNA was isolated from batch 22.09.05 (0.610.sup.7 MDCK cells/ml; manufactured in the laboratories of the Cell Culture Technology (TDM)). To produce a concentrated influenza virus solution and soluble MDCK host cell proteins, the F110829 B1 sample was centrifuged for 2 hours at 55,000 g. The pellet was resuspended in 5 ml PF/CDM media.

(5) The inhibitory substances and all investigated biological samples were characterized according to DNA, total protein content and influenza virus concentrations. The analytical data is summarized in table 3.

(6) TABLE-US-00003 TABLE 3 Summary of the analytical data for the three inhibitory substances and the matrices that were used during this evaluation. The DNA, the protein content and influenza virus concentration are the average of three determinations. Inhibitory DNA Protein Influenza viruses substances/matrices (ng/mL) (g/mL) (copies/mL) Inhibitory substances: Influenza viruses 3937 1129 1.80 10.sup.12 (F110829_B1) MDCK host cell 86539 ND ND DNA Matrices: F110711_B1 1206 102 1.54 10.sup.10 F110714_B1 644 83 2.54 10.sup.10 F110717_B1 757 51 6.12 109 Seed virus 1014 56 9.79 10.sup.9 522SSV0805 Seed virus 1080 45 1.84 10.sup.10 522SSV0809 Seed virus 300 23 6.35 10.sup.9 522SSV0811 ND Not detectable

(7) The Pico Green assay was used to quantify the DNA content of the inhibitory substances, the B1 and seed virus samples.

(8) The total protein content of the inhibitory substances and the matrices used was determined by the Bradford method. The test principle is as for a normal Bradford but with low protein concentrations. The samples were pre diluted with PBS buffer and measured against a BSA standard curve at 595 nm absorption. The dye reagent is the quick start Bradford dye reagent (150 l) from BioRad which was incubated with the samples (150 l) for 15 minutes before measurement.

(9) To quantify the influenza virus copy number in a sample, a quantitative one step RT-PCR was used. The samples were pretreated with 1.5 l RNase A/T1 (3 g RNase A and 7.5 U RNase T1) for 60 minutes at room temperature (about 22 C.) to digest free ssRNA in a sample to quantify only influenza RNA protected by virus particles. Afterwards, the RNA was extracted with a RNA specific nucleic acid kit (MagNA Pure Compact Nucleic Acid Isolation Kit ILarge Volume).

(10) For the quantitative RT-PCR, 5 l of sample was used. The influenza virus RNA was reverse transcribed (RT) and amplified (RT for 15 minutes at 50 C., Taq activation for 2 minutes at 95 C.) and detected by PCR (denaturing for 15 seconds at 94 C. for 45 cycles; annealing/elongation for 45 seconds at 45 C. for 45 cycles) using influenza A or influenza B specific primers and probes in a SmartCycler Cepheid. The samples were measured against a standard to quantify the influenza virus copy number. The standard is a ssRNA fragment that was synthesized and cloned into the KpnI and SacI sites of a T3/T7 transcription vector (pGA4-ampR). It was prepared as final ssRNA solutions of 10 ng/ml of ssRNA (1 ml per aliquot). To prevent a non-specific absorption of the low concentration of ssRNA to the tube, 100 ng/l yeast tRNA in 1TE (pH 8.0) was added.

(11) The effect of the choice of biological sample on variability of repeat assays was evaluated. The duplicate determination of MRV-1 in three different B1 samples showed only slightly differences between the determinations with a standard deviation of 0.72 Ct-values for the 6-FAM probe and 0.17 Ct-values for the Cy5 probe, respectively (table 4).

(12) TABLE-US-00004 TABLE 4 Ct-values of the evaluation of the influence of inhibitory substances in three different B1 samples. No significant difference was observed. Probe F110711_B1 F110714_B1 F110717_B1 Average StDev 6-FAM 29.33 29.94 30.94 30.14 0.72 30.59 29.29 30.74 Cy5 27.77 27.95 27.68 27.73 0.17 27.57 27.53 27.90
Primers, Probes and Reagents

(13) To show the robustness of the primer and probe concentrations, slightly differences in the given concentrations were used. The investigated concentrations were 0.5, 0.6 and 0.7 M for the MRV and ApTV primers (AV F primer: 5 CCC TGC TCC TAC TCA CAA TCT CC 3-SEQ ID NOs: 2 and AV R primer: 5 AGC TTT CCT CTC CCA CAT CA 3-SEQ ID NO: 3), 0.18, 0.20 and 0.22 M for the MRV probe and 0.08, 0.10 and 0.12 M for the ApTV probe. All combinations of the investigated concentrations were measured in duplicate.

(14) The investigation of slight differences in the primer and probe concentrations showed a standard deviation below 0.7 Ct-values (table 5). Additionally, the maximal deviation from the mean Ct value was below 1.5 Ct-values.

(15) TABLE-US-00005 TABLE 5 Results of the investigation of slightly differences in the primer and probe concentrations. No differences in the Ct-values of the MRV detection greater than 1.43 from the mean value detectable. Primer MRV 0.6 Primer ApTV 0.6 M/Probe 0.2 M M/Probe 0.1 M Ct-values Probe Ct-values Primer AV Probe AV MRV Primer MRV MRV (M) (M) (6-FAM) MRV (M) (M) (6-FAM) 0.5 0.08 28.26 0.5 0.18 28.70 0.5 0.08 27.84 0.5 0.18 29.45 0.6 0.08 29.03 0.6 0.18 27.10 0.6 0.08 33.79* 0.6 0.18 29.04 0.7 0.08 27.96 0.7 0.18 27.90 0.7 0.08 27.83 0.7 0.18 28.64 0.5 0.12 28.00 0.5 0.22 29.40 0.5 0.12 28.66 0.5 0.22 28.84 0.6 0.12 27.25 0.6 0.22 29.53 0.6 0.12 27.37 0.6 0.22 28.82 0.7 0.12 28.00 0.7 0.22 28.87 0.7 0.12 27.38 0.7 0.22 28.62 Average 27.96 Average 28.74 Standard deviation 0.54 Standard deviation 0.68 Max Ct-value 29.03 Max Ct-value 29.53 (difference) (+1.07) (difference) (+0.79) Min Ct-value 27.25 Min Ct-value 27.10 (difference) (0.71) (difference) (1.43)

(16) To show the robustness of the method, three different operators, three days, three batches of each primer and probe, three extraction kits and PCR kits were tested with two MagNA Pure LC extractors and two LightCycler 480 PCR machines. The mean Ct-value of the MRV detection (8 single determinations per sample) was investigated to show the robust detection of the virus.

(17) MRV could be detected in the 24 determinations (mean of 8 replicates) in the investigation of different reagent lots, with a standard deviation of 1.00 Ct-value (table 6). In three cases, one or two of the eight replicates per sample failed. However, overall the samples are termed positive.

(18) TABLE-US-00006 TABLE 6 Results of the investigation of different reagent lots. In all 24 determinations the MRV could be detected. Additionally, the determined Ct-values showed a standard deviation of 1.00 Ct-value. Ct- value PCR/ Primer and probe lot MRV extraction AV AV MRV MRV (6- Assay kit lot no. primer probe primer probe FAM) Average StDev 09110DW 10710420/ 1 1 1 1 28.33 28.95 1.00 24 Feb. 2009 14288500 2 2 1 1 31.34* 1 2 2 1 28.21 1 1 3 2 30.34 2 2 2 2 27.81 2 2 2 2 28.08 3 2 2 3 28.88 3 3 3 3 28.97 09112SG 13633721/ 1 1 1 1 28.66 25 Feb. 2009 14237900 2 2 1 1 27.70 1 2 2 1 30.96 1 1 3 2 28.78 2 2 2 2 29.15* 2 2 2 2 26.96** 3 2 2 3 27.96 3 3 3 3 28.89 09122GS 14532820/ 1 1 1 1 28.85 02 Mar. 2009 13632100 2 2 1 1 29.34 1 2 2 1 29.91 1 1 3 2 29.22 2 2 2 2 28.90 2 2 2 2 28.90 3 2 2 3 29.07 3 3 3 3 29.68 *Only 7 of 8 values positive **Only 6 of 8 values positive
Internal Positive Control for Nucleic Acid Extraction

(19) To control the efficiency of every extraction the ApTV extraction-internal positive control (EX-IPC) was spiked into every sample. The nucleic acids of the EX-IPC and MRV were amplified and detected by a different primer and probe set in a one step RT-PCR. Therefore, a competitive inhibition of one of the two targets is possible, when the concentration of one of the two targets is too high. Therefore, the concentration of the EX-IPC has to be adjusted to a concentration, that guaranteed the robust detection of the EX-IPC and also a sensitively detection of MRV.

(20) To show the robustness of the EX-IPC, 100, 200 and 300 pg/ml of the ApTV were used, with MRV concentrations of 10.sup.2 and 10.sup.3 TCID.sub.50/ml. Additionally, one sample without MRV was used. Per MRV and ApTV concentration, one determination was performed, in total nine determinations (six with MRV and three without MRV). The influence on the EX-IPC determination within MRV-free B1 samples (NGK-EX-IPC) was investigated.

(21) Only slightly differences in the MRV determination were observed with a maximal standard deviation of 0.56 Ct-values with EX-IPC concentrations in the range of 100-300 pg/mL. The EX-IPC determination in samples without MRV (NGK-EX-IPC) had an equal low standard deviation (0.46 Ct-values; table 7). The evaluation of 25 NGK-EX-IPC controls showed a mean Ct-value of 28.39 (Standard Deviation 1.37).

(22) TABLE-US-00007 TABLE 7 Ct-values of the MRV determinations with different EX-IPC concentrations. Conc. of the Ct-values at different MRV concentrations (TCID50/mL) EX-IPC 10.sup.3 (6-FAM) 10.sup.2 (6-FAM) NGK-EX-IPC (Cy5) 300 pg/ml 26.74 29.03 26.66 200 pg/ml 26.24 29.17 27.12 100 pg/ml 26.20 30.06 27.57 Average 26.39 29.42 27.12 StDev 0.30 0.56 0.46
Other Controls

(23) The second control (MRV-PC) controls the function of the 6-FAM labeled probe for the detection of MRV. The MRV-1 strain will be used at a concentration of 102 TCID50/mL in a MRV free B1 sample.

(24) The MRV-PC will be used for every assay. No ApTV will be spiked into the MRV-PC sample. The Ct-values for the MRV-PC will be monitored in a control chart to see slightly differences during time for the performance of the assay. The MRVPC will be spiked into a MRV-free B1 sample (NGK). The control is called NGK-MRV-PC.

(25) The third RTD-PCR control (MRV-IPC) is necessary to check the accurate performance of the amplification and detection during the RTD-PCR. The MRV-IPC is a 256 base pairs long ssRNA construct. The construct was produced by the company Panomics (Fremont, Calif.). The fragment was synthesized and cloned into the KpnI and SacI sites of a T3/T7 transcription vector (pGA4-ampR). It was prepared as final ssRNA solutions of 10 ng/mL of ssRNA. To prevent a non-specific absorption of the low concentration of ssRNA to the tube, 100 ng/L yeast tRNA in 1TE (pH 8.0) were added. The MRV-IPC will be amplified and detected by the ApTV specific primers and probe. The control demonstrates the functionality of the RTD-PCR in every assay. This control can distinguish between extraction and PCR errors.

(26) The forth control is the NTC (no template control). Here, only PCR water is used as template in the RTD-PCR. This control is a negative control for the assay and is used to show any contaminations during the performance of the assay.

(27) Detection of MRV and EX-IPC

(28) To show that there are no false positive results resulting from cross contamination during the nucleic acid extraction, 10.sup.5 TCID.sub.50/ml MRV samples and MRV free samples were extracted crosswise. Only the samples spiked with MRV should show a positive detection of MRV.

(29) MRV was detected in all samples where MRV was added. All samples without MRV were analysed as negative for MRV. The EX-IPC was detected in each case.

CONCLUSIONS

(30) The evaluation of the EX-IPC showed a robust performance. The investigation of slightly differences in primer and probe concentrations showed no significant influence. Additionally, the use of different batches of reagents showed also no influence on the RTD-PCR performance.

(31) There were no false positive or false negative results, and no cross contamination during the nucleic acid extraction between samples spiked with 10.sup.5 TCID.sub.50/ml and samples without a MRV was observed.

(32) It will be understood that the invention has been described by way of example only and modifications may be made whilst remaining within the scope and spirit of the invention.