Compositions and methods for detecting biological contaminants - Patents.com

Abstract

To provide compositions and methods useful to the determination of whether a microbial contaminant is present in a biological therapeutic production process.SOLUTION: Provided are an artificial positive amplification control plasmid and unique quantitative PCR detection probe are provided, which enables the rapid and real-time detection of a false positive result. Also provided is a composition comprising an artificial nucleotide sequence, a fluorophore, and a quencher. Here, the artificial nucleotide sequence comprises at its three-prime end no more than five consecutive nucleic acids that are identical to any one sequence in a group of sequences.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. §119(e) to U.S. Provisional Patent Application No. 62 / 139,321, filed March 27, 2015, which is specifically incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates generally to processes for producing biological molecules by cell culture. The present invention more specifically, but not exclusively, relates to compositions and methods for detecting biological contaminants in cell cultures. [Background technology]

[0003] 2. Background of the Invention Biopharmaceuticals, especially therapeutic antibodies, are produced by mammalian cell culture. Chinese Hamster Ovary (CHO) cells are the most commonly used host cells. These production systems are prone to exogenous and endogenous viral infections, which present potential safety issues for biopharmaceuticals. Therefore, to improve drug safety, viral clearance procedures and viral load measurements are used. Steps employed to reduce viral load include nanofiltration, viral inactivation by heat or pH retention, and chromatography. Viral load, and viral clearance efficacy, can be monitored by time-consuming infectivity assays or by rapid quantitative assays such as real-time PCR or quantitative polymerase chain reaction (Q-PCR).

[0004] Q-PCR requires proper negative and positive controls to be reliable. Nucleic acid extraction controls are added to test samples to control for proper nucleic acid extraction. If the nucleic acid extraction control is negative or outside the expected recovery range during Q-PCR, the sample is rejected. Conversely, if the nucleic acid extraction control is positive or within the expected recovery range during Q-PCR, the nucleic acid extraction from the test sample is considered reliable. A negative control, such as no sample buffer, is usually included in the Q-PCR assay. The presence of a positive signal in the negative control may signify contamination of the Q-PCR or nucleic acid extraction reagents with viral material.

[0005] A positive growth control may also be included in the Q-PCR viral load assay. Such a positive control may include a conserved viral nucleic acid sequence that is amplified using primers that amplify authentic viral contaminants. The inability of Q-PCR to detect the positive control may indicate that the amplification procedure would not have been able to detect the viral contaminant if it was present in the test sample.

[0006] The use of positive amplification controls that mimic the target contaminants creates its own problems. Given the excellent sensitivity of Q-PCR, if a test sample is contaminated with even a small amount of positive control, the test sample may show a false positive. False positives can be mitigated to some extent by using low levels of positive amplification controls, using an isolated room for positive control work, using the UNG / dUTP system that selectively degrades dUTP-containing PCR products, using single-use containers and displacement pipettes, and by thoroughly cleaning the work area and equipment. Despite the meticulous use of these mitigation measures, false positive results still occur during PCR testing.

[0007] In biopharmaceutical manufacturing, the risk of false positives for biological contaminants is not negligible and can result in costly corrective measures.There is a great need for a system and method to determine whether a given positive PCR result is a true positive or a false positive caused by cross-contamination of positive controls.Applicants have developed and herein disclose positive control compositions, systems and methods that allow real-time determination of false positive Q-PCR signals. Summary of the Invention

[0008] The applicants have solved the problem of identifying in real time whether a positive Q-PCR signal for a target contaminant in a test sample is a true positive or a false positive due to cross-contamination. The applicants have created a positive amplification control (PAC) plasmid that includes a biological contaminant target sequence (i.e., a positive control sequence) and a unique artificial plasmid-specific sequence. This unique artificial plasmid-specific sequence allows the assay technician to specifically identify the plasmid if it is present in the sample. Thus, if a positive contaminant signal is detected and the artificial plasmid-specific sequence is determined to be absent, the technician can be confident that the result is a true positive result. Conversely, in the event of a false positive, the presence of the unique artificial plasmid sequence allows the technician to quickly eliminate the ostensible positive result as a false positive.

[0009] In some embodiments, the positive control unique artificial plasmid-specific sequence ("unique sequence"; aka PACP or positive growth control polynucleotide) is detected using a fluorescently labeled artificial oligonucleotide detection probe ("unique detection probe" or "UDP") included in the Q-PCR reaction mixture. The unique detection probe comprises a nucleic acid polymer covalently attached to a fluorophore and a quencher. The "unique" nucleic acid polymer is designed to specifically anneal to the unique sequence and be incorporated into the amplicon copy of the unique sequence during PCR. The "unique" nucleic acid polymer is designed not to recognize or anneal to any naturally occurring parvovirus under the hybridization conditions employed in performing the subject assay. In one embodiment, the "unique" nucleic acid polymer comprises 17-20 nucleotides, where no more than 7-10 consecutive internal nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence. In one embodiment, a "unique" nucleotide polymer comprises 17-20 nucleotides, where no more than 7-10 consecutive nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence set forth in SEQ ID NOs:9 and 12-37. If the nucleic acid polymer of the unique detection probe is not incorporated into an amplicon copy, the quencher remains in close proximity to the fluorophore. If the fluorophore is excited, the quencher absorbs the emitted light and prevents it from being detected (by FRET or contact quenching). If the nucleic acid polymer is incorporated into an amplicon copy (i.e., if the unique sequence is present in the sample), the fluorophore and quencher are released from the unique detection probe and are therefore spatially separated. In this case, when the fluorophore is excited, the quencher is far enough away that it cannot efficiently quench the emitted light. Thus, the emitted light can be detected. Thus, if the unique sequence is present in the sample, the detectable emission wavelength increases in intensity as the PCR proceeds. If the unique sequence is absent, the quencher performs its function and no emission wavelength is detected as the PCR proceeds.In one embodiment, the fluorophore is attached at or near the 5' end of the nucleic acid polymer and the quencher is attached at or near the 3' end. In an alternative embodiment, the fluorophore is attached at or near the 3' end of the nucleic acid polymer and the quencher is attached at or near the 5' end.

[0010] Any fluorophore-quencher pair now known or later discovered may be used in the practice of the present invention. (See, e.g., SA Marras, "Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes", Methods Mol. Biol. 2006; 335:3-16.) In some embodiments, the fluorophore has an excitation wavelength of 495 nm, 538 nm, or 646 nm, and an emission wavelength of 520 nm, 554 nm, or 669 nm, respectively. In some embodiments, the quencher is a dye with an absorption peak between 430 nm and 672 nm. In some embodiments, the quencher is selected from the group consisting of DDQ-I, Dabcyl, Eclipse, Iowa Black FQ, BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black RQ, QSY-21, and DHQ-3. In one embodiment, the fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 520 nm (e.g., FAM) and the quencher is BHQ-1. Contains the nucleic acid sequence of TIFF0007669448000001.tif4128.

[0011] One aspect of the present invention is the unique detection probe itself described above, which contains a nucleic acid polymer and a linked fluorophore and quencher. Another aspect of the present invention includes the positive growth control (PAC) plasmid itself, which contains a biological contaminant target sequence and a unique artificial plasmid-specific sequence, and the use of that plasmid as a positive control to evaluate the presence of the target biological contaminant in a cell culture. The PAC plasmid is used to control the success of a PCR amplification reaction designed to amplify the target biological contaminant sequence. For example, a separate and parallel PCR reaction is performed, which contains the same components and is performed under the same parameters as the test sample, but contains the positive control plasmid instead of the test sample. If the positive control plasmid-containing sample generates a positive "contaminant" signal, but the test sample does not, it can be concluded that the test sample lacks the target biological contaminant. In some embodiments, the test sample is obtained from a mammalian cell culture, such as a bioreactor culture containing CHO cells modified to produce a therapeutic protein of interest.

[0012] In one embodiment, the PAC plasmid contains (a) a target amplification polynucleotide (TAP) sequence, such as a parvovirus nucleic acid sequence or another target contaminant sequence, and (b) a plasmid amplification control polynucleotide (PACP) sequence. The PACP sequence (sense or Crick strand) is complementary to the nucleic acid polymer of the unique sequence probe (antisense or Watson strand).

[0013] In one embodiment, the PAC plasmid is employed in a separate Q-PCR reaction in parallel with the Q-PCR reaction containing the test sample. The TAP sequence is designed to represent the target biological contaminant. For example, if the Q-PCR reaction of the test sample cannot produce a TAP amplicon and the Q-PCR of the positive control (i.e., the PAC plasmid-containing sample) cannot produce a TAP amplicon, the Q-PCR reaction can be assumed to have failed. In one embodiment, the target biological contaminant is a rodent parvovirus, and the TAP sequence comprises a rodent parvovirus sequence. In one embodiment, the TAP sequence comprises all or part of the parvovirus NS1 sequence that is conserved across several rodent parvovirus lineages. See Cotmore, et al., "Replication Initiator Protein NS1 of the parvovirus Minute Virus of Mice Binds to Modular Divergent Sites Distributed throughout Duplex Viral DNA," J. Virol. 2007 Dec; 81(23):13015-13027. In some embodiments, some rodent parvovirus strains include minute virus of mice prototype strain (MVMp), minute virus of mice immunosuppressed strain (MVMi), minute virus of mice Cutter strain (MVMc), mouse parvovirus 1b (MPV-1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MVP-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a (RPV-1a), minute virus of rat (RMV), and University of Massachusetts strain of rat virus L (RV-Umass). See O.-W. Merten, "Virus Contaminations of Cell Cultures - A Biotechnological View", Cytotechnology. 2002 July; 39(2):91-116.In one embodiment, the TAP sequence comprises the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, and the complement of SEQ ID NO:4.

[0014] In other aspects, the present invention is directed to PCR cocktail compositions and methods of using the PCR cocktails to detect target contaminants in a test sample and eliminate false positives due to contamination of the test sample with PACs.

[0015] In one embodiment, the PCR cocktail contains, among others, a target contaminant-specific forward oligonucleotide primer, a target contaminant-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the unique detection probe (UDP) described above, and a target contaminant-specific reverse oligonucleotide primer. In an embodiment in which the target contaminant is a rodent parvovirus, the PCR cocktail contains, among others, a rodent parvovirus-specific forward oligonucleotide primer, a rodent parvovirus-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the unique detection probe (UDP) described above, and a rodent parvovirus-specific reverse oligonucleotide primer. Each detection probe (i.e., the target contaminant-specific oligonucleotide detection probe, such as the rodent parvovirus-specific oligonucleotide detection probe, and the artificial oligonucleotide detection probe) contains a nucleic acid sequence linked to a fluorophore at one end (either 5' or 3') and a quencher at the other end (either 3' or 5', respectively).

[0016] In the above case, the nucleic acid sequence of the rodent parvovirus-specific forward oligonucleotide primer and the rodent parvovirus-specific oligonucleotide detection probe hybridize to the antisense strand of the parvovirus. The rodent parvovirus-specific reverse oligonucleotide primer hybridizes to the sense strand of the parvovirus. In one embodiment, the parvovirus sequence to which the primer and the parvovirus-specific oligonucleotide probe hybridize is a conserved rodent parvovirus sequence. In some cases, the conserved parvovirus sequence is a parvovirus NS1 sequence, such as the nucleic acid sequence set forth in SEQ ID NO:9. By using a conserved sequence, a single probe is believed to be effective in detecting multiple strains of rodent parvovirus.

[0017] In one embodiment, the artificial oligonucleotide detection probe does not hybridize to parvovirus nucleic acid or any biological contaminant sequence. The nucleic acid of the artificial oligonucleotide detection probe is synthetic and is not expected to hybridize to any biological contaminant nucleic acid sequence under any stringency. In one embodiment, the nucleic acid of the artificial oligonucleotide detection probe (also known as a "unique" nucleic acid polymer or unique sequence) is designed not to recognize or anneal to any naturally occurring parvovirus under hybridization conditions employed in performing the subject assay. In one embodiment, a "unique" nucleic acid polymer comprises 17-20 nucleotides, where no more than 7-10 consecutive internal nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence. In one embodiment, a "unique" nucleotide polymer comprises 17-20 nucleotides, where no more than 7-10 consecutive nucleotides and no more than 6 consecutive 3' nucleotides are identical to any parvovirus sequence set forth in SEQ ID NOs:9 and 12-37. However, the nucleic acid (i.e., unique sequence) of the artificial oligonucleotide detection probe hybridizes to the PACP sequence of the PAC plasmid, and therefore the artificial oligonucleotide detection probe detects the PAC plasmid but not the parvovirus or other biological contaminant sequence.

[0018] In one embodiment, the PCR cocktail can be used to determine whether the PAC plasmid that gives a false positive result is present in the test biological sample. In this case, if the test sample shows a positive Q-PCR signal for the parvovirus-specific oligonucleotide probe and a negative Q-PCR signal for the artificial oligonucleotide detection probe, the test sample is presumed to be free of PAC contamination (i.e., true positive).

[0019] In one embodiment, the target contaminant-specific forward oligonucleotide primer comprises the sequence of SEQ ID NO:1; the target contaminant-specific oligonucleotide detection probe nucleic acid comprises the sequence of SEQ ID NO:2; the artificial oligonucleotide detection probe nucleic acid (i.e., UDP) comprises the sequence of SEQ ID NO:3; and the target contaminant-specific reverse oligonucleotide primer comprises the sequence of SEQ ID NO:4.

[0020] In another aspect, the present invention provides a system and method for detecting biological contaminants in a test sample. In this case, the test sample is a cell culture, such as an industrial-scale mammalian cell culture for the production of therapeutic proteins. Mammalian cells useful in the practice of the present invention include, but are not limited to, CHO cells, CHO-K1 cells, and EESYR cells (see U.S. Patent No. 7,771,997). The system and method include the use of a nucleic acid extraction control (NEC) in addition to the primers, probes, cocktails, and PAC plasmids used as described above. In one embodiment, the NEC is an M13K07 phage, which is included in the test sample before nucleic acid extraction. If the nucleic acid extraction is suitable for the purpose of detecting contaminant DNA or RNA, the NEC nucleic acid (e.g., M13K07 nucleic acid) is detected by Q-PCR in the "spiked" test sample. In certain embodiments, the Q-PCR reaction mix contains a target contaminant-specific forward oligonucleotide primer, a target contaminant-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe such as the unique detection probe (UDP) described above, a target contaminant-specific reverse oligonucleotide primer, a NEC-specific forward oligonucleotide primer, a NEC-specific oligonucleotide detection probe, and a NEC-specific reverse oligonucleotide primer. In one embodiment, the test sample is taken from a therapeutic protein-producing cell culture to which an NEC (e.g., M13K07 phage) has been added prior to nucleic acid extraction and subsequent Q-PCR analysis.

[0021] In a specific embodiment, the target contaminant is a rodent parvovirus, in which case (1) the target contaminant-specific forward oligonucleotide primer is a rodent parvovirus-specific forward oligonucleotide primer, more specifically comprising the sequence of SEQ ID NO:1; (2) the target contaminant-specific oligonucleotide detection probe is a rodent parvovirus-specific oligonucleotide detection probe, more specifically comprising the nucleic acid sequence of SEQ ID NO:2; (3) the artificial oligonucleotide detection probe is a unique detection probe (UDP), more specifically comprising the nucleic acid sequence of SEQ ID NO:3; (4) the target contaminant-specific reverse oligonucleotide primer is a rodent parvovirus-specific reverse oligonucleotide primer, more specifically comprising the nucleic acid sequence of SEQ ID NO:4; (5) the NEC-specific forward oligonucleotide primer is an M13 forward oligonucleotide primer, more specifically comprising the nucleic acid sequence of SEQ ID NO:5; (6) the NEC-specific oligonucleotide detection probe is an M13 detection probe, more specifically comprising the nucleic acid sequence of SEQ ID NO:6. and (7) the NEC-specific reverse oligonucleotide primer is an M13 reverse oligonucleotide primer, more specifically comprising the nucleic acid of SEQ ID NO:8. In one embodiment of the method, if the NEC Q-PCR signal is detected (i.e., the signal is within the expected recovery range), it can be assumed that the nucleic acid extraction of the test sample was successful. On the other hand, if the NEC signal is not detected (i.e., the signal is outside the expected recovery range), it can be assumed that the nucleic acid extraction of the test sample was unsuccessful, and any negative Q-PCR target contaminant signals are considered invalid (i.e., false negatives). [The present invention 1001] A composition comprising an artificial nucleotide sequence, a fluorophore, and a quencher, wherein the artificial nucleotide sequence comprises, at its 3' end, five or fewer consecutive nucleic acids identical to any one of SEQ ID NOs:9 and 12 to 37. [The present invention 1002] The composition of the present invention 1001, wherein the fluorophore is selected from the group of fluorophores having an excitation wavelength in the range of 495 nm to 680 nm, and an emission wavelength in the range of 515 nm to 710 nm. [The present invention 1003] The composition of claim 1002, wherein the quencher is selected from the group of dyes having an absorption peak within the range of 430 nm to 672 nm. [The present invention 1004] 1001. The composition of matter of the present invention, wherein the fluorophore has an excitation wavelength of 495 nm and an emission wavelength of 520 nm. [The present invention 1005] The composition of the present invention 1004, wherein the fluorophore is FAM. [The present invention 1006] The composition of the present invention 1005, wherein the quencher is BHQ-1. [The present invention 1007] 1001. The composition of claim 1001, wherein a fluorophore is attached to the 5' end of the artificial nucleotide sequence and a quencher is attached to the 3' end of the artificial nucleotide sequence. [The present invention 1008] The artificial nucleotide sequence is The composition of the present invention 1001, comprising the nucleic acid sequence of TIFF0007669448000002.tif4128. [The present invention 1009] a. rodent parvovirus-specific forward oligonucleotide primer; b. Rodent parvovirus-specific oligonucleotide detection probe; c. artificial oligonucleotide detection probes; d. a rodent parvovirus-specific reverse oligonucleotide primer; e. M13-specific forward oligonucleotide primer; f. M13-specific oligonucleotide detection probe; and g. M13-specific reverse oligonucleotide primer A composition comprising: [The present invention 1010] The composition of the present invention 1009, wherein the oligonucleotide sequences of a, b, and d each comprise an NS-1 sequence of a rodent parvovirus selected from the group consisting of minute virus of mice prototype strain (MVMp), minute virus of mice immunosuppressed strain (MVMi), minute virus of mice Cutter strain (MVMc), mouse parvovirus 1b (MPV-1b), mouse parvovirus 1a (MPV-1a), mouse parvovirus 1c (MPV-1c), hamster parvovirus (HaPV), Toolan's parvovirus (H-1), Kilham rat virus (KRV), rat parvovirus 1a, rat minute virus, and rat virus L Umass strain (RV-Umass). [The present invention 1011] The composition of the present invention 1009, wherein the artificial oligonucleotide detection probe comprises a nucleotide sequence having at its 3' end not more than 5 consecutive nucleic acids that are identical to any one of SEQ ID NOs:9 and 12 to 37. [The present invention 1012] The composition of the present invention 1010, wherein the oligonucleotide sequences a, b, and d hybridize to SEQ ID NO:37. [The present invention 1013] The composition of claim 1009, wherein each of the detection probes b, c, and f comprises a fluorophore and a quencher such that the fluorophore for each detection probe emits light at a different wavelength. [The present invention 1014] The composition of the present invention 1013, wherein the fluorophore of each probe is selected from the group of fluorophores having an excitation wavelength within the range of 495 nm to 680 nm, and an emission wavelength within the range of 515 nm to 710 nm. [The present invention 1015] The composition of claim 1014, wherein the quencher is selected from the group of dyes having an absorption peak within the range of 430 nm to 672 nm. [The present invention 1016] The composition of the present invention 1013, wherein the artificial oligonucleotide detection probe (c) comprises the fluorophore FAM and the quencher BHQ-1; the rodent parvovirus-specific oligonucleotide detection probe (b) comprises the fluorophore VIC and the minor groove binding non-fluorescent quencher (MGBNFQ); and the M13-specific oligonucleotide detection probe comprises the fluorophore Cy5 and the quencher BHQ2. [The present invention 1017] The composition of the present invention 1012, wherein the rodent parvovirus-specific forward oligonucleotide primer (a) comprises the nucleic acid sequence of SEQ ID NO:1; the rodent parvovirus-specific reverse oligonucleotide primer (d) comprises the nucleic acid sequence of SEQ ID NO:4; and the rodent parvovirus-specific oligonucleotide detection probe (b) comprises the nucleic acid sequence of SEQ ID NO:2. [The present invention 1018] The composition of the present invention, wherein the artificial oligonucleotide detection probe (c) comprises the nucleic acid sequence of SEQ ID NO:3. [The present invention 1019] The composition of the present invention 1009, wherein the M13-specific forward oligonucleotide primer (e) comprises the nucleic acid sequence of SEQ ID NO:5; the M13-specific oligonucleotide detection probe (f) comprises the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer (g) comprises the nucleic acid sequence of SEQ ID NO:7. [The present invention 1020] a. mixing a plurality of components to form a reaction mixture, the components comprising: (i) a nucleic acid sample derived from a test sample, (ii) an oligonucleotide, and (iii) a DNA polymerase; b. subjecting the reaction mixture to a polymerase chain reaction (PCR); c. monitoring the production of (i) target amplified polynucleotides (TAPs), (ii) nucleic acid extraction control amplified polynucleotides (NACPs), and (iii) plasmid amplified control polynucleotides (PACPs) during PCR; and d. Comparing the production of TAP with the production of NACP and PACP wherein the presence of TAP, the presence of NACP, and the absence of PACP produced during PCR indicates that the test sample contains the biological contaminant and does not contain the positive amplification control plasmid. [The present invention 1021] The method of the present invention 1020, wherein the oligonucleotides comprise (a) a rodent parvovirus-specific forward oligonucleotide primer, (b) a rodent parvovirus-specific oligonucleotide detection probe, (c) an artificial oligonucleotide detection probe, (d) a rodent parvovirus-specific reverse oligonucleotide primer, (e) an M13-specific forward oligonucleotide primer, (f) an M13-specific oligonucleotide detection probe, and (g) an M13-specific reverse oligonucleotide primer. [The present invention 1022] The method of claim 1020, wherein the test sample is obtained from a mammalian cell culture or a purified fraction thereof. [The present invention 1023] The method of claim 1020, wherein the test sample is spiked with M13K07 phage. [The present invention 1024] The method of claim 1020, wherein the nucleic acid sample is prepared by subjecting approximately 1 mL of a test sample to lysis, proteolysis, and heat denaturation, followed by mixing the sample with an extraction control sample, and then extracting the nucleic acid from the sample. [The present invention 1025] 1024. The method of claim 1024, wherein the extraction control sample is M13K07 phage. [The present invention 1026] The method of the present invention 1025, wherein (a) the rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:1; (b) the rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binding non-fluorescent quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2; (c) the artificial oligonucleotide detection probe comprises a FAM fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO:3; (d) the rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:4; (e) the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5; (f) the M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7. [The present invention 1027] The method of claim 1020, wherein the component comprises uracil-N-glycosylase (UNG). [The present invention 1028] The method of claim 1020, comprising incubating the reaction mixture at 50° C. for at least 2 minutes. [The present invention 1029] The PCR step (b) (i) incubating the reaction mixture at 95° C. for 2 minutes; followed by (ii) eight cycles of (1) denaturation at 95° C. for 10 seconds, followed by (2) annealing for 30 seconds, wherein the annealing temperature in the first of the eight cycles is 70° C., the annealing temperature is decreased by 1° C. per cycle, and the annealing temperature in the last of the eight cycles is 62° C.; (iii) at least 40 cycles of DNA amplification comprising (1) a denaturation step at 95° C. for 10 seconds followed by (2) an annealing step at 62° C. for 30 seconds, wherein the rate of temperature change from the denaturation temperature to the annealing temperature is about 4.4° C. per second and from the annealing temperature to the denaturation temperature is about 2.2° C. per second. The method of the present invention 1028, comprising: [The present invention 1030] The method of the present invention, wherein (a) the production of TAP is monitored by measuring fluorescence at 533-580 nm at each amplification cycle; (b) the production of NACP is monitored by measuring fluorescence at 618-660 nm at each amplification cycle; and (c) the production of PACP is monitored by measuring fluorescence at 465-510 nm at each amplification cycle. [The present invention 1031] The method of the present invention 1020, comprising a step of comparing the results obtained from the method performed on the test sample with an external positive control and an external negative control, and if the negative control or the positive control fails, the results obtained from the method performed on the test sample are rejected. [The present invention 1032] An external positive control is a. mixing a plurality of positive control components to create a positive control mixture, the positive control components comprising: (i) a positive amplification control (PAC) plasmid, and (ii) a positive control oligonucleotide cocktail, and (iii) no test sample; b. subjecting the positive control mixture to a positive control polymerase chain reaction (PCR); and c. monitoring the production of (i) target amplified polynucleotide (TAP), (ii) nucleic acid extraction control amplified polynucleotide (NACP), and (iii) plasmid amplified control polynucleotide (PACP) during PCR. wherein the presence of TAP, NACP, and PACP produced during PCR indicates that the PCR is functioning properly, and the absence of any one or more of TAP, NACP, or PACP indicates that the positive control has failed. [The present invention 1033] The method of claim 1032, wherein the PAC plasmid comprises (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid. [The present invention 1034] The method of claim 1033, wherein (i) the parvovirus nucleic acid sequence comprises the sequence of SEQ ID NO:37; (ii) the M13K07 nucleic acid sequence comprises the sequence of SEQ ID NO:8; and (iii) the artificial nucleic acid sequence unique to the plasmid comprises the sequence of SEQ ID NO:10. [The present invention 1035] The method of claim 1034, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:11. [The present invention 1036] A positive control oligonucleotide cocktail was a rodent parvovirus-specific forward oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:1; a rodent parvovirus-specific oligonucleotide detection probe comprising a VIC fluorophore, an MGBNFQ quencher, and the nucleic acid sequence of SEQ ID NO:2; an artificial oligonucleotide detection probe comprising a FAM fluorophore, a BHQ quencher, and the nucleic acid sequence of SEQ ID NO:3; a rodent parvovirus-specific reverse oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:4; An M13-specific forward oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:5; an M13-specific oligonucleotide detection probe comprising a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and M13-specific reverse oligonucleotide primer comprising the nucleic acid sequence of SEQ ID NO:7 The method of the present invention 1033, comprising: [The present invention 1037] Positive control PCR: a. Incubating the positive control mixture at 95° C. for 2 minutes; followed by b. 8 cycles of (i) denaturation at 95°C for 10 seconds followed by (ii) annealing for 30 seconds, such that the annealing temperature at the beginning of the 8 cycles is 70°C, the annealing temperature is decreased by 1°C per cycle, and the annealing temperature at the end of the 8 cycles is 62°C; and c. 40 cycles of DNA amplification including (i) a denaturation step at 95°C for 10 seconds followed by (ii) an annealing step at 62°C for 30 seconds. The method of the present invention 1036, comprising: [The present invention 1038] The method of the present invention 1036, wherein the production of TAP in the positive control PCR is monitored by measuring fluorescence at 533-580 nm at each amplification cycle, the production of NACP in the positive control PCR is monitored by measuring fluorescence at 618-660 nm at each amplification cycle; and the production of PACP in the positive control PCR is monitored by measuring fluorescence at 465-510 nm at each amplification cycle. [The present invention 1039] a. Positive Amplification Control (PAC) plasmid; b. Rodent parvovirus-specific forward oligonucleotide primer; c. Rodent parvovirus-specific oligonucleotide detection probe; d. artificial oligonucleotide detection probes; e. a rodent parvovirus-specific reverse oligonucleotide primer; f. M13-specific forward oligonucleotide primer; g. M13-specific oligonucleotide detection probe; h. M13-specific reverse oligonucleotide primer; and i. Buffer A composition comprising: [The present invention 1040] The composition of the present invention 1039, wherein the PAC plasmid comprises (i) a parvovirus nucleic acid sequence, (ii) an M13K07 nucleic acid sequence, and (iii) an artificial nucleic acid sequence unique to the plasmid. [The present invention 1041] The composition of claim 1040, wherein the parvovirus nucleic acid sequence comprises the sequence of SEQ ID NO:37, the M13K07 nucleic acid sequence comprises the sequence of SEQ ID NO:8, and the unique nucleic acid sequence comprises the sequence of SEQ ID NO:10. [The present invention 1042] The composition of the present invention 1041, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:11. [The present invention 1043] The rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:1; a rodent parvovirus-specific oligonucleotide detection probe comprising a VIC fluorophore, a minor groove binding quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2; the artificial oligonucleotide detection probe comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and a nucleic acid sequence of SEQ ID NO:3; The rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:4; The M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5; the M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6; and The M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7. The composition of the present invention 1042. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, since such methods and conditions may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.As used herein, the term "about" when used in relation to a specific stated numerical value means that the value can vary from the stated value by at most 1%.For example, as used herein, the expression "about 100" includes 99 and 101, and all values ​​therebetween (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0024] Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described below. All publications mentioned herein are incorporated by reference in their entirety. Other aspects will become apparent from review of the following detailed description.

[0025] In order that the invention described herein may be fully understood, the following detailed description is set forth.

[0026] The present invention relates to improved materials and methods for detecting any biological contaminants in any cell culture producing a therapeutic protein. In particular, the present invention relates to materials and methods for quantitative polymerase chain reaction (Q-PCR) that employ an easily detected positive amplification control element or step to rule out false positives.

[0027] PCR and quantitative PCR As used herein, the phrase "polymerase chain reaction" ("PCR") refers to a method for making copies of a nucleic acid (e.g., DNA) by employing multiple cycles of denaturation (separation of a template DNA strand), annealing (hybridization of a single-stranded oligonucleotide to the single-stranded template DNA strand), and DNA synthesis (DNA polymerase catalyzes the synthesis of a new DNA strand primed from the 3' end of the hybridized oligonucleotide, using the template DNA strand as a template). To allow amplification to occur, at least two different oligonucleotide primers (known as "primers" for short) are used in a PCR reaction. One primer, commonly called the forward primer, hybridizes to the antisense strand of the template DNA and forms the 5' end of the newly synthesized sense strand. The other primer, commonly called the reverse primer, hybridizes to the sense strand of the template DNA and forms the 5' end of the newly synthesized antisense strand. In each cycle, each template strand is copied to form a new double-stranded DNA molecule, also known as an "amplicon." Thus, using non-limiting amounts of oligonucleotide primers, DNA polymerase (i.e., Taq polymerase or other thermostable DNA polymerase; see Innis et al., DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA, 85(24) Proc Natl Acad Sci US A. 9436-40 (1988)), and nucleotide triphosphates, the number of DNA molecules (templates and amplicons) is doubled in each cycle. PCR is described in U.S. Patent No. 4,683,202 (issued July 28, 1987). See also PCR Primer: A Laboratory Manual (Carl W. Dieffenbach & Gabriela S. Dveksler eds., 1995).

[0028] As used herein, the term "cycle" refers to one round of (1) melting of DNA strands, called "denaturation," followed by (2) hybridization of an oligonucleotide primer to the resulting single-stranded DNA according to the rules of base pairing, a process called "annealing," and (3) polymerization of the new strand of DNA starting at the 3' end of the oligonucleotide primer and proceeding in the 5' to 3' direction, a process called "amplification" or "extension." Polymerization typically uses a DNA polymerase enzyme, such as Taq polymerase, to catalyze the formation of phosphodiester bonds between adjacent deoxynucleotide triphosphates ("dNTPs") along the exposed single-stranded template DNA by hydrogen bonding according to the rules of base pairing. Denaturation, annealing, and amplification are performed at certain temperatures based in part on the GC content of the DNA template and oligonucleotide primers, and the length of the DNA strand to be copied. The temperatures of denaturation and annealing, as well as the ionic strength of the reaction buffer, control the stringency of hybridization and the fidelity of DNA copying.

[0029] "Quantitative PCR" or "qPCR" or "Q-PCR" (also known as "real-time PCR") is a type of PCR that allows for the monitoring of amplicon formation during the PCR cycling process. Q-PCR can be used to quantify the amount of a specific template DNA in a sample. Q-PCR incorporates at least one oligonucleotide detection probe in the reaction mixture in addition to a forward oligonucleotide primer and a reverse oligonucleotide primer. The detection probe is a single-stranded oligonucleotide that hybridizes to the sense or antisense strand of the target template DNA somewhere between the forward and reverse primer binding sites. During the annealing step, the oligonucleotide detection probe anneals to the single-stranded template. As polymerization occurs, the probe is cleaved and degraded by the 5' nuclease activity of the DNA polymerase. Thus, as amplification of a specific template sequence occurs, the detection probe is degraded at an exponential rate.

[0030] Q-PCR oligonucleotide detection probes are generally constructed with an attached fluorophore (also known as a reporter fluorescent dye or simply a "reporter") and an attached quencher. In most cases, the fluorophore is attached at or near the 5' end of the oligonucleotide, and the quencher is attached at or near the 3' end of the oligonucleotide. However, any viable configuration can be used in the practice of the present invention. When the oligonucleotide detection probe is intact, the fluorophore and quencher are in close proximity such that the quencher absorbs the light emitted by the excited fluorophore, thereby greatly reducing the detectable fluorophore emission. When the oligonucleotide detection probe is cleaved or degraded, the fluorophore and quencher are released and consequently separated in space. The quencher is no longer close enough to quench the fluorophore emission. As specific amplicons are formed, more oligonucleotide detection probes are cleaved, more fluorophores and quenchers are released, and therefore more fluorophore / quencher pairs are separated, increasing the amplitude of the fluorescence emission. In other words, increasing fluorophore emission signal correlates with the amount of specific target DNA in the sample. For a review of Q-PCR, see Ian M. Mackay et al., Survey and Summary: Real-Time PCR in Virology, 30(6) Nucleic Acids Research 1292-1305 (2002).

[0031] Fluorescence quenching can occur by direct contact between the reporter and quencher (also known as static quenching), or by fluorescence resonance energy transfer (FRET) between the reporter and quencher when both are within the Förster radius of each other. A specific fluorophore can be excited by light of one or more specific wavelengths, or a range of wavelengths with a local maximum. This is called the excitation wavelength. After a fluorophore is excited, it returns to the ground state and emits light at a wavelength longer than the excitation wavelength. This is called the emission wavelength. During FRET, a second fluorophore, dye, lanthanide series molecule, etc., with an absorption spectrum that matches or overlaps with the emission spectrum of the fluorophore, absorbs the light emitted by the excited fluorophore within the Förster radius, thereby quenching or decreasing the wavelength of the fluorophore emission. Contact or static quenching occurs when the reporter and quencher form a ternary complex with the ground state of the fluorophore. This ternary complex is non-fluorescent, ie, essentially non-excitable, and therefore does not emit light at the expected emission wavelength.

[0032] For a review of static quenching and FRET, see Salvatore AE Marras et al., Efficiencies of Fluorescence Resonance Energy Transfer and Contact-Mediated Quenching in Oligonucleotide Probes, 30(21) Nucleic Acids Research e122, pp.1-8 (2002). Marras et al. also discuss the selection of reporter / quencher pairs for use in Q-PCR applications.

[0033] nucleic acid The terms "polynucleotide", "oligonucleotide", "probe", "primer" or "nucleotide primer" or "oligonucleotide primer", "template" or "template nucleic acid" or "template DNA" are used herein according to their ordinary meaning to those skilled in the art of molecular biology. For a detailed description of each, see, for example, PCR Primer: A Laboratory Manual (Carl W. Dieffenbach & Gabriela S. Dveksler eds., 1995).

[0034] As used herein, "amplicon" refers to the DNA product resulting from the amplification of a template nucleic acid sequence by PCR. As PCR proceeds and the template is amplified, the newly formed DNA amplicon serves as a template for subsequent rounds of DNA synthesis.

[0035] cell culture The present invention is directed to an improved Q-PCR method for detecting biological contaminants in cell cultures. Cell cultures are often used to produce complex biological molecules for therapeutic use, such as antibodies, capture molecules, and Fc fusion proteins. These cultures must remain free of biological contaminants. Detection of contaminants is important to determine whether a particular batch should be discarded or subjected to repair.

[0036] Cell culture includes culture medium and cells that are usually derived from a single cell line. In this case, the cell line includes cells that can produce biotherapeutic proteins. Examples of cell lines that are routinely used to produce proteinaceous biologics include, among others, primary cells, BSC cells, HeLa cells, HepG2 cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK cells, BHK-21 cells, CHO cells, CHO-K1 cells, NS-1 cells, MRC-5 cells, WI-38 cells, 3T3 cells, 293 cells, Per.C6 cells, and chicken embryo cells. Chinese hamster ovary (CHO) cell line, or one or more of several specific CHO cell variants, such as CHO-K1 cell line, are optimized for large-scale protein production. EESYR® cell line is a specialized CHO cell line optimized for enhanced production of a protein of interest. For a detailed description of EESYR® cells, please refer to U.S. Patent No. 7,771,997 (issued August 10, 2010).

[0037] "Cell culture" or "culture" refers to the growth and proliferation of cells outside a multicellular organism or tissue. Suitable culture conditions for mammalian cells are known in the art. For example, Animal cell culture: A practical approach(D. Rickwood, ed., 1992). Mammalian cells can be cultured in suspension or attached to a solid substrate. Fluidized bed bioreactors, hollow fiber bioreactors, roller bottles, shake flasks, or stirred tank bioreactors, with or without microcarriers and run in batch, fed-batch, continuous, semi-continuous, or perfusion modes, are available for mammalian cell culture. Cell culture medium or concentrated feed medium can be added to the culture continuously or at intervals during the culture (i.e., batch-fed). For example, the culture can be fed once a day, every other day, every second day, or when the concentration of a particular medium component, which is monitored directly or indirectly, falls outside the desired range.

[0038] Animal cells such as CHO cells or EESYR® cells can be grown in small scale cultures, such as in 125 ml vessels with about 25 ml of medium, 250 ml vessels with about 50-100 ml of medium, 500 ml vessels with about 100-200 ml of medium. Alternatively, cultures can be large scale, such as in 1000 ml vessels with about 300-1000 ml of medium, 3000 ml vessels with about 500-3000 ml of medium, 8000 ml vessels with about 2000-8000 ml of medium, and 15000 ml vessels with about 4000-15000 ml of medium. Cultures for production can contain 10,000 L of medium or more. Large scale cell cultures, such as for clinical production of proteinaceous therapeutics, are typically maintained for days or weeks while the cells produce the desired protein. During this period, samples of the culture may be removed and tested for the presence of biological contaminants.

[0039] Therapeutic Protein Production The cell culture monitored for biological contamination may be used to produce a protein or other biological molecule of interest, such as a therapeutically effective antibody or other biopharmaceutical material. The protein product (protein of interest) may be, inter alia, an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single-chain antibody, a diabody, a triabody, or a tetrabody, a Fab fragment or a F(ab')2 fragment, an IgA antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody.

[0040] The protein of interest can be a recombinant protein (e.g., Fc fusion protein) that contains an Fc portion and another domain. The Fc fusion protein can be a receptor Fc fusion protein that contains one or more of one or more extracellular domains of a receptor bound to the Fc portion. In some cases, the Fc portion includes a hinge region followed by the CH2 and CH3 domains of IgG. In some cases, the receptor Fc fusion protein contains two or more individual receptor chains that bind to either a single ligand or multiple ligands. For example, the Fc fusion protein is a capturer such as an IL-1 capturer (e.g., rilonacept, which contains the IL-1RAcP ligand binding region fused to the Il-1R1 extracellular domain fused to the Fc domain of hIgG1; see U.S. Patent No. 6,927,004), or a VEGF capturer (e.g., aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc domain of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

[0041] The present invention is not limited to any particular type of cell or cell line for protein production. Examples of cell types suitable for protein production include mammalian cells, such as CHO-derived cells like EESYR®, insect cells, avian cells, bacterial cells, and yeast cells. The cells can be stem cells, or recombinant cells transformed with vectors for recombinant gene expression, or cells transfected with viruses to produce viral products. The cells can contain a recombinant heterologous polynucleotide construct that encodes a protein of interest. The construct can be episomal (such as an extrachromosomal plasmid or fragment) or physically integrated into the genome of the cell. The cells can also produce a protein of interest without having the protein encoded on a heterologous polypeptide construct. In other words, the cells can naturally encode a protein of interest, such as B cells that produce antibodies. Methods and vectors for genetically modifying cells or cell lines to express a protein of interest are well known to those skilled in the art. For example, various techniques include: Current Protocols in Molecular Biology , Ausubel et al., eds. (Wiley & Sons, New York, 1988, and quarterly revisions);Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989);Kaufman, RJ, Large Scale Mammalian Cell Culture , 1990, pp. 15-69. A wide variety of cell lines suitable for growth in culture are available from the American Type Culture Collection (Manassas, Va.) and commercial suppliers.

[0042] The cell may also be a primary cell, such as a chicken embryo cell, or a primary cell line. Examples of useful cells include BSC cells, LLC-MK cells, CV-1 cells, COS cells, VERO cells, MDBK cells, MDCK cells, CRFK cells, RAF cells, RK cells, TCMK-1 cells, LLCPK cells, PK15 cells, LLC-RK cells, MDOK cells, BHK-21 cells, chicken embryo cells, NS-1 cells, MRC-5 cells, WI-38 cells, BHK cells, 293 cells, Per.C6 cells, and CHO cells. In various embodiments, the cell line is a CHO cell derivative, such as CHO-K1, CHO DUX B-11, CHO DG-44, Veggie-CHO, GS-CHO, S-CHO, CHO lec mutant line, or EESYR® cell line.

[0043] In one particular scenario, the cell is a CHO cell derivative, such as an EESYR® cell, that ectopically (heterologously) expresses a protein. The protein comprises an immunoglobulin heavy chain region, such as a CH1, CH2, or CH3 region. In one embodiment, the protein comprises a human or rodent immunoglobulin CH2 and CH3 region. In one embodiment, the protein comprises a human or rodent immunoglobulin CH1, CH2, and CH3 region. In one embodiment, the protein comprises a hinge region, and a CH1, CH2, and CH3 region. In a specific embodiment, the protein comprises an immunoglobulin heavy chain variable domain. In a specific embodiment, the protein comprises an immunoglobulin light chain variable domain. In a specific embodiment, the protein comprises an immunoglobulin heavy chain variable domain and an immunoglobulin light chain variable domain. In a specific embodiment, the protein is an antibody, such as a human antibody, a rodent antibody, or a chimeric human / rodent antibody (e.g., human / mouse, human / rat, or human hamster).

[0044] The protein production phase of cell culture can be carried out in any scale of culture, from individual flasks and shake flasks or wave bags, to 1 liter bioreactors and large-scale industrial bioreactors. Large-scale processes can be carried out in volumes of about 100 liters to 20,000 liters or more. Protein production may be controlled using one or more of several means, such as temperature shifts or chemical induction. The growth phase of the cells can occur at a higher temperature than the production stage, in which the protein is expressed and / or secreted. For example, the growth phase can occur at a first temperature of about 35°C to 38°C, and the production phase can occur at a second temperature of about 29°C to 37°C, optionally about 30°C to 36°C, or about 30°C to 34°C. In addition, chemical inducers of protein production, such as caffeine, butyrate, tamoxifen, estrogen, tetracycline, doxycycline, and hexamethylene bisacetamide (HMBA), can be added simultaneously with, before, or after the temperature shift. If inducers are added after the temperature shift, they can be added 1 hour to 5 days after the temperature shift, such as 1 to 2 days after the temperature shift. The production cell culture can be carried out as a continuous feed culture system, such as in a chemostat (see C. Altamirano et al., Biotechnol Prog. 2001 Nov-Dec; 17(6):1032-41), or according to a fed-batch (batch-fed) process (Huang, 2010).

[0045] Therapeutic Protein Products As used herein, "peptide", "polypeptide" and "protein" are used interchangeably throughout and refer to molecules that contain two or more amino acid residues linked together by peptide bonds. Peptides, polypeptides and proteins may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation and ADP-ribosylation. Peptides, polypeptides and proteins may be of scientific or commercial interest, such as protein-based drugs. Peptides, polypeptides and proteins include, among others, antibodies and chimeric or fusion proteins. Peptides, polypeptides and proteins are produced by recombinant animal cell lines using cell culture methods.

[0046] "Antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody. The term antibody also includes bispecific antibodies, including heterotetrameric immunoglobulins that can bind to more than one epitope. Bispecific antibodies are generally described in U.S. Patent Application Publication No. 2010 / 0331527, which is incorporated herein by reference.

[0047] The term "antigen-binding portion" (or "antibody fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment, which consists of the VH and CH1 domains; (iv) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment (Ward et al. (1989) Nature 241:544-546), which consists of the VH domain; (vi) an isolated CDR; and (vii) an scFv, which consists of the two domains of the Fv fragment, VL and VH, connected by a synthetic linker to form a single protein chain, where the VL and VH domains are paired to form a monovalent molecule. Other forms of single chain antibodies, such as diabodies, are also encompassed under the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123).

[0048] An antibody or its antigen-binding portion may be part of a larger immunoadhesion molecule formed by covalent or non-covalent binding of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab')2 fragments, may be prepared from whole antibodies using conventional techniques, such as by papain or pepsin digestion of whole antibodies. Moreover, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques (see Sambrook et al., 1989).

[0049] The term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include, for example, in the CDRs, particularly CDR3, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0050] As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo chromosomal mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally exist within the human antibody germline repertoire in vivo.

[0051] An "Fc fusion protein" includes any or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, that are not found together in nature and are not fusion proteins. Preparation of fusion proteins containing certain heterologous polypeptides fused to various portions of antibody-derived polypeptides, including Fc domains, has been described, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991;Byrn et al., Nature 344:677, 1990;and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", Current Protocols in Immunology, Suppl. 4, pp.10.19.1-10.19.11, 1992. "Receptor Fc fusion protein" comprises one or more extracellular domains of a receptor bound to an Fc portion, which in some embodiments comprises a hinge region followed by the CH2 and CH3 domains of an immunoglobulin. In some embodiments, an Fc fusion protein comprises two or more individual receptor chains that bind to one or more ligands. For example, an Fc fusion protein is a trap, such as an IL-1 trap (e.g., rilonacept, which comprises an IL-1RAcP ligand binding domain fused to an IL-1R1 extracellular domain fused to an Fc of hIgG1; see U.S. Patent No. 6,927,004), or a VEGF trap (e.g., aflibercept, which comprises an Ig domain 2 of the VEGF receptor Flt1 fused to an Ig domain 3 of the VEGF receptor Flk1 fused to an Fc of hIgG1; see U.S. Patent Nos. 7,087,411 and 7,279,159).

[0052] Biological Contaminants As used herein, the term "biological contaminant" refers to any unwanted, undesirable, harmful, or potentially harmful biological entity. These entities include, among others, prions (the etiological cause of bovine / transmissible spongiform encephalopathies), virions, viruses, mycoplasma, other bacteria, contaminating metazoan cells, DNA, RNA, transposons, other transposable elements, yeast, other fungi, algae, protozoa, and other exogenous and endogenous agents. Of particular concern for biopharmaceutical production processes using rodent cells (such as CHO cells and derivatives of CHO cells) are adventitious viruses associated with the cells or media or manufacturing raw materials. Contamination of cell culture bulk processing materials and the resulting drug product poses direct risks to patients as well as indirect risks of impeding the supply of medicines.

[0053] A non-exhaustive list of exogenous and / or endogenous agents that can infect CHO cell cultures includes single-stranded (-) RNA viruses, such as Cachet Valley virus, influenza A / B virus, parainfluenza 1 / 2 / 3, simian virus 5, mumps virus, bovine respiratory syncytial virus, and vesicular stomatitis virus; single-stranded (+) RNA viruses, such as bovine coronavirus, vesivirus 2117, encephalomyocarditis virus, coxsackievirus B-3, Semliki Forest virus, and Sindbis virus; double-stranded RNA viruses, such as bluetongue virus, epizootic hemorrhagic disease virus, and reovirus 1 / 2 / 3; single-stranded DNA viruses, such as porcine circovirus 1, and the particularly problematic parvoviruses, including minute virus of mice (also known as minute virus of mice); and double-stranded DNA viruses, such as adenovirus and pseudorabies virus. Among these potential exogenous agents, four types of viruses predominate in bulk harvest samples from CHO cell cultures across various manufacturers. These viruses are Reovirus type 2, Cachet Valley virus, Enzootic Hemorrhagic Virus, and Rodent Parvovirus Minute Virus of Mice. For a detailed review of adventitious virus contamination of CHO cell cultures, see Andreas Berting et al., Virus Susceptibility of Chinese Hamster Ovary (CHO) Cells and Detection of Viral Contaminations by Adventitious Agent Testing, 106(4) Biotechnology and Bioengineering 598-607 (2010), and Andrew Kerr & Raymond Nims, Adventitious Viruses Detected in Biopharmaceutical Bulk Harvest Samples over a 10 Year Period, 64(5) PDA Journal of Pharmaceutical Science & Technology 481-485 (2010).

[0054] Exogenous agent testing is broadly divided into two categories. The first is classical virology techniques using in vitro virus assays, in which test samples are applied to indicator cell lines, the cells are incubated and passaged for 14-28 days, and then endpoints such as cytopathic effect or hemagglutination are measured (Berting, 2010). The second is PCR-based assays, which measure the presence of nucleic acids associated with exogenous or endogenous agents in real time. See, for example, Zhan et al., Detection of Minute Virus of Mice Using Real Time Quantitative PCR in Assessment of Virus Clearance during the Purification of Mammalian Cell Substrate Derived Biotherapeutics, 30(4) Biologicals 259-270 (2002).

[0055] Minute virus of mice (also known as MMV, Minute Virus of Mice, or MVM) poses special problems for biopharmaceutical manufacturing. Both the U.S. Food and Drug Administration (FDA) and the European Medicines Agency require specific testing for MVM. The virus is a member of the Parvoviridae family (Parvovirus) and is common in mice. It is excreted in urine and feces, is hardy, and persists in the environment. It can be easily introduced into biopharmaceutical manufacturing processes. See Moody et al., Mouse Minute Virus (MMV) Contamination - A Case Study: Detection, Root Cause Determination, and Corrective Actions, 65(6) PDA Journal of Pharmaceutical Science and Technology 580-288 (2011). Other rodent parvoviruses can negatively impact cell culture-based biopharmaceutical production. In addition to the prototype MVM strain, these rodent parvoviruses include, inter alia, the MVM immunosuppressant strain and the Cutter strain, mouse parvovirus 1a (MPV-1a), MPV-1b, MPV-1c, hamster parvovirus, Toolan's parvovirus (parvovirus H-1), Kilham rat virus, rat parvovirus 1a, rat minute virus, and the Umass strain of rat virus L. See S. F. Cotmore & P. ​​Tattersal, The Autonomously Replicating Parvoviruses of Vertebrates, 33 Advances in Virus Research 91-174 (1987), and Jacoby et al., Rodent Parvovirus Infections, 46(4) Lab Anim Sci. 370-80 (1996).

[0056] These parvoviruses share a conserved nucleic acid sequence called NS-1 (NS1), which encodes a large nonstructural protein involved in the amplification of the viral genome. The conserved NS1 nucleotide sequence from MVM is depicted in SEQ ID NO:9. Nucleotides 875-956 of that sequence are at least 97% conserved across the diverse rodent parvovirus NS1 sequences and therefore serve as a good target sequence for PCR-based adventitious and endogenous agent testing. PCR-based testing for rodent parvoviruses (as well as other adventitious and endogenous agents) can be performed on raw materials, pre-harvested culture media, at various points along the bulk processing purification of biopharmaceutical molecules, as well as at the formulation and packaging stages. Detection of contaminants may necessitate remediation steps such as disposal of contaminated materials, replacement of raw materials, and decontamination of facilities.

[0057] In addition to testing for adventitious agents, endogenous agents, and other biological contaminants that allow for corrective and preventive actions (CAPA) during production, specialized manufacturing and bulk processing steps (i.e., unit operations) may be employed to eliminate, reduce, or inactivate viral contaminants. Chemical inactivation, virus-retentive filtration, and chromatography have been shown to be effective in reducing herpesviruses, retroviruses, and parvoviruses in harvested or partially purified cell culture fluids. The most frequently used chemical inactivation step is low pH treatment, a treatment that some in the art believe is due to denaturation of viral envelope proteins. Protein A, hydroxyapatite, cation exchange, and anion exchange chromatography steps have all been shown to remove viruses to some extent. See, e.g., Miesegaes et al., Analysis of Viral Clearance Unit Operations for Monoclonal Antibodies, 106(2) Biotechnology and Bioengineering 238-246 (2010), and Liu et al., Recovery and Purification Process Development for Monoclonal Antibody production, 2(5) mAbs 480-499 (2010).

[0058] Q-PCR Testing: Positive and Negative Controls Tests for biological contaminants should be properly controlled to ensure accuracy, reliability, and authenticity. As used herein, a "negative control" includes most or all of the experimental reagents and conditions, but does not include the test sample. The test sample may be replaced with a buffer or mock culture medium that is known not to contain the biological contaminant of interest. As used herein further, a "negative control" should produce a negative result for biological contaminants. If the negative control produces a positive biological contaminant result, a skilled technician or scientist may infer that a positive result from a parallel test sample may not accurately reflect whether the test sample contains a biological contaminant.

[0059] As used herein, one or more "positive controls" are used to assess whether the experimental conditions function properly to detect biological contaminants. Positive controls can be used at any step along the experimental process to ensure that each step is working and to determine at which step the process breaks down.

[0060] In some embodiments, a positive control is used in the nucleic acid extraction step to evaluate whether the intended extraction of any biological contaminant nucleic acid was efficient enough to detect the biological contaminant. Such a positive control is called a "nucleic acid extraction control" or "NEC". In some cases, the NEC is selected to mimic the target biological contaminant in terms of protein-nucleic acid structure. If the NEC is extracted in a manner sufficient to be detected, the technician can infer that the target biological nucleic acid was also extracted in a manner sufficient to be detected. In one embodiment, the NEC is a single-stranded DNA phage that is not entirely different from a parvovirus.

[0061] In a specific embodiment, the NEC is the M13 bacteriophage, which is composed of a circular single-stranded DNA of about 6407 nucleotides. In a more specific embodiment, the NEC is the M13K07 strain, which is a commonly available molecular biology reagent used for cloning and other laboratory purposes. See van Wezenbeek et al., Nucleotide Sequence of the Filamentous Bacteriophage M13 DNA Genome: Comparison with Phage fd, 11(1-2) Gene 129-148 (1980). The nucleotide sequence of the M13K07 phage is depicted in SEQ ID NO:8. Although the M13 bacteriophage or the M13K07 bacteriophage strain may be used as the NEC in a specific embodiment, the present invention is by no means limited to using that particular reagent as the NEC. One of skill in the art may substitute other reagents as the NEC in the practice of the invention without departing from the scope of the invention (e.g., MS2 phage for reverse transcriptase PCR assays; see Kothapalli et al., Problems associated with product enhancement reverse transcriptase assay using bacteriophage MS2 RNA as a template, 109(2) J. Virol. Methods 203-207 (2003)).

[0062] In some embodiments of the present invention, a positive control is used in the PCR amplification step to assess whether the PCR reagents (including primers) and PCR steps are / were sufficient to detect the biological contaminant template nucleic acid. Such a positive control is called a "plasmid amplification control" or "PAC." In some cases, the PAC is selected or designed to match the target biological contaminant nucleic acid sequence and to match exactly with the forward and reverse oligonucleotide primers of the test sample. In a specific embodiment, the PAC further contains a "unique sequence" that is not found in the target biological contaminant nucleic acid. In a more specific embodiment, the unique sequence is not found in nature. The "unique" nucleic acid polymer is designed to specifically anneal to this unique sequence and be incorporated into the amplicon copy of the unique sequence during PCR. The "unique" nucleic acid polymer is designed not to recognize or anneal to any naturally occurring parvovirus under the hybridization conditions employed in performing the subject assay. In one embodiment, a "unique" nucleic acid polymer comprises 17-20 nucleotides, wherein no more than 7-10 contiguous internal nucleotides and no more than 6 contiguous 3' nucleotides are identical to any parvovirus sequence. In one embodiment, a "unique" nucleotide polymer comprises 17-20 nucleotides, wherein no more than 7-10 contiguous nucleotides and no more than 6 contiguous 3' nucleotides are identical to any parvovirus sequence set forth in SEQ ID NOs:9 and 12-37.

[0063] This unique sequence allows a skilled technician to distinguish between a bona fide target biological contaminant sequence and cross-contamination of a test Q-PCR reaction with a PAC plasmid.

[0064] In one embodiment, the PAC (as a plasmid, also known as the PAC plasmid) is included in an identical but separate Q-PCR reaction to control PCR amplification. To accurately reflect the amplification of the actual target biological contaminant nucleic acid, the PAC reaction uses oligonucleotide primers and probes that are exactly the same as those used in the test sample Q-PCR reaction. The test sample Q-PCR reaction and the separate Q-PCR reaction of the PAC plasmid contain a target detection probe and a unique sequence probe. If the PAC reaction is functioning properly, it is expected to produce a positive target signal and a positive unique sequence signal. Obtaining a positive target signal and a positive unique sequence signal in the PAC plasmid-containing reaction indicates that the test sample Q-PCR reagents and conditions are working properly to produce a positive target signal in the test sample whenever the target biological contaminant nucleic acid is present.

[0065] Therefore, PAC serves as a control for correct PCR amplification.If negative target signal is obtained in test sample, but PAC shows positive target signal, skilled technician can infer that there is no detectable target contaminant DNA in test sample.On the contrary, if positive target sequence signal and positive unique sequence signal are obtained in test sample, skilled technician can infer that PAC plasmid cross-contaminates test sample, and therefore positive target sequence signal may be false positive.

[0066] DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS In some embodiments, improved positive control systems (ie, compositions and methods) are provided for detecting biological contaminants by use of the polymerase chain reaction, more specifically Q-PCR.

[0067] In one aspect, the present invention provides a unique sequence probe (USP) for detecting a positive proliferation control plasmid (PAC plasmid). The USP is a unique artificial plasmid-specific sequence ( u niquea Artificial p Laser-specific s In a specific embodiment, the artificial nucleotide sequence capable of hybridizing to the UAPS comprises the nucleic acid sequence of SEQ ID NO:3, a fluorophore, and a quencher. TIFF0007669448000003.tif4128, which contains UAPS sense strand sequences (e.g., It is antisense to the gene encoding the nucleotide sequence TIFF0007669448000004.tif4128.

[0068] As described above, the Q-PCR detection probe contains a fluorophore and a quencher. Quenching can be by contact quenching or by FRET, depending on the fluorophore / quencher pair. In this case, the fluorophore and the quencher are covalently attached to the USP oligonucleotide. In one embodiment, the fluorophore has an excitation wavelength of 495 nm to 680 nm and an emission wavelength of 515 nm to 710 nm. In some embodiments, the fluorophore has an excitation wavelength of 495 nm, 538 nm, or 646 nm, and an emission wavelength of 520 nm, 554 nm, or 669 nm, respectively. In some embodiments, the quencher is a dye with an absorption peak of 430 nm to 672 nm. Examples of useful quenchers include DDQ®-I, Dabcyl, Eclipse®, Iowa Black FQ®, BHQ®-1, QSY®-7, BHQ®-2, DDQ®-II, Iowa Black RQ®, QSY®-21, and DHQ®-3. In a specific embodiment, the fluorophore is a fluorescein amidite (FAM; described in U.S. Pat. No. 5,583,236, issued Dec. 10, 1996), which has an absorption maximum at about 495 nm and an emission maximum at about 520 nm, and the quencher is Black Hole Quencher®-1 (BHQ®-1, Biosearch Technologies, Inc., Petaluma, Calif.), which absorbs at 480 nm to 580 nm. BHQ®-1 quenches by FRET and contact quenching. Typically, but not always, the quencher is attached to the 3′ hydroxyl group of the oligonucleotide via an ether bond and the fluorophore is attached to the 5′ phosphate group of the oligonucleotide via an ester bond.

[0069] In another aspect, the present invention provides a mixture of Q-PCR reagents comprising multiple oligonucleotides and probes useful for the detection of biological contaminants in cell cultures, raw materials, partially purified and purified biological molecules, and the like. In one embodiment, the biological contaminant is a DNA virus, more specifically a parvovirus, and even more specifically a rodent parvovirus, such as MVM. The parvovirus contains an NS1 gene having a nucleic acid sequence that is at least 88% identical to any sequence listed in Table 1. In another embodiment, the parvovirus contains an NS1 gene having a nucleic acid sequence that is at least 97% identical to the sequence set forth in SEQ ID NO:9 (i.e., the minute virus of mice (MVM) NS1 gene). In yet another embodiment, the parvovirus contains an NS1 gene comprising the consensus sequence of SEQ ID NO:37.

[0070] Table 1 Parvovirus NS1 sequences TIFF0007669448000005.tif110170

[0071] In a specific embodiment, the mixture contains (1) a rodent parvovirus-specific forward oligonucleotide primer; (2) a rodent parvovirus-specific oligonucleotide detection probe; (3) an artificial oligonucleotide detection probe, such as USP; and (4) a rodent parvovirus-specific reverse oligonucleotide primer. Such a mixture can be used in both test samples and positive control samples. In a more specific embodiment, the oligonucleotide primer and the parvovirus-specific oligonucleotide detection probe hybridize to an NS1 sequence, such as the NS1 sequence set forth in SEQ ID NO:9. In a more specific embodiment, (1) the forward primer comprises the sequence of SEQ ID NO:1; (2) the parvovirus-specific oligonucleotide detection probe comprises the sequence of SEQ ID NO:2; (3) the artificial probe is USP and comprises the sequence of SEQ ID NO:3; and (4) the reverse primer comprises the sequence of SEQ ID NO:4.

[0072] As described above, USP contains a fluorophore and a quencher, so that the amplicons containing this sequence, i.e., PAC DNA, can be detected in real time as Q-PCR proceeds. In a similar manner, parvovirus-specific detection probes contain oligonucleotides with covalently attached fluorophores and quenchers. In fact, to distinguish between true parvovirus positive signals and false positives caused by contamination of test samples with PAC plasmids or other PACPs (e.g., PACP-contaminated amplicons), the fluorophore emission wavelength of the parvovirus detection probe should be different from the emission wavelength of the USP. Thus, in an embodiment in which the USP fluorophore is FAM, the fluorophore attached to the parvovirus detection probe oligonucleotide should have an emission wavelength other than about 520 nm. In a specific embodiment, the fluorophore attached to the rodent parvovirus-specific oligonucleotide detection probe is a VIC® dye (Life Technologies, Inc., Carlsbad, CA), which has an absorption maximum at 538 nm and an emission maximum at about 554 nm. Here, the quencher can be a FRET quencher or a contact quencher. In one embodiment, the quencher is a minor groove binding non-fluorescent quencher (MGBNFQ) (see Sylvain et al., Rapid Screening for HLA-B27 by a TaqMan-PCR Assay Using Sequence-Specific Primers and a Minor Groove Binder Probe, a Novel Type of TaqMan™ Probe, 287(1-2) Journal of Immunological Methods 179-186 (2004)).

[0073] The mixture of primers and probes described above is used to test and distinguish between positive amplification control constructs and bona fide parvovirus contaminants. In some embodiments, the mixture of primers also includes a set of primers and probes to detect a nucleic acid extraction control (NEC). In certain embodiments, the NEC is an M13 bacteriophage, such as M13K07 strain (SEQ ID NO:8). Thus, in some embodiments, in addition to the parvovirus primers and probes and the UPS probe, the mixture of primers and probes contains (5) an M13-specific forward oligonucleotide primer; (6) an M13-specific oligonucleotide detection probe; and (7) an M13-specific reverse oligonucleotide primer.

[0074] In some embodiments, the M13 primer and probe hybridize to the sequence of SEQ ID NO:8. In a specific embodiment, the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5; the M13-specific oligonucleotide detection probe comprises the nucleic acid sequence of SEQ ID NO:6; and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7. As in the case of non-overlapping fluorophore emission spectra for the parvovirus probe and USP, the NEC probe contains a fluorophore that emits light in a non-overlapping spectrum. In a specific embodiment, the fluorophore attached to the M13-specific oligonucleotide detection probe is Cy5, a cyanine dye with an absorption maximum at about 650 nm and an emission maximum at about 670 nm (see Southwick et al., Cyanine Dye Labeling Reagents: Carboxymethylindocyanine Succinimidyl Esters, 11 Cytometry, 418-430 (1990)). In this case, the quencher may function by FRET or contact quenching. In one embodiment, the quencher is Black Hole Quencher®-2 (BHQ®-2, Biosearch Technologies, Inc., Petaluma, Calif.), which has an absorption maximum at about 579 nm and quenches in the range of about 550 nm to about 650 nm.

[0075] In another aspect, the present invention provides a method for detecting biological contaminants in a biological molecule production process. The biological contaminants more specifically include parvoviruses, even more specifically include rodent parvoviruses, and most specifically include parvoviruses that share at least 97% identity with the MVM NS1 gene. In some embodiments, the NS1 gene includes the sequence of SEQ ID NO:9. In one embodiment, the biological molecule production process is a mammalian cell culture process for producing antibodies, capture molecules, or other therapeutic antibodies. A test sample is taken from the cell culture (or bulk components) and extracted for nucleic acid. In some cases, a NEC such as M13 (e.g., SEQ ID NO:8) is added to the test sample prior to Q-PCR to serve as a control for proper extraction of nucleic acid. The method includes the steps of (1) mixing (a) a nucleic acid sample extracted from a test sample, (b) oligonucleotide primers and probes (described above), and (c) a DNA polymerase, preferably a thermostable DNA polymerase with 5' exonuclease activity, such as Taq polymerase; (2) subjecting the combination to polymerase chain reaction (PCR); and (3) monitoring the production of various amplicons by the magnitude of fluorescent emission.

[0076] The formation of specific amplicons correlates with the presence and amount of various template nucleic acids in test samples.Specific amplicons include (1) target amplification polynucleotides (TAP), such as rodent parvovirus sequences (e.g., biological contaminants containing NS1 sequences), (2) nucleic acid extraction controls (NEC), such as M13 (e.g., M13K07) polynucleotides (NECP), and (3) plasmid amplification control polynucleotides (PACP), such as unique artificial plasmid specific sequences (UAPS).If TAP and NECP are produced and PACP is not produced, it can be concluded that the test sample contains biological contaminants and does not contain cross-contaminated positive amplification control plasmids.However, if both TAP and PACP (i.e., UAP) are produced in test sample Q-PCR reaction, it can be concluded that the test sample is cross-contaminated with PAC plasmid and the TAP result may be false positive.

[0077] The primers and probes include (1) a rodent parvovirus-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO:1; (2) a rodent parvovirus-specific oligonucleotide detection probe comprising the sequence of SEQ ID NO:2, a VIC fluorophore, and an MGBNFQ quencher; (3) an artificial oligonucleotide detection probe, such as USP, comprising the sequence of SEQ ID NO:3 and labeled with 6-FAM and BHQ®-1; (4) a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of SEQ ID NO:4; (5) an M13-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO:5; (6) an M13-specific oligonucleotide detection probe comprising the sequence of SEQ ID NO:6 and labeled with Cy5 and BHQ®-2; and (7) a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of SEQ ID NO:7 and labeled with Cy5 and BHQ®-2. In a specific embodiment comprising an M13-specific reverse oligonucleotide primer comprising the sequence of NO:7, production of TAP is monitored at about 533 nm to about 580 nm, production of PACP is monitored at about 465 nm to about 510 nm, and production of NECP is monitored at about 618 nm to about 660 nm.

[0078] In one embodiment, a test sample is taken from a production CHO cell culture, e.g., a production culture of EESYR® cells transformed with a nucleic acid encoding a protein of interest, 96-72 hours prior to culture harvest time. If TAP and NECP are detected in the test sample, but PACP is not detected in the test sample, a confirmatory test can be performed on a second test sample obtained from the same producer cell culture 48 hours prior to harvest time. If TAP and NECP are again detected in the second test sample, but PACP is not detected in the test sample, the cell culture will be considered contaminated and will not be processed further. Alternatively, a second test will not be performed, but the cell culture will be considered contaminated and the culture will not be processed further.

[0079] In one embodiment, nucleic acid is extracted from the test sample taken from the production cell culture. In this case, one milliliter of the test sample is subjected to cell lysis, proteolysis, and heat denaturation, followed by mixing the sample with a nucleic acid extraction control (NEC) sample, and then extracting nucleic acid from the sample. In one embodiment, nucleic acid is extracted from the test sample (or the NEC additional test sample) using an automated nucleic acid extraction system such as the QIAsymphony® instrument (Qiagen, Inc., Valencia, CA) (see Lee et al., Comparative evaluation of the QIAGEN QIAsymphony® SP system and bioMerieux NucliSens easyMAG automated extraction platforms in a clinical virology laboratory, 52(4) J. Clin. Virol. 339-43 (2011)).

[0080] In one embodiment, uracil-N-glycosylase (UNG) enzyme is added to the Q-PCR reaction mixture prior to performing PCR to selectively degrade contaminating amplicons (see Taggart et al., Use of heat labile UNG in an RT-PCR assay for enterovirus detection, 105(1) J. Virol. Methods. 57-65 (2002)). The reaction mixture is incubated at 50° C. for at least 2 minutes, more specifically in some cases for 2 minutes or 5 minutes.

[0081] In a specific embodiment, after any UNG treatment, the reaction mixture is incubated at 95° C. for 2 minutes, followed by 8 cycles of (1) denaturation at 95° C. for 10 seconds, followed by (2) annealing and extension for 30 seconds, such that the initial annealing temperature is 70° C., and then the annealing temperature is decreased by 1° C. per cycle, such that the eighth annealing is at 62° C. After the first 8 cycles, 40 cycles of DNA amplification are performed, including (1) a denaturation step at 95° C. for 10 seconds, followed by (2) an annealing and extension step at 62° C. for 30 seconds. In a specific embodiment, the rate of temperature change from the denaturation temperature to the annealing temperature is about 4.4° C. per second, and from the annealing temperature to the denaturation temperature is about 2.2° C. per second.

[0082] In some embodiments, a method for detecting biological contaminants in a production cell culture medium or product thereof includes performing an external positive amplification control (PAC) assay performed separately from the test sample assay, and performing an external negative control assay performed separately from the test sample assay and the PAC assay. If either the negative control or the positive control fails, the results obtained from the test sample assay are rejected.

[0083] In one embodiment, the external positive control is (1) inter alia and in the absence of a test sample, (a) a positive amplification control (PAC) plasmid, which in a specific embodiment comprises the sequence of SEQ ID NO:11; (b) a rodent parvovirus-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO:1; (c) a rodent parvovirus-specific oligonucleotide detection probe comprising the sequence of SEQ ID NO:2, labeled with VIC and MGBNFQ; (d) an artificial oligonucleotide detection probe, such as USP, comprising the sequence of SEQ ID NO:3, labeled with 6-FAM and BHQ®-1; (e) a probe comprising the sequence of SEQ ID NO:4, labeled with 6-FAM and BHQ®-2, labeled with 6-FAM and BHQ®-1 ... The method includes the steps of: (1) mixing a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of NO:4, and (f) a DNA polymerase, preferably a thermostable DNA polymerase having 5' exonuclease activity, such as Taq polymerase; (2) subjecting the positive control mixture to a positive control polymerase chain reaction (PCR); and (3) monitoring during PCR the production of (a) target amplified polynucleotide (TAP), (b) nucleic acid extraction control amplified polynucleotide (NECP), and (c) plasmid amplified control polynucleotide (PACP). The production of TAP is monitored at about 533 nm to about 580 nm, the production of PACP is monitored at about 465 nm to about 510 nm, and the production of NECP is monitored at about 618 nm to about 660 nm.

[0084] The PCR reaction of the positive amplification control is carried out identically to the PCR reaction of the test sample (described above). If TAP and PACP are produced in the reaction of the positive control, it can be concluded that the PCR amplification procedure is functioning properly. If TAP is not produced in the reaction of the positive amplification control, any negative TAP in the test sample will be ignored as a failed PCR reaction. In one embodiment, NEC (i.e., for example, M13K07) is included in the positive amplification control. A properly functioning control should also show a positive NECP signal (see Table 1).

[0085] In one embodiment, the external negative control is (1) a blank, inter alia, without test sample and without PAC plasmid, (a) a buffer mimicking the test sample buffer system, or which may simply be water; (b) a rodent parvovirus-specific forward oligonucleotide primer comprising the sequence of SEQ ID NO:1; (c) a rodent parvovirus-specific oligonucleotide detection probe comprising the sequence of SEQ ID NO:2, labeled with VIC and MGBNFQ; (d) an artificial oligonucleotide detection probe, such as USP, comprising the sequence of SEQ ID NO:3, labeled with 6-FAM and BHQ®-1; (e) a probe comprising the sequence of SEQ ID NO:4, labeled with 6-FAM and BHQ®-2; The method includes the steps of: (1) mixing a rodent parvovirus-specific reverse oligonucleotide primer comprising the sequence of NO:4, and (f) a DNA polymerase, preferably a thermostable DNA polymerase having 5' exonuclease activity, such as Taq polymerase; (2) subjecting the positive control mixture to a positive control polymerase chain reaction (PCR); and (3) monitoring during PCR the production of (a) target amplified polynucleotide (TAP), (b) nucleic acid extraction control amplified polynucleotide (NECP), and (c) plasmid amplified control polynucleotide (PACP). The production of TAP is monitored at about 533 nm to about 580 nm, and the production of PACP is monitored at about 465 nm to about 510 nm.

[0086] The negative control PCR reaction is performed identically to the test sample PCR reaction (described above). If TAP and PACP are produced in the negative control reaction, it can be concluded that the PCR reagents are contaminated with PAC plasmid. If TAP is produced in the negative control reaction but PACP is not produced, it can be concluded that the PCR reagents are contaminated with parvovirus. In both cases, the test sample result is discarded. However, if the production of TAP and PACP is negative in the negative control reaction, the production of TAP and PACP is positive in the positive amplification control, and the production of TAP (and optionally NECP) is positive and the production of PACP is negative in the test sample reaction, the technician can conclude that the test sample is contaminated (see Tables 2 and 3).

[0087] In another aspect, the present invention provides a positive amplification control plasmid (PAC plasmid) and a mixture of positive control reagents comprising the positive control plasmid. In one embodiment, the PAC plasmid comprises (1) a parvovirus nucleic acid sequence, (2) an M13K07 nucleic acid sequence, and (3) an artificial nucleic acid sequence unique to the plasmid (also known as a UAPS or "unique" sequence). In a specific embodiment, the parvovirus nucleic acid sequence comprises the sequences of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:4; the M13K07 nucleic acid sequence comprises the sequences of SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7; and the unique sequence comprises the antisense sequence of SEQ ID NO:3. In a more specific embodiment, the nucleotide sequence of the PAC plasmid consists of the sequence set forth in SEQ ID NO:11.

[0088] In some embodiments, the positive control reagent mixture includes, inter alia, the PAC plasmid described above, a rodent parvovirus-specific forward oligonucleotide primer, a rodent parvovirus-specific oligonucleotide detection probe, an artificial oligonucleotide detection probe (i.e., USP), a rodent parvovirus-specific reverse oligonucleotide primer, an M13-specific forward oligonucleotide primer, an M13-specific oligonucleotide detection probe, and an M13-specific reverse oligonucleotide primer, and a buffer. The mixture optionally contains dNTPs and Taq polymerase.

[0089] Table 2: Assay controls and test session status TIFF0007669448000006.tif33170

[0090] (Table 3) Test session status TIFF0007669448000007.tif31170

[0091] In specific embodiments, the rodent parvovirus-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:1, the rodent parvovirus-specific oligonucleotide detection probe comprises a VIC fluorophore, a minor groove binding quencher (MGBNFQ), and the nucleic acid sequence of SEQ ID NO:2, the USP comprises a VIC fluorophore, a non-fluorescent quencher BHQ, and the nucleic acid sequence of SEQ ID NO:3, the rodent parvovirus-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:4, the M13-specific forward oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:5, the M13-specific oligonucleotide detection probe comprises a Cy5 fluorophore, a BHQ-2 quencher, and the nucleic acid sequence of SEQ ID NO:6, and the M13-specific reverse oligonucleotide primer comprises the nucleic acid sequence of SEQ ID NO:7. EXAMPLES

[0092] Example 1: Oligonucleotide and Nucleic Acid Reagents Rodent parvovirus, M13K07, and artificial unique oligonucleotides (oligos) were obtained in various sizes and formats from various suppliers listed in Table 4. All oligos were given a 3-year expiration date upon receipt from the supplier.

[0093] Master stocks were made by reconstituting oligonucleotides in water to a concentration of 100 μM. Prior to setting up the qPCR reactions, the master stocks were further diluted to make 10× stock solutions. Table 5 shows the 10× and 1× concentrations of qPCR oligonucleotides (primers and probes).

[0094] Table 4. Primers and probes for parvoviruses, M13, and unique artificial sequences. TIFF0007669448000008.tif134170

[0095] Table 5: Oligonucleotide concentrations TIFF0007669448000009.tif58170

[0096] Example 2: Positive Amplification Control Plasmid Parvo-M13 positive amplification control (PAC) plasmid was prepared in pUC57-Kan plasmid (GeneWiz, Inc., South Plainfield, NJ). This PAC plasmid consists of the sequence set forth in SEQ ID NO:11. Parvovirus-M13 PAC plasmid has the following formula: [number=(amount * number / mol) / (bp * ng / g * g / mol of bp); where amount = ng, number / mol = 6.022 x 10 23 , bp=4372, ng / g=1×10 9 , and bp g / mol = 650, calculated as 2.1 x 10 8 The plasmid concentration was calculated and expressed as ng / μL and is expressed as 10 copies / ng. 2Serial dilutions were made to a 10x concentration of copies / μL. 2 From the copies / μL dilution, 50 μL aliquots were prepared and stored in 2 mL sterile screw-cap tubes. All aliquots were stored at ≦−60° C.

[0097] Example 3: Preparation of M13K07 phage Ten-fold serial dilutions of K13K07 phage were performed using MEM as diluent to obtain a titer of 100 pfu / μL of M13K07. Aliquots of 200 μL were prepared and stored at ≦−60° C. and given a 3-year expiration date.

[0098] Example 4: Preparation of Q-PCR reaction reagents The rodent parvovirus real-time PCR detection assay is a fully automated TaqMan® PCR process consisting of automated DNA purification using a QIAsymphony® (Qiagen), followed by nucleic acid amplification, and real-time PCR product detection on a LightCycler® 480 instrument (Roche Diagnostic). Phage M13K07 was automatically added to each test article as an internal control (IC) to assess the presence of PCR inhibitors. Rodent parvovirus primer oligonucleotides were designed to hybridize within a highly conserved region (NS-1 region) of the rodent parvovirus genome to ensure broad-range detection. The assay was performed in a duplex (i.e., two-target) format, and the rodent parvovirus and M13K07 primers generated PCR products of approximately 110 and 97 bp, respectively. PCR products were detected in real time by cleavage of two probes labeled with different reporter dyes: VIC fluorophore for rodent parvovirus and Cy5 fluorophore for M13K07 phage. A positive amplification control (PAC) plasmid was used at a concentration of 100 copies / uL. This plasmid contains a unique (Flag) sequence (USP) that distinguishes it from wild-type parvovirus using a specific probe labeled with the fluorophore FAM.

[0099] The master mix was assembled in 2 mL tubes according to Table 6, with sufficient volume for at least 3x the number of test articles, including controls. Tubes were stored at 2-8 °C. Negative amplification control (NAC) vials were prepared by adding 50 uL of water to a 2 mL tube.

[0100] Table 6. Reaction mixture TIFF0007669448000010.tif46170

[0101] Example 5: Preparation of test samples Aseptic practices were adhered to for all sample preparation steps since all samples have the potential to contain or be contaminated with adventitious agents. The following steps were performed in a clean biosafety cabinet. Test articles (samples) were obtained from EESYR® cell cultures producing antibodies or capture molecules and were either frozen or used directly.

[0102] 1000 μL of PBS was aliquoted into approximately 2 mL tubes to act as negative extraction controls (NECs). When PCR was used as the endpoint for the cell culture step, cell culture positive and negative controls were used as positive and negative extraction controls, respectively.

[0103] Each test article was thawed at room temperature. The samples in the 60 mL bags were transferred to 50 mL Falcon tubes and then aliquoted. 1000 μL of each sample was pipetted into 2 mL tubes for pretreatment. 400 μL of lysis buffer (Charge Switch® Lysis Buffer L13) (Invitrogen, Cat. No. CS11202 or equivalent, Carlsbad, CA) was added to each tube and vortexed for at least 10 seconds. Then, 20 μL of proteinase K (≧10 mg / mL, ≧800 units / mL in 40% glycerol (v / v) containing 10 mM Tris-HCl, pH 7.5 with 1 mM calcium acetate; SAFC, Cat. No. P4850-5ML, Sigma Aldrich, St. Louis, MO) was added to each sample and vortexed for at least 10 seconds. The samples were then incubated at 65° C. for 30 minutes. After incubation, the samples were vortexed and centrifuged at 17,000 xg for 10 minutes.

[0104] At the same time, positive amplification controls (PACs; a minimum of 50 uL of 10 2 copies / μL plasmid) and M13K07 (10 2 PFU / mL) internal control (IC; minimum 150 μL / 12 samples) was thawed.

[0105] Example 6: Nucleic Acid Extraction An internal control (IC) was required for the programmed extraction protocol on the QIASymphony® instrument (Qiagen, Valencia, CA). The instrument automatically added 120 μL of reconstituted IC to each sample. For every 12 samples, 1.8 mL of IC (i.e., 1 vial) was required by the instrument. Vials of IC were prepared by adding 1650 μL of AVE buffer (RNase-free water containing 0.04% sodium azide) to 150 μL of M13K07 IC.

[0106] Each prepared sample was loaded onto the instrument's sample carrier in a biosafety cabinet (BSC). Vials of IC were loaded onto separate (dedicated) sample carriers in the BSC, with a ratio of 1 vial of IC per 12 samples. All drawers were closed and the instrument was run according to the manufacturer's recommended protocol (see QIAsymphony DNA Handbook, 09 / 2010, available at http: / / www.algimed.by / download / EN-QIAsymphony-DNA-Handbool.pdf).

[0107] Reagent prep cartridges were prepared with the QIAsymphony® DSP Virus / Pathogen Reagent Kit, which contained all the reagents required to perform the extraction (see QIAsymphony® DSP Virus / Pathogen Kit Instructions for Use (Handbook, April 2013, available at https: / / www.qiagen.com / us / resources / download.aspx?id=f8bc0b3c-0aff-46ee-8807-5ed145f9e969&lang=en). The reagent prep cartridge contained proteinase K and had a shelf life of approximately 2 weeks.

[0108] Nucleic acid extraction was performed in a 96-well format reaction plate to facilitate integration with Q-PCR. After the automated nucleic acid extraction procedure was completed and passed the status check, the reaction plate was cooled and sealed with a LightCycler® 480 Sealing Foil (Roche, Branchburg, NJ). The 96-well plate was placed in a plate spinner balanced with an appropriate counterweight (e.g., another 96-well plate) and spun for 30-60 seconds. The wells were checked for air bubbles and the spin was repeated if necessary.

[0109] Example 7: Q-PCR Q-PCR was performed on a LightCycler® 480 instrument (Roche, Branchburg, NJ) using one of two programs: TaqMan Triplex and / or Veriquest Triplex. The TaqMan Triplex protocol used TaqMan Fast Advance Custom Master Mix without the reference dye (ROX). Three fluorescent channels (FAM, VIC, and CY5) were selected, and the duration of the UNG step was 2 minutes. The reaction parameters used are summarized in Table 7.

[0110] Similarly, when using the ROX-free VeriQuest® Master Mix, a 5 minute UNG step time was used. The reaction parameters used are summarized in Table 8.

[0111] Table 7. TaqMan Triplex Program Steps for Rodent Parvovirus PCR TIFF0007669448000011.tif51170

[0112] The crossing point (Cp) fluorescent signal for each of the M13 internal control, rodent parvovirus, and positive amplification control plasmid was determined by one or both of two algorithms. The first algorithm is an automatic second derivative method. This method does not require user input and generally results in greater consistency, and was therefore considered to be the preferred method. The second algorithm is a Fit Point method. This method allows the user to set a threshold line in the case of a dispersed background. The point at which the log-linear curve crosses the threshold line is the crossing point. The following filter combinations were used to monitor the fluorescent signal: 533-580 nm (VIC signal) for rodent parvovirus (subset "Sample-Parvo"); 618-660 nm (Cy5 signal) for the internal control M13K07 (subset "Sample-M13"); and 465-510 nm (FAM signal) for the positive amplification control plasmid (subset "Sample-FAM"). In case of unclear fluorescent signals, fit point analysis was used to determine the fluorescent background level. An acceptable background fluorescent signal was considered to be ≦1 unit on the amplification plot scale. Any fluorescent signal of ≦1 unit was within the acceptable background and therefore considered negative.

[0113] Table 8. VeriQuest Triplex Program Steps for Rodent Parvovirus PCR TIFF0007669448000012.tif51170

[0114] Example 8: Conditions for a valid test session For a test session to be considered valid, the following conditions must be met: The negative amplification control (NAC, i.e., water) must be negative for the fluorescent signal in all three channels. The negative extraction control (NEC, i.e., PBS) or cell culture negative control flask must be negative for the fluorescent signal in the [533–580] channel (i.e., rodent parvovirus probe-VIC signal), negative for the fluorescent signal in the [465–510] channel (i.e., positive amplification control [PAC] antisense Flag probe-FAM signal), and positive for the fluorescent signal in the [618–660] channel (i.e., M13K07 probe-CY5 signal). The PAC should be positive for the fluorescent signal in all three channels. The Cp value of M13K07 in the NEC was used as a reference to evaluate the presence of inhibitory substances in the samples.

[0115] When using PCR as an endpoint for test articles derived from the cell culture step with a rodent parvovirus positive control, the positive virus control should be positive for a fluorescent signal in the [533-580] channel (i.e., rodent parvovirus probe VIC signal), positive for a fluorescent signal in the [618-660] channel (i.e., M13K07 probe-CY5 signal), and negative for a fluorescent signal in the [465-510] channel (i.e., PAC antisense Flag probe-FAM signal). The Cp value of M13K07 in the cell culture positive control sample was expected to be within ±4 cycles of the Cp value of NEC-M13.

[0116] For all PAC plasmid-containing control reactions, the fluorescent signal should be positive in all three channels.

[0117] Example 9: Conditions for an Invalid Test Session An assay was considered invalid if any one or more of the following conditions were met: (1) a very low amplification curve for PAC (<1 unit on the fluorescence scale), (2) a determinant error (machine, software, or human error) was identified, (3) the NAC was positive for amplification in any of the three channels, (4) the NEC was positive for amplification in the [533-580] VIC channel, positive for amplification in the [465-510] FAM channel, or negative for amplification in the [618-660] Cy5 channel, and (5) the PAC was negative for an amplification signal in any of the three channels. Whenever an assay was determined to be invalid, the test samples were investigated and retested.

[0118] Example 10: Conditions for a negative sample result in a valid test For a valid negative parvovirus test result, all of the following conditions must be met: The sample must be positive for M13K07 DNA amplification signal in the M13 [618-660] channel within the expected Cp value range of NEC-M13 Cp ± 4 cycles, suggesting the absence of PCR inhibitors. The sample must be negative for parvovirus DNA amplification signal in the Parvovirus [533-580] channel. Fluorescence signals below 1 unit on the fluorescence scale were reported as negative for parvovirus DNA amplification, regardless of whether the Cp value was considered to be within the acceptable fluorescence background level. The sample must be negative for PAC amplification signal in the Antisense Flag [465-510] channel. Please note that fluorescence signals below 1 unit on the fluorescence scale were reported as negative for PAC plasmid DNA amplification, regardless of whether the Cp value (automatically provided by the instrument) was considered to be within the acceptable fluorescence background level.

[0119] Example 11: Conditions for a "No Sample" Result in a Valid Test Session A "no sample" result occurred when any one or more of the following conditions were met: Whenever a sample was negative for M13K07 DNA amplification signal in the Cy5 channel [618-660], negative for parvovirus DNA amplification signal in the parvovirus channel [533-580], and negative for PAC amplification in the antisense Flag FAM channel [465-510], the sample or PCR reagents were investigated according to standard operating procedures. This condition suggests the presence of PCR inhibitors or failure of proper nucleic acid extraction. To overcome inhibition, the sample could be diluted (1:2, 1:5, 1:10). Cp values ​​of the fluorescent signal in the M13 channel [618 660] that were outside the range (NEC M13 Cp ± 4 cycles) indicated partial inhibition of PCR or an error in phage addition, and required a repeat test. Dilution of the sample (1:2, 1:5, 1:10) could be considered to overcome any inhibition of PCR. Any evidence of systematic error or unexpectedly very low fluorescence signals (<1 unit on the fluorescence scale) observed in the M13 channel [618-660] that did not allow a conclusive assessment of sample suitability was considered a "no sample result" and suggested a failure during amplification or DNA extraction, which required repeat testing.

[0120] Example 12: Conditions for an Initial Out-of-Specification (iOOS) Sample Result in a Validated Testing Session A sample was considered to be iOOS if it (i) was positive for parvovirus DNA amplification signal (i.e., rodent parvovirus probe VIC signal) in the parvovirus channel [533-580] with a fluorescent signal above 1 unit on the fluorescence scale (for at least one of the two duplicate wells) and (ii) was negative for PAC amplification in the antisense Flag FAM channel [465-510] (indicating no cross-contamination with PAC). As a result, the technician must (i) initiate a GLIF (a form of common laboratory investigation), (ii) notify the department that the sample has been sent to QC (virology) for testing, (iii) initiate a NOE, (iv) store the amplification tubes (frozen at -20°C) for further investigation (e.g., Flag sequence screening or sequencing), and (v) repeat the assay and retest the sample.

[0121] Example 13: Replication and Retest Plan Repeat testing was initiated whenever the assay was invalid or the sample result was deemed "no result." Retesting was performed for confirmation of iOOS events (i.e., the initial sample result was positive for DNA amplification signal in the parvovirus channel [533-580]). Retests or repeat testing must be performed with fresh reagent aliquots (i.e., master mix reagents, extraction kits).

[0122] If the NAC was positive for fluorescence in any channel, the entire test session was repeated starting from the amplification (PCR) step with the already purified DNA sample with a freshly made master mix. If the NEC was negative for fluorescence in the M13 channel [618-660], the entire test session was repeated starting from the DNA extraction step. If the NEC was positive for fluorescence in the parvovirus channel [533-580] or the antisense Flag channel [465-510], the entire test session was repeated starting from the DNA extraction step. If the PAC was negative for fluorescence in any channel, the test session was repeated starting from the amplification (PCR step) with the same purified DNA sample with a freshly made master mix.

[0123] For samples with a "No Sample" result, the test session starting with the DNA extraction step was repeated for the affected sample. As part of the investigation and problem-solving process, samples may be diluted (1:2, 1:5, or 1:10) prior to DNA extraction (in addition to the undiluted sample) to demonstrate the presence of inhibitors.

[0124] Retesting to confirm the initial positive result (iOOS) was performed with four separate aliquots: two aliquots of the test article from the original sampling event (e.g., one day after the last feeding) and two aliquots of the test article from a different sampling event (e.g., two days after the last feeding, if possible) or from a different sample bag. If any of the additional tests on the four aliquots yielded a positive signal without evidence of determinate error (demonstrated by investigation), the lot was considered to have failed the requirement for the absence of rodent parvovirus genomic material. The final disposition for such a positive Q-PCR result required an infectivity assay. CHO-K1 cells are used as an indicator cell line to determine the infection status of the detected nucleic acid.

[0125] Whenever the retest result is negative, a confirmatory test is required at a different sampling event (eg, 3 days after the last feeding) to confirm the absence of rodent parvovirus genomic material.

[0126] Example 14: Retesting Plan for Other Sample Types with iOOS Other sample types that had initial positive results (iOOS), including unprocessed bulk material, end-of-production cells, cells at the limit of in vitro life span, and soy materials, were retested using four separate aliquots as follows.

[0127] Two aliquots of the test article from the original sample container (i.e., e.g., bag) and two aliquots from a different sample container were retested according to standard operating procedures. CHO-K1 indicator cells were inoculated with one sample aliquot of the test article from the original sample container. The inoculated indicator cells CHO-K1 were harvested after 1-3 days in culture according to standard operating procedures to determine the infection status of the detected nucleic acid. A standard curve for quantification of the re-extracted nucleic acid may be used.

[0128] If any of the additional PCR tests on the four aliquots are positive without evidence of clear error (demonstrated by investigation), the infectivity assay will determine the ultimate disposition of the test material. Upon confirmation of initial OOS, nucleic acid sequencing and transmission electron microscopy (TEM) may be considered to identify the organism and rule out any laboratory error.

[0129] Infectivity assays using CHO-K1 as indicator cells are required for suspected contaminated material to determine the infectious state of detected nucleic acids. Whenever the investigation failed to support possible viral contamination with parvovirus and additional PCR tests and CHOK1 cultures on four aliquots of the test material were all negative, the lot was considered as meeting the requirement for the absence of infectious rodent parvovirus.

[0130] Example 15: Parvovirus testing during recombinant protein production The Q-PCR procedure described above was performed as a critical in-process control for testing on cell culture fluid, i.e., unprocessed bulk material from the production bioreactor containing CHO cell derivatives containing heterologous antibody heavy and light chain constructs. Each heterologous monoclonal antibody (mAb) binds to a different target or epitope. Good manufacturing practice (GMP) testing was performed by QC (virology) scientists in a large-scale bioprocess production facility. Sixteen of those tests are listed in Table 9. In each case, the test session was valid, the assay system suitability criteria were met, and no false positive detections were observed. In test sessions #13 and #14, false negative detections occurred, which suggested the presence of PCR inhibitors or failure of nucleic acid extraction.

[0131] Table 9. Rodent parvovirus PCR test TIFF0007669448000013.tif120168UPB=Unprocessed Bulk Material;EA=Early Alert;Rodent ParvoPCR=Q-PCR procedure as described above

[0132] Sequence information SEQUENCE LISTING <110> Regeneron Pharmaceuticals, Inc. <120> COMPOSITIONS AND METHODS FOR DETECTING A BIOLOGICAL CONTAMINANT <150> US 62 / 139,321 <151> 2015-03-27 <160> 37 <170> PatentIn version 3.5 <210> 1 <211> twenty four <212> DNA <213> Parvovirus <400> 1 tgcataaaag agtaacctca ccag 24 <210> 2 <211> 18 <212> DNA <213> Parvovirus <400> 2 actggatgat gatgcagc 18 <210> 3 <211> 19 <212> DNA <213> Parvovirus <400> 3 tgtcgatggc gaatggcta 19 <210> 4 <211> 18 <212> DNA <213> Parvovirus <400> 4 ccacctggtt gagccatc 18 <210> 5 <211> 20 <212> DNA <213> Bacteriophage M13 <400> 5 aagcctcagc gaccgaatat 20 <210> 6 <211> 24 <212> DNA <213> Bacteriophage M13 <400> 6 tatgcgtggg cgatggttgt tgtc 24 <210> 7 <211> 22 <212> DNA <213> Bacteriophage M13 <400> 7 tcagcttgct ttcgaggtga at 22 <210> 8 <211> 6407 <212> DNA <213> Bacteriophage M13 <400> 8 aacgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60 atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120 cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 180 gttgcatatt taaaacatgt tgagctacag caccagattc agcaattaag ctctaagcca 240 tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300 ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 360 tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420 cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480 tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540 aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600 ggttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 660 aattccttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 720 atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780 tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840 caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900 ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960 aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1020 tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080 gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140 caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctggggt 1200 caaagatgag tgttttagtg tattctttcg cctctttcgt tttaggttgg tgccttgta 1260 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320 caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440 tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560 tttttggaga ttttcaacat gaaaaaatta ttattcgcaa ttcctttagt tgttcctttc 1620 tattctcact ccgctgaaac tgttgaaagt tgtttagcaa aaccccatac agaaaattca 1680 tttactaacg tctggaaaga cgacaaaact ttagatcgtt acgctaacta tgagggttgt 1740 ctgtggaatg ctacaggcgt tgtagtttgt actggtgacg aaactcagtg ttacggtaca 1800 tgggttccta ttgggcttgc tatccctgaa aatgagggtg gtggctctga gggtggcggt 1860 tctgagggtg gcggttctga gggtggcggt actaaacctc ctgagtacgg tgatacacct 1920 attccgggct atacttatat caaccctctc gacggcactt atccgcctgg tactgagcaa 1980 aaccccgcta atcctaatcc ttctcttgag gagtctcagc ctcttaatac tttcatgttt 2040 cagaataata ggttccgaaa taggcagggg gcattaactg tttatacggg cactgttact 2100 caaggcactg accccgttaa aacttattac cagtacactc ctgtatcatc aaaagccatg 2160 tatgacgctt actggaacgg taaattcaga gactgcgctt tccattctgg ctttaatgag 2220 gatccattcg tttgtgaata tcaaggccaa tcgtctgacc tgcctcaacc tcctgtcaat 2280 gctggcggcg gctctggtgg tggttctggt ggcggctctg agggtggtgg ctctgagggt 2340 ggcggttctg agggtggcgg ctctgaggga ggcggttccg gtggtggctc tggttccggt 2400 gattttgatt atgaaaagat ggcaaacgct aataaggggg ctatgaccga aaatgccgat 2460 gaaaacgcgc tacagtctga cgctaaaggc aaacttgatt ctgtcgctac tgattacggt 2520 gctgctatcg atggtttcat tggtgacgtt tccggccttg ctaatggtaa tggtgctact 2580 ggtgattttg ctggctctaa ttcccaaatg gctcaagtcg gtgacggtga taattcacct 2640 ttaatgaata atttccgtca atatttacct tccctccctc aatcggttga atgtcgccct 2700 tttgtcttta gcgctggtaa accatatgaa ttttctattg attgtgacaa aataaactta 2760 ttccgtggtg tctttgcgtt tctttatat gttgccacct ttatgtatgt atttctacg 2820 tttgctaaca tactgcgtaa taaggagtct taatcatgcc agttcttttg ggtattccgt 2880 tattattgcg tttcctcggt ttccttctgg taactttgtt cggctatctg cttacttttc 2940 ttaaaaaggg cttcggtaag atagctattg ctatttcatt gtttcttgct cttattattg 3000 ggcttaactc aattcttgtg ggttatctct ctgatattag cgctcaatta ccctctgact 3060 ttgttcaggg tgttcagtta attctcccgt ctaatgcgct tccctgtttt tatgttattc 3120 tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg 3180 attgggataa ataatatggc tgtttatttt gtaactggca aattaggctc tggaaagacg 3240 ctcgttagcg ttggtaagat tcaggataaa attgtagctg ggtgcaaaat agcaactaat 3300 cttgattaa ggcttcaaaa cctcccgcaa gtcgggaggt tcgctaaaac gctcgcgtt 3360 cttagaatac cggataagcc ttctatatct gatttgcttg ctattgggcg cggtaatgat 3420 tcctacgatg aaaataaaaa cggcttgctt gttctcgatg agtgcggtac ttggtttaat 3480 acccgttctt ggaatgataa ggaaagacag ccgattattg attggttct acatgctcgt 3540 aaattaggat gggatattat ttttcttgtt caggacttat ctattgttga taaacaggcg 3600 cgttctgcat tagctgaaca tgttgtttat tgtcgtcgtc tggacagaat tactttacct 3660 tttgcggta ctttatattc tcttattact ggctcgaaaa tgcctctgcc taaattacat 3720 gttggcgttg ttaaatatgg cgattctcaa ttaagcccta ctgttgagcg ttggctttat 3780 actggtaaga atttgtataa cgcatatgat actaaacagg cttttctctag taattatgat 3840 tccggtgttt attcttattt aacgccttat tttcacacg gtcggtattt caaaccatta 3900 aatttaggtc agaagatgaa attaactaaa atatatttga aaaagttttc tcgcgttctt 3960 tgtcttgcga ttggatttgc atcagcattt acatatagtt atataaccca acctaagccg 4020 gaggttaaaa agtagtctc tcagacctat gattttgata aattcactat tgactcttct 4080 cagcgtctta atctaagcta tcgctatgtt ttcaaggatt ctaagggaaa attaattaat 4140 agcgacgatt tacagaagca aggttattca ctcacatata ttgattatg tactgtttcc 4200 attaaaaaag gtaattcaaa tgaaattgtt aaatgtaatt aatttgttt tcttgatgtt 4260 tgttcatca tcttctttg ctcaggtaat tgaaatgaat aattcgcctc tgcgcgattt 4320 tgtaacttgg tattcaaagc aatcaggcga atccgttatt gttctcccg atgtaaaagg 4380 tactgttact gtatattcat ctgacgttaa acctgaaaat ctacgcaatt tctttattc 4440 tgttttacgt gctaataatt ttgatatggt tggttcaatt ccttccataa ttcagaagta 4500 taatccaaac aatcaggatt atattgatga attgccatca tctgataatc aggaatatga 4560 tgaattcc gctccttctg gtggttctt tgttccgcaa aatgataatg ttactcaaac 4620 ttttaaaatt aataacgttc gggcaaagga tttaatacga gttgtcgaat tgtttgtaaa 4680 gtctaatact tctaaatcct caaatgtatt atctattgac ggctctaatc tattagttgt 4740 tagtgcacct aaagatattt tagataacct tcctcaattc ctttctactg ttgatttgcc 4800 aactgaccag atattgattg agggtttgat atttgaggtt cagcaaggtg atgctttaga 4860 tttttcattt gctgctggct ctcagcgtgg cactgttgca ggcggtgtta atactgaccg 4920 cctcacctct gttttatctt ctgctggtgg ttcgttcggt atttttaatg gcgatgtttt 4980 agggctatca gttcgcgcat taaagactaa tagccattca aaaatattgt ctgtgccacg 5040 tattcttacg ctttcaggtc agaagggttc tatctctgtt ggccagaatg tcccttttat 5100 tactggtcgt gtgactggtg aatctgccaa tgtaaataat ccatttcaga cgattgagcg 5160 tcaaaatgta ggtatttcca tgagcgtttt tcctgttgca atggctggcg gtaatattgt 5220 tctggatatt accagcaagg ccgatagttt gagttcttct actcaggcaa gtgatgttat 5280 tactaatcaa agaagtattg ctacaacggt taatttgcgt gatggacaga ctcttttact 5340 cggtggcctc actgattata aaaacacttc tcaagattct ggcgtaccgt tcctgtctaa 5400 aatcccttta atcggcctcc tgtttagctc ccgctctgat tccaacgagg aaagcacgtt 5460 atacgtgctc gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg 5520 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt 5580 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 5640 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 5700 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 5760 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 5820 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 5880 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa 5940 tttaaatatt tgcttataca atcttcctgt ttttggggct tttctgatta tcaaccgggg 6000 tacatatgat tgacatgcta gttttacgat taccgttcat cgattctctt gtttgctcca 6060 gactctcagg caatgacctg atagcctttg tagacctctc aaaaatagct accctctccg 6120 gcatgaattt atcagctaga acggttgaat atcatattga tggtgatttg actgtctccg 6180 gcctttctca cccttttgaa tctttaccta cacattactc aggcattgca tttaaaatat 6240 atgagggttc taaaaatttt tatccttgcg ttgaaataaa ggcttctccc gcaaaagtat 6300 tacagggtca taatgttttt ggtacaaccg atttagcttt atgctctgag gctttattgc 6360 ttaattttgc taattctttg ccttgcctgt atgatttatt ggatgtt 6407 <210> 9 <211> 2019 <212> DNA <213> Minute virus of mouse <400> 9 atggctggaa atgcttactc tgatgaagtt ttgggagcaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaaatgaaa atgttcaact gaatggaaaa 120 gatatcggat ggaatagtta caaaaaagag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacttg ggaccaaagc gaggacatgg aatgggaaac cacagtggat 240 gaaatgacca aaaagcaagt attcattttt gattctttgg ttaaaaaatg tttatttgaa 300 gtgcttaaca caaagaatat atttcctggt gatgttaatt ggtttgtgca acatgaatgg 360 ggaaaagacc aaggctggca ctgccatgta ctaattggag gaaaggactt tagtcaagct 420 caagggaaat ggtggagaag gcaactaaat gtttactgga gcagatggtt ggtaacagcc 480 540 gagtgggtta ctctacttac ttataagcat aagcaaacca aaaaagacta taccaagtgt 600 gttctttttg gaaacatgat tgcttactat tttttaacta aaaagaaaat aagcactagt 660 ccaccaagag acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg agcgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 1020 tttgacttaa ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgcctgac 1080 acaagaacct gcagaatttt tgcttttcat ggctgggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttttaaacag aaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg caatgttggt 1260 tgctataatg cagccaatgt aaactttcca tttaatgact gtaccaacaa gaacttgatt 1320 tgggtagaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca taccttgcct 1560 ggtgactttg gtttggttga caaaaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc aaaggtgcca actcctataa attactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 10 <211> 19 <212> DNA <213> Artificial <220> <223> synthetic <400> 10 tagccattcg ccatcgaca 19 <210> 11 <211> 1707 <212> DNA <213> Artificial <220> <223> synthetic <400> 11 aagcctcagc gaccgaatat atcggttatg cgtgggcgat ggttgttgtc attgtcggcg 60 caactatcgg tatcaagctg tttaagaaat tcacctcgaa agcaagctga cacgcctacc 120 gcgatgctga atgacccgga ctagagtggc gaaatttatg gcgtgtgacc cgttatgctc 180 catttcggtc agtgggtcat tgctagtagt cgattgcatt gccattctcc gagtgattta 240 gcgtgacagc cgcagggaac ccataaaatg caatcgtagt ccacctgatc gtacttagaa 300 atgagggtcc ccttttgccc acgcacctgt tcgctcgtcg tttgctttta agaaccgcac 360 gaaccacaga gcataaagag aacctctagc tcctttacaa ggtactggtt ccctttccag 420 cgggatgcct tatctaaacg caatgacaga cgtattcctc aggccacatc gcttcctact 480 tccgctggga tccatcattg gcggccgaag ccgccattcc atagtgagtc cttcgtctgt 540 gtctttctgt gccagatcgt ctagcaaatt gccgatccag tttatctcac gaaactatag 600 tcgtacagac cgaaatctta agtcaaatca cgcgactagg ctcagctcta ttttagtggt 660 catgggtttt ggtccgcccg agcggtgcaa ccgattagga ccatgtaaaa catttgttac 720 aagtcttct ttaaacacaa tcttcctgct cagtggcgca tgattatcgt tgttgctagc 780 cagcgtggta agtaacagca ccactgcgag cctaatgtgc cctttccacg aacacagggc 840 tgtccgatcc tatattagga ctccgcaatg gggttagcaa gtcgcaccct aaacgatgtt 900 gaagactcgc gatgtacatg ctctggtaca atacatacgt gttccggctg ttatcctgca 960 tcggaacctc aatcatgcat cgcaccagcg tattcgtgtc atctaggagg ggcgcgtagg 1020 fathers gtcgttatgc tagtatacgc ctacccgtca ccggccatct gtgtgcagat ggggcgacga gttactggcc ctgatttctc cgcttctaat accacacact gggcaatacg agctcaagcc agtctcgcag taacgctcat cagctaacga aagagttaga 1260. ggctcgctaa cggagacgag ttaaagacac gagttcccaa aaccaggcgg gctcgccacg acggctaatc ctggtagttt acgtgaacaa tgttctgaag aaaatttgtg aaagaaggac ccgtcaccgc ctacaattac ctacaacggt cggccgcacc ttcgattgtc gtggccaccc 1380 tcggattaca cggcagaggt ggttgtgtcc cgacaggcca gcatattatc ctgaggcgtt 1440 accccaatcg ttctccgtcg gatttgctac agcccctgag cgctacatgc acgaaaccaa gttatgtatg cactgggccg tcaataggac gtagccttgt agttagcacg tagcccggcc 1560. gcattagtac agtagcct ccgccggcat cctgtttatt aagttatttc tgcataaaag 1620 agtaacctca ccagaggact ggatgatgat gcagccagac agttagccat tcgccatcga 1680. cattgaatg atggctcaac caggtgg <210> 12 <211> 2019 <212> DNA <213> Mvm lymphotropic variant <400> 12 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaaaaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaatagc agaagacagt 540 gagtgggtta ctttactcac ttataaacat aagcaaacca aaaaggacta tactaaatgt 600 gttctttttg gaaatatgat tgcttactac ttttaacca aaaagaaaat aagcaccagt 660 ccgccaaggg acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ttatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctagtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acgctactc tagccagaac aaaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatttt tgcttttcat ggctgggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgtttttt tcacggacca 1200 gccagtcag gcaaatctat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagctaatgt gaactttcca tttaatgact gtaccaacaa gaacttgatt 1320 tgggtagaag aagctggtaa ctttggacag gaattaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacaaattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaattag agacagaatg ctcaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caagaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgcgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc aaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcaat aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 13 <211> 2019 <212> DNA <213> Mouse parvovirus 4b <400> 13 atggctggaa atgcttactc tgatgaagtt ttgggagcaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtattctc atttgttttt aaaaatgagg atgttcaact gaatggaaaa 120 gatattggat ggaatagcta caaaaaagag ctacaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaagtaacca aaaagcaagt attcattttt gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttaaca caaagaacat agctcctagt gatgttaatt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct 420 caaggaaagt ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctatcacc agctgaaaga attaaactaa gagaatagc agaagacaat 540 gagtgggtta gcttgctcac ttataagcat aagcaaacca aaaaggacta tactaagtgt 600 gttctttttg gcaacatgat tgcttactac ttttaacca aaaagaaaat aagcactagt 660 ccaccaaggg acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcacaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctggtgcata aaagagtac ctcaccagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acactactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ctaacttttc actgccggac 1080 acaagaacct gcaagatttt tgcttttcat ggctgggaact acataaagt ttgccatgct 1140 atttgctgtg ttttaaacag aaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagtcag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt aaactttcca ttcaatgact gtaccaacaa gaacttgatt 1320 tgggtggaag aagctggtaa ctttggacag gaagtaaacc agttcaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcagaa taggctgtga agaaagacca 1500 gagcacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgtg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 14 <211> 2042 <212> DNA <213> Mouse parvovirus 4a <400> 14 atggctggaa atgcttactc tgatgaagtt ttgggagcaa ccaactggtt aaaggaaaaa 60 aataatcagg aagtgttctc atttgttttt aaaaatgagg atgttcaact gaatggaaaa 120 gatatcggat ggaatagtta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tgcagtggat 240 gaagtgacca aaaagcaagt attcattttt gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttaaca caaagaacat agctcctgct gatgttaatt ggtttgtgca gcatgaatgg 360 ggaaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaagt ggtggagaag gcagctaaat gtttactgga gcagatggtt agtaacagcc 480 tgtaatgtac agctatcacc agctgaaaga attaaactaa gagaatagc agaagacaat 540 gagtgggtta ccttgctcac ttataagcat aagcaaacca aaaaggacta tactaagtgt 600 gttctttttg gcaacatgat tgcttactac ttttaacca aaaagaaaat aagcactagt 660 ccaccaaggg acggaggcta tttctgagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatatactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcaa ttaaaacac acttaaaagag ctggtgcata aaagagatac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa caaacttttc actgcctgat 1080 acaagaacct gcaagatttt tgcttttcat ggctgggaact acataaagt ttgccatgct 1140 atttgctgtg ttttaaacag aaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagtcag gcaaatctat cattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa gaagtaaacc agtttaaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacatcaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcaacga gccgctgaac ttggactaag gtacgatggc gcctccagct 2040 aa 2042 <210> 15 <211> 2019 <212> DNA <213> Mouse parvovirus 1b <400> 15 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaagaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaatagc agaagacagt 540 gagtgggtta ctttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttactac ttttaacca agaagaaaat aagcactagt 660 ccgccaaggg acggaggcta tttctgagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctggtgcata aaagagtac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatctgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatctt tgcttttcat ggctgggaact acataaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagtcag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttgatt 1320 tgggtggaag aagctggtaa ctttggacag bagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcaaaa taggctgcga ggagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaagtgag tggcccatga tctgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 16 <211> 2019 <212> DNA <213> Mouse parvovirus minute virus immunosuppressive variant <400> 16 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaaaaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaatagc agaagacagt 540 gagtgggtta ctttactcac ttataaacat aagcaaacca aaaaggacta tactaaatgt 600 gttctttttg gaaatatgat tgcttactac ttttaacca aaaagaaaat aagcaccagt 660 ccgccaaggg acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ttatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctagtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatttgt acgctactc tagccagaac aaaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatttt tgcttttcat ggctgggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgtttttt tcacggacca 1200 gccagtcag gcaaatctat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagctaatgt gaactttcca tttaatgact gtacgaacaa aaacttgatt 1320 tgggtagaag aagctggtaa ctttggacag gaattaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacaaattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaattag agacagaatg ctcaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caagaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgcgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagcga gccgctgaac ttggactaa 2019 <210> 17 <211> 2019 <212> DNA <213> Mouse parvovirus 1 <400> 17 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta caaaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaaatgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttagca caaagaatat agctcctgct gatgttactt ggtttgtgca gcatgaatgg 360 gggaaagacc aaggctggca ctgccatgta ctaattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtgc agctaacacc agctgaaaga attaaactaa gagaatagc agaagacagt 540 gagtgggtta ctttacttac ttataaacat aagcaaacca aaaaggacta tactaaatgt 600 gttctttttg gaaatatgat tgcttactac ttttaacca aaaaaaaaat aagcaccagt 660 ccgccaagag acggaggcta ttttctaagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg accagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaaccac acttaaagag ctggtgcata aaagagtac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatctgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatctt tgcttttcat ggctgggaact acataaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagtcag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa gaagtaaacc agtttaaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 18 <211> 2019 <212> DNA <213> Mouse parvovirus 5a <400> 18 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgtttt aaaactgaag atgttcaact gaatggaaaa 120 gatattggat ggaataatta cagaaaggag ctgcaagagg acgagctaaa atctttacaa 180 240 gaagtgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtacttagca caaagaacat agctcctagt gatgttaatt ggtttgtgca gcatgaatgg 360 ggaagagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct 420 caaggaaagt ggtggagaag gcagctaagt gtttactgga ccagatggtt ggtaacagct 480 540 gaatgggtta ccttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt 600 gttctttttg gaacataat tgcttactac tttttaacta aaaagaaaat aagcaccagt 660 ccgccaagag acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcaa ttaaaacac acttaaaagag ctggtgcata agagagaaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgccggac 1080 acaagaacct gcaaaatttt tgcttttcat ggctgggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aagagaaata ctgttttatt tcatggacca 1200 gccagcacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa gaagtaaacc agtttaaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 19 <211> 2019 <212> DNA <213> Mouse parvovirus UT <400> 19 atggctggaa acgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgagg atgttcaact aaatggaaaa 120 gatatcggat ggaataatta cagaaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcag aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaagtgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttaaca caaagaacat atctcctggt gatgttaatt ggtttgtgca gcatgaatgg 360 ggaaaagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagccaagct 420 caaggaaagt ggtggagaag gcagctaagt gtttactgga gcagatggtt agtaacagcc 480 tgtaatgtgc agctatcacc agctgaaaga attaaactaa gagaatagc agaagacagt 540 gagtgggtta ccttgctcac ttataagcat aagcaaacca aaaaagacta tactaagtgt 600 gttctttttg gcaacataat tgcttactac ttttaacca agaagaaaat aagcactagt 660 ccgccaaggg acggaggcta tttctgagc agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ctaacttttc actgcctgac 1080 acaagaacct gcaagatttt tgcttttcat ggctgggaact acataaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagtcag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttgatt 1320 tgggtggaag aagctggtaa ctttggacag bagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcaaaa taggctgcga ggagagacca 1500 gaacacactc aaccaataag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaagtgag tggcccatga tctgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 20 <211> 2009 <212> DNA <213> Mouse parvovirus 1e <400> 20 atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa agtaaccagg 60 aagtgttctc atttgttttt aaaactgaag atattcaact gaatggaaaa gatattggat 120 ggaataatta cagaaaggag ctgcaagagg acgagctaaa atctttacaa cgaggagcgg 180 aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat gaagtgacca 240 aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa gtgcttaaca 300 caaagaacat atctcctggt gatgttaatt ggtttgtgca gcatgaatgg ggaagagacc 360 aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct caaggaaagt 420 ggtggagaag gcagctaagt gtttactgga gcagatggtt ggtaacagcc tgtaatgtac 480 agctaacacc agctgaaaga attaaactaa gagaatagc agaagacagt gaatgggtta 540 ccttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt gttctttttg 600 gcaacataat tgcttactac ttttaacca agaagaaaat aagcaccagt ccgccaaggg 660 acggaggcta ttttcttagt agtgactctg gctggaaaac taacttttta aaagagggcg 720 aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg gttgaaacca 780 cattaaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa gaggtttcta 840 900 tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa aacctgctga 960 1020 ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgcctgac acaagaacct 1080 gcaagatctt tgcttttcat ggctgggaact acattaaaagt ttgccatgct atttgctgtg 1140 ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca gccagtacag 1200 gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt tgctataatg 1260 cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt tgggtggaag 1320 aagctggtaa ctttggacaa cagtaaacc agtttaaagc catttgctct ggtcaaacaa 1380 ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca gtcatcatga 1440 ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca gaacacactc 1500 aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct ggtgactttg 1560 gtttggttga caaaaatgag tggcccatga tttgtgcttg gttggtaaag aatggttacc 1620 aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg acggaaaact 1680 gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc tcaccattca 1740 cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt gcatcggatc 1800 tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg ggcactgcag 1860 aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa ctgagcccaa 1920 cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg ttgaagagag 1980 acttcagcga gccgctgaac ttggactaa 2009 <210> 21 <211> 2019 <212> DNA <213> Mouse parvovirus 1c <220> <221> misc_feature <222> (609)..(609) <223> n is a, c, g, or t <220> <221> misc_feature <222> (1590)..(1590) <223> n is a, c, g, or t <400> 21 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaactgaag atgttcaact aaatggaaaa 120 gatattggat ggaataatta cagaaaggag ctgcaagagg acgagctaaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tacagtggat 240 gaagtgacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgcttaaca caaagaacat atctcctggt gatgttaatt ggtttgtgca gcatgaatgg 360 ggaagagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagtcaagct 420 caaggaaagt ggtggagaag gcagctaagt gtttactgga gcagatggtt ggtaacagcc 480 tgtaatgtac agctaacacc agctgaaaga attaaactaa gagaatagc agaagacagt 540 gaatgggtta ccttgctcac ttataagcat aagcaaacca aaaaggacta taccaagtgt 600 gttcttttng gcaacataat tgcttactac tttttaacca agaagaaat aagcaccagt 660 ccgccaaggg acggaggcta ttttcttagt agtgactctg gctggaaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaacac acttaaaagag ctagtgcata agagagatac ctcaccagagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 aacctgctga aaaatacgct agagatctgt acactaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgcctgac 1080 acaagaacct gcaagatctt tgcttttcat ggctgggaact acataaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagtcag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgttataatg cagcaaatgt gaactttcca ttcaatgact gtaccaacaa gaacttaatt 1320 tgggtggaag aagctggtaa ctttggacaa caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacaa ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgcga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaacatgan tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgaaag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagga gccgctgaac ttggactaa 2019 <210> 22 <211> 2019 <212> DNA <213> Hamster parvovirus <400> 22 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaaagagaaa 60 agtaaccagg aagtgttctc atttgttttt aaaaatgaag atgttcagct caatggaaaa 120 gatatcggat ggaatagtta caaaaaggag ctgcaagagg aagagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccagagc gaggacatgg aatgggaatc ttcagtggat 240 gaactaacca aaaagcaagt attcatttt gactctttag ttaaaaaatg tttgtttgaa 300 gtgctggagat caaaagacat agcacctagt gatgttactt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgta ctaattggag gcaaggactt tagccaagct 420 caaggaaaat ggtggagaag ccagttaaat gtttactgga ccagatggtt ggtaacagcc 480 tgtagtgtgc agctattacc agctgaaaga attaagctga gagagatagc ggaagaccaa 540 gatgggtca ctttgcttac ttataagcat aagcaaacca aaaagacta taccaagtgt 600 gtttgctttg gaaatagt tgcttactac ttttttacca agaagaaaat atgcaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taacttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca actaaaaaa 840 gaggtctcta ttaaaaccac acttaaagag ctggtgcata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaatga tggctcacc aggtggagaa 960 aaacctgctga aaatacgct aggatttgt acactactc tagccagaac aaaaacagca 1020 tttgacttaa ttttagaaa agctgaaacc agcaacta ccaactttc actgccggac 1080 acaagaacct gcaagactt tgctttcat gggctgaact atattaaagt tgccatgct 1140 atttgctgtg ttctaacag aaaggaggc aaaagaata ctgttttatt tcatggacca 1200 gccagtacag gcaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca tttaatgact gcaccaacaa aaacctgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgtga agaaagacca 1500 gaacacactc aaccaatcag agacagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaacatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagga gccgctgaac ttggactaa 2019 <210> 23 <211> 2019 <212> DNA <213> Mouse parvovirus 3 <400> 23 atggctggaa acgcttactc tgatgaagtt ttaggaacaa ccaactggtt aaaggaaaaa 60 agtaaccagg aagtgttctc atttgttttt aaaaatgaag atgttcaact gaatggaaaa 120 gatatcggat ggataatta cagaaggag ctgcaggagg acgagctgaa atctttacaa 180 cgaggagcgg aaactacctg ggaccaaagc gaggacatgg aatgggaatc tgcagtggat 240 gaactaacca aaaagcaagt attcatttat gactctttag ttaaaaaatg tttgtttgaa 300 gtgctggagat caaaagacat agctcctagt gatgttactt ggtttgtacg gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgtg ctcattggag gcaaggactt tagccaagct 420 caaggaaaat ggtggagaag ccagttaaat gtttactgga ccagatggtt agtaacagcc 480 tgtaatgtgc agttatcacc agctgaaaga attaagctga gagagatagc ggaagaccaa 540 gatgggtca ctttgcttac ttataagcat aagcaaacca aaaggacta taccaagtgt 600 gttctttttg gaaatagt tgcttactac ttttacca agaagaaaat aagcaccagt 660 ccaccaaggg acggacta tttctgagc agtgactctg gctggaaac taacttttta 720 aaagagggcg aacgccatct agtgagcaa ctatacactg atgacatgcg accagaaacg 780 gttgaaacca cagtaaccac tgcgcaggaa actaagcgcg gcagaattca aactaagaaa 840 gaggtctcta ttaaaaccac acttaaagag ctggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaatga tggctcacc aggtggagaa 960 aaacctgctga aaatacgct aggatttgt acactactc tagccagaac aaaaacagca 1020 tttgacttga tttgaaaa agctgaaacc agcaacta ccaactttc actgccggac 1080 acaagaacct gcaagactt tgctttcat gggctgaact atattaaagt tgccatgct 1140 atttgctgtg ttctaacag aaaggaggc aaaagaata ctgttttatt tcatggacca 1200 gccagtacag gcaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gcaccaacaa aaacctgatt 1320 tgggtagaag aagctggtaa ctttggacag caagtaaacc aatttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcaaaa taggctgtga agaaagacca 1500 gaacacactc aaccaatcag agatagaatg cttaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaacatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 acggaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagagag acttcagga gccgctgaac ttggactaa 2019 <210> 24 <211> 2019 <212> DNA <213> Mouse minute virus <400> 24 atggctggaa atgcttactc tgatgaggtt ttgggagcaa ccaactggtt aaaggaaaaa 60 agtaaccagt tagtattctc atttgttttt aaaaatgaag atgttcaatt gaatggaaaa 120 180 cgaggagcgg aaactacctg ggaccagagc gaggacatgg aatgggaatc ttcagtggat 240 gaactaacca caaagcaagt attcattttt gactctttag ttaaaaaagtg tttatttgaa 300 gtgctaagta caagaacat agctcctagt gatgttaatt ggtatgtgca gcatgaatgg 360 ggaaaagacc aaggctggca ttgccatgta ctaattggag gcaaagactt tagccaagct 420 caaggaaagt ggtggagaag gcagctaaat gtttactgga ccagatggtt ggtaacagcc 480 tgcagtgtgc agctatcacc agccgaaaga attaagctga gagaaatagc ggaagaccaa 540 gaatgggtca ctttgcttac ttataagcat aagcaaacca aaaaagacta taccaaatgt 600 gtttgctttg gaaatatgat tgcttactac tttttaacca agaagaaat atgcactagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca cagataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtttcta ttaaaacac acttaaaagag ctggtgcata agagagatac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcagcc aggtggagaa 960 aacctgctta aaaatacgct agagatctgt acgctactc tagctagaac caaaacagcc 1020 tttgacttga ttttagaaaa agctgaaacc agcaactaa ccaacttttc actgccggac 1080 acaagaacct gtaagatttt tgcttttcat ggctgggaact acataaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgtttttt tcatggacca 1200 gccagtcag gcaaatccat cattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca ttcaatgact gcaccaacaa aaacctgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc aatttaaagc catttgctct 1380 ggtcaaacta ttcgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga gaacattaca gtggtcagaa taggctgcga agagagacca 1500 gaacacactc aaccaattag agacagaatg ctcaacattc atctaacaca tacattgcct 1560 ggtgactttg gtttggttga caaaaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaaaact gggcggagcc gaaggtgccg actcctataa attcactagg ttcggcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaag acttcagcga gccgctgaac ttggactaa 2019 <210> 25 <211> 2019 <212> DNA <213> MVM strain M <400> 25 atggctggaa atgcttactc tgatgaagtt ttgggaacaa ccaactggtt aaagagaaa 60 agtaaccagg aagtgttctc atttgtttttt aaaaatgaag atgttcagct caatggaaaa 120 gatatcggat ggatagtta aaaaggg ctgcaagagg agagctgaa atctttaca 180 cgaggagcgg aaactaccctg ggaccagagc gaggacatgg aatgggaatc ttcagtggt 240 gaactaacca aaagcaagt attcatttt gactctttag ttaaaaaatg ttgttttgaa 300 gtgctgagta caaacat agctcctagt gatgttactt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaagctggca ctgtcatgta ctattggag gcaaggactt tagccagct 420 caggaaaat gtggagaag gcagttaat gtttactgga gcagatggtt ggtaacaccc 480 tgtagtgtgc agctatcacc agctgaaaga attaagctga gagagatagc ggaagaccaa 540 gatgggtca ctttgcttac ttataagcat aagcaaacca aaaagacta taccaagtgt 600 gtttgctttg gaaatatagt tgcttactac tttttatcca agaagaaaat atgcaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taacttttta 720 aaagaaggcg aacgccatct agtgagcaaa ctatacactg atgacatgcg gccagaaacg 780 gttgaaacca caataaccac tgcgcaggaa actaagcgcg gcagaattca aactaaaaaa 840 gaggtctcta ttaaaaccac acttaaagag ctggtacata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacctgctta aaaatacgct agaaatctgt acgctaactc tagctagaac caaaacagca 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaacttttc actgccggac 1080 acaagaacct gcaagatctt tgcttttcat ggctggaact atgttaaagt ttgccatgct 1140 atttgctgtg ttctaaacag acaaggaggc aaaagaaata ctgttttatt tcatggacca 1200 gccagtacag gcaaatccat tattgcacaa gccatagcac aggcagttgg taatgttggt 1260 tgctataatg cagcaaatgt gaactttcca tttaatgact gcaccaacaa aaacctgatt 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgctct 1380 ggtcaaacta tccgcattga tcaaaaagga aaaggcagca agcagattga accaacacca 1440 gtcatcatga ccacaaatga aaacattaca gtggtcagaa taggctgcga ggagagacca 1500 gagcacactc aaccaatcag agacagaatg ctcaacattc atctgacaca tacattgcct 1560 ggtgactttg gtttggttga caagaatgaa tggcccatga tttgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcaagctac tgtgctaaat ggggcaaagt acctgattgg 1680 tcagaaaact gggcagagcc gaaggtaccg actcctataa attcactagg ttcagcacgc 1740 tcaccattca cgacaccgaa aagtacgcct ctcagccaga actatgcact aactccactt 1800 gcatcggatc tcgaggacct ggctttagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag aaacccagaa cactggggaa gctggttcca aagcctgcca agatggtcaa 1920 ctgagcccaa cttggtcaga gatcgaggag gatttgagag cgtgcttcgg tgcggaaccg 1980 ttgaagaaag acttcagcga gccgctgaac ttggactaa 2019 <210> 26 <211> 1966 <212> DNA <213> Parvovirus LuII <400> 26 atggctggaa acgcgtactc tgatgaagtt ttgggacaa ctaactggtt gaaggataag 60 agcaaccagg aagtattctc atttgtttttt aaaaatgagg atgttcagct caatggaaaa 120 atatcggat ggaacagtta cagaaggag ctgcagagg aggagctgaa atctttacaa 180 cgaggagctg aaactacctg ggaccagagc gaggacatgg aatgggaatc ttcagtgt 240 gaactgacca aaagcaagt attcatttt gactctttag ttaaaaagtg tctctttgaa 300 gtactgagca caagaacat agctcctagt gatgttactt ggtttgtaca gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgtg ctcattggag gcaagaactt tagccaggct 420 caggaaaat ggtggaggag acaattaat gtttactgga gtagatggtt ggtaacaccc 480 tgtagcgtgc agctatcacc agctgaaaga attackaacta gagaatagc agagaccaa 540 gatgggtta ctctgcttac ttataagcat aagcaaacca aaaagacta tactaagtgt 600 gtttgctttg gaaatatggt tgcttactac ttttacca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttctcagt agtgactctg gctggaaac taactttttg 720 aaagaaggcg aacgccatct agtgagcaaa ctatatactg atgacatgcg gccagaaacg 780 gttgagacca fotaaccac agcgcaggaa actaagcgcg gcagaattca aactaagaag 840 gaagtctcta ttaagactac acttaaagag ctggtacata agagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggggagaa 960 aacctactta agaatacgct agagatctgt acgctgactc tagccagaac caaaacagcc 1020 tttgacttga ttttagaaaa agctgaaacc agcaaactaa ccaactttt actggctgat 1080 acaagaacct gtagaatctt tgctttcat ggctggaact acatcaaagt ctgtcatgct 1140 atttgttgtg tcttgaacag acagggaggc aaaagaaata ctgttctgtt tcatggacca 1200 gccagtacag gcaaatcaat cattgcacag gccatagcac aggcagttgg taatgttggt 1260 tgttataacg cagccaatgt gaactttcca tttaatgact gtaccaacaa gaacttaatc 1320 tgggtggaag aagctggtaa ctttggacag caagtaaacc agtttaaagc catttgttct 1380 ggtcagacca ttcgcattga ccaaaagga aaaggcagca aacagattga accacacca 1440 gtgatcatga ccacaatga aacatcaca gtggtcaaa taggtgtga agagagacca 1500 gaacacactc aaccaatcag agacagaatg ttaacattc atctgacaca tacattgcct 1560 ggtgactttg gtttggttga taaaaacgaa tggcttga tatgtgcttg gttggtaaag 1620 aacggttacc aatcgaccat ggcaagttac tgtgctaaat ggggcaagt tcctgattgg 1680 acagaaaact gggcggagcc aaaagtaacg actgaaataa attcggtagg ttcaccaac 1740 tcaccatctc cgaaagtac gcctcagc cagaactacg cactactcc gtcggatc 1800 gaggacctgg ctctggagcc tggagcaca ccaagtactc ctgttgtggg cactgtcaa 1860 accccgaaca ctggggaac tggttcaca gcctgtcaag aagctcaacg gagcccaact 1920 tggtccgaga tcgaggagg ttgagagcg tgcttcagtt cggaac 1966 <210> 27 <211> 2019 <212> DNA <213> Rat parvovirus UT <400> 27 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaacggct aaaggacaaa 60 agtagccaag aggtgttctc atttgtttttt aaaaatgaga acgtccagct aaatgggaag 120 gatacggtt ggaatagtta cagaaaggag ctacagatg acgagctgaa gtctctaca 180 cgaggagcgg aaaccacttg ggaccaagc gaggacatgg aatgggag cgcagtggat 240 gatagacca aaaagcaagt attcatttt gattctttgg ttaagaatg ttgttttgaa 300 gtgctcagca caaacat aactcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctattggag gcaagactt tagtcaagct 420 caggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaga aaaactga gagaatagc agaggacagt 540 gatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttcttttg gaaacatgat tgcttattac tttttaagca aaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taactttg 720 aaagagggcg agcgccatct agtgagcaag ctgtatactg atgagatgaa accagaaacg gttgagacca cagtgaccac agcacagga gctaagcgcg gcagaattca aactagaaag gaggtctcta ttaaaaccac acttaaagag ttggtacata aaagagtaac ctcaccagaa gactggatga tgatgcagcc agacagttac attgaatga tggctcaacc aggtggagaa aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagca tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctaac accagaacct gtagaatctt tgctgaacat ggctggaact attack ctgtcatgcc atctgttgtg tgctaatag acaaggaggc aaaaggaca ctgtgctctt tcatggacca gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg taatgttggt 1320. tgttataatg ctgccaatgt gaactttcca tttaatgact gcaccaacaa aaacttgatt tgggtggaag aagctggtaa ctttggccag caagtaacc aattcaaagc tatttgttct ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca gttatcatga ccaccaacga gaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaagact gggcggagcc gaagctagag actcctataa attcactagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgtg 1860 ggcactgcag caagccagaa cactggggag gctggtttca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgacc ttggactaa 2019 <210> 28 <211> 2019 <212> DNA <213> Kilham rat virus <400> 28 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gacatcggtt ggaatagtta cagaaaggag ctacaagatg acgagctgaa gtctctacaa 180 cgaggggcgg agaccacttg ggaccaaagc gaggacatgg aatgggagag cgcagtggat 240 gacatgacca aaaagcaagt attcattttt gattctttgg ttaagaagtg tttgtttgaa 300 gtgctcagca caaagaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaggacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt tagtcaagct 420 caaggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 540 gaatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac tttctaagca aaaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta ttttcttagc agtgactctg gctggaaaac taactttttg 720 aaagagggcg agcgccatct agtgagcaaa ctatatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaatga tggctcacc aggtggagaa 960 aaacttgctta aaatacact agagatctgt acacctgactc tagcagac caaacaccc 1020 tttgacttga ttctgaaa agctgaacc agcaactag ccaacttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctaaatag aaaggaggc aaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaatctat cattgcacaa gccatagcac aaggattgg taatgttggt 1260 tgttataatg ctgccaatgt gaactttcca tttaatgact gtaccaca aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc attcaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaagga aaaggcagca aacagattga accaacca 1440 gttatcatga ccaccaacga gaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagag actcctataa tttcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccagaa cactggggag gctggtttca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgaca ttggactaa 2019 <210> 29 <211> 2024 <212> DNA <213> Rat minute virus 1c <400> 29 atggctggaa acgcttactc cgatgaagtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aagtgttctc atttgtttttt aaaaatgaga acgtccaact aaatgggaag 120 gatacggtt ggaatagtta cagaaagag ctacagatg acgagctgaa gtctctaca 180 cgaggggcgg agaccacttg ggaccaagc gaggacatgg aatgggagag cgcagtggat 240 gatagacca aaaagcaagt attcatttt gattctttgg ttaagaagtg tctgtttgaa 300 gtgctcagca caaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt 420 caggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt agtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaactga gagaatagc agaggacagt 540 gatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac ttctagca aaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taactttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaatga tggctcacc aggtggagaa 960 aaacttgctta aaatacact agagatttgt acacctgactc tagcagac caaacaccc 1020 tttgacttga ttctgaaa agctgaacc agcaactag ccaacttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tactaatag aaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaatctat cattgcacaa gccatagcac aaggattgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaca aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc attcaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaagga aaaggcagca aacagattga accaacca 1440 gttatcatga ccaccaacga gaacattaca gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctaa tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agccctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccagaa cactggggag gctggtttca cagcctgtca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgacc ttggactaag gtac 2024 <210> 30 <211> 2019 <212> DNA <213> Rat minute virus 1b <400> 30 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaacggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gatacggtt ggaatagtta cagaaaggag ctacagatg acgagctgaa gtctctgcaa 180 cgaggggcgg agaccacttg ggaccaagc gaggacatgg aatgggagag cgcagtggat 240 gatagacca aaaagcaagt attcatttt gattctttgg ttaagaagtg tctgtttgaa 300 gtgctcagca caaacat agctcctagt gatgttactt ggtttgtgca gcatgaatgg 360 ggaaaagacc aaggctggca ctgtcatgtg ctgattggag gcaggactt 420 caggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaactga gagaatagc agaggacagt 540 gatgggtga ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac ttctagca aaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taactttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaatga tggctcacc aggtggagaa 960 aaacttgctta aaatacact agagatctgt acacctgactc tagcagac caaacaccc 1020 tttgacttga ttctgaaa agctgaaacc agcaactag ccaactttt catggctaac 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgtcatgcc 1140 atctgttgtg tgctaaatag aaaggaggc aaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaatctat cattgcacaa gccatagcac aaggattgg taatgttggt 1260 tgttataatg ctgccaatgt gaactttcca tttaatgact gtaccaca aaacttgatt 1320 tgggtggaag aagctggtaa ctttggccag caagtaaacc attcaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaagga aaaggcagca aacagattga accaacca 1440 gttatcatga ccaccaacga gaacattacc gtggtcaaaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtaaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggatc tcgcggacct ggcactggaa ccttggagca caccaaatac tcctgttgtg 1860 gacactgtac aaaccccgaa cactggggag gctggtttca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatctgagag cgtgcttcag ccaggaacag 1980 ctggagaaag acttcagcga ttcactgacc ttggactaa 2019 <210> 31 <211> 1997 <212> DNA <213> Rat minute virus 1a <400> 31 atgaggtttt gggagcaacc aactggctaa aggacaaaag tagccaggag gtgttctcat 60 ttgtttttaa aaatgagaac gtccaactaa atgggaagga catcggttgg aatagttaca 120 gaaaggagct acaagatgac gagctgaagt ctttgcaacg aggggcggag accacttggg 180 accaaagcga ggacatggaa tgggagagcg cagtggatga catgaccaaa aagcaagtat 240 tcatttttga ttctttggtt aaaaagtgtc tgtttgaagt gctcagcaca aagaacatag 300 ctcctagtga tgttacttgg ttcgtgcagc atgaatgggg aaaggaccaa ggctggcact 360 gtcatgtgct gattggaggc aaggacttta gtcaacctca aggaaagtgg tggagaaggc 420 agctaaatgt gtactggagt agatggttgg tgactgcctg taatgttcaa ctaacaccag 480 ctgaaagaat taaactgaga gaaatagcag aggacagtga atgggtcact ttgcttacct 540 ataagcataa acacaccaag aaggactata ccaagtgtgt tttttttgga aacatgattg 600 cttattactt tctaagcaaa aagaaaatat gtaccagtcc accaagggac ggaggctatt 660 ttcttagcag tgactctggc tggaaaacta actttttgaa agagggcgag cgccatctag 720 tgagcaaact gtatactgat gagatgaac cagaacggt cgagaccaca gtgaccactg 780 cgcaggaagc tagcgcggc agaattcaaa ctagaaagga ggtctcgatt aaaaccacac 840 tcaaagagtt ggtgcataaa agagaacct caccagaga ctggatgatg atgcagccag 900 acagttacat tgaaatgatg gctcaaccag gtggagaaaa cttgcttaaa aatacactag 960 agatctgtac actgactcta gcaagaacca aaacagcctt tgacttgatt ctagaaaaag 1020 ctgaaaccag caaactagcc aacttttcca tggctaacac cagaacctgt agaatctttg 1080 ctgagcatgg ctggaactat attaaagtct gccatgccat ctgttgtgtg ctgaatagac 1140 aaagggcaa aaagacact gtgctctttc acggaccagc cagcacaggc aaatctatca 1200 ttgcacaagc catagcacaa ggagttggta atgttggttg ttacaatgct gccaatgtga 1260 actttccatt taatgactgt accaacaaaa acttgatttg ggtggaagaa gctggtaact 1320 ttggccagca agtaaccaa ttcaaagcta tttgttctgg ccaaaccata cgcattgatc 1380 aaaaaggaaa aggcagcaaa cagattgaac caacaccagt tatcatgacc accaacgaga 1440 acattaccgt ggtcagaata ggctgtgagg aaaggccaga acacactcaa ccaatcagag 1500 acagaatgct caacattcac ctgacacgta cactgcctgg tgactttggt ctggtggata 1560 agcacgaatg gcctctgatc tgtgcttggt tggtgaagaa tggttaccaa tctaccatgg 1620 cttgttactg tgctaaatgg ggcaaagttc ctgattggtc agaggactgg gcggagccga 1680 agctagac tcctataaat tcgctaggtt caatgcgctc accatctctg actccgagaa 1740 gtacgcctct cagccagaac tacgctctta ctccacttgc atcggacctt gcggacctag 1800 ctctagagcc ttggagcaca ccaaatactc ctgttgcggg cactgcagca agccagaaca 1860 ctggggaggc tggtttcaca gcctcccaag gtgctcaacg gagcccaacc tggtccgaga 1920 tcgaggcgga tctgagagcg tgcttcagcc aggaacagct ggagaaagac ttcagcgatt 1980 cactgacctt ggactaa 1997 <210> 32 <211> 2019 <212> DNA <213> H-1 parvovirus <400> 32 atggctggaa acgcttactc cgatgaggtt ttgggagtaa ccaactggct gaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaaa acgtccaact aaatggaaag 120 gatacggtt ggaatagtta cagaaaggag ctacagatg acgagctgaa gtctctaca 180 cgaggggcgg agaccacttg ggaccaagc gaggacatgg aatgggagag cgcagtggat 240 gatagacca aaaagcaagt attatttt gattctttgg ttaagaagtg ttgttttgaa 300 gtgctcagca caaacat agctcctagt aatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctgattggag gcaggactt tagtcaacct 420 caggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaactga gagaatagc agaggacagt 540 gatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttcttttg gaaacatgat tgcttattac tttttaagca aaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taactttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcacaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctgaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aaggctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttattatga ccaccaacga gaacattacc gtggttagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactacct 1560 ggtgactttg gtttggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagac actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaaa actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccaaaa cactggggag gctggttcca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatttgagag cttgcttcag tcaagaacag 1980 ttggagagcg acttcaacga ggagctgacc ttggactaa 2019 <210> 33 <211> 2019 <212> DNA <213> Rat minute virus isolate NTU1 <400> 33 atggctggaa acgcttactc cgatgaagtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gatacggtt ggaatagtta cagaaggag ctacagatg acgagctgaa gtctctaca 180 cgaggggcgg agaccacttg ggaccaagc gaggacatgg aatgggagag cgcagtggat 240 gatagacca aaaagcaagt attcatttt gattctttgg ttaagaagtg ttgttttgaa 300 gtgctcagca caaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg tgattggag gcaggactt tagtcaagct 420 caggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaactga gagaatagc agaggacagt 540 gatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttctttttg gaaacatgat tgcttattac ttctagca aaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taactttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcacaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttat attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatatgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc tatggctaac 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctaaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat cattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aaggctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga gaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctacaat ggctgttac tgtgctaaat ggggcaagt tcctgattgg 1680 tcagaagact gggcggagcc gaagctagag actctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactcactt 1800 gcatcggacc ttgcggacct agctctggag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccagaa cactggggag gctgtttcg cagcctgcca aggtgctca 1920 cggagcccaa cctggtccga gatgaagca gacttgagag cttgcttcag tcagacag 1980 ttggagagcg acttcaacga ggaactgacc ttggactaa 2019 <210> 34 <211> 2019 <212> DNA <213> Parvovirus h-1 <400> 34 atggctggaa acgcttactc cgatgaggtt ttgggagtaa haactggct gaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaaa acgtccaact aaatggaaag 120 gatacggtt ggaatagtta cagaaaggag ctacagatg acgagctgaa gtctctaca 180 cgaggggcgg agaccacttg ggaccaagc gaggacatgg aatgggagag cgcagtggat 240 gatagacca aaaagcaagt attatttt gattctttgg ttaagaagtg ttgttttgaa 300 gtgctcagca caaacat agctcctagt aatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc caggctggca ctgtcatgtg ctgattggag gcaggactt tagtcaacct 420 caggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaactga gagaatagc agaggacagt 540 gatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttcttttg gaaacatgat tgcttattac tttttaagca aaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taactttg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcacaggaa gctaagcgcg gcagaattca aactagagag 840 gaggtctcga ttaaaaccac actcaaagag ttggtacata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatctgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgctgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tgctgaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat tattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aaggctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttattatga ccaccaacga gaacattacc gtggttagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactacct 1560 ggtgactttg gtttggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctaccat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaggact gggcggagcc gaagctagac actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaaa actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctagag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgcag caagccaaaa cactggggag gctggttcca cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatcgaggcg gatttgagag cttgcttcag tcaagaacag 1980 ttggagagcg acttcaacga ggagctgacc ttggactaa 2019 <210> 35 <211> 1997 <212> DNA <213> Rat minute virus isolate NTU2 <400> 35 atgaggtttt gggagcaacc aactggctaa aggacaaaag tagccaggag gtattctcat 60 ttgtttttaa aaatgagaac gtccaactaa atgggaagga catcggttgg aatagttaca 120 gaaaggagct acaagatgac gagctgaagt ctctacaacg aggggcggag accacttggg 180 accaaagcga ggacatggaa tgggagagcg cagtggatga catgaccaaa aagcaagtat 240 tcatttttga ttctttggtt aagaagtgtc tgtttgaagt gctcagcaca aagaacatag 300 ctcctagtga tgttacttgg ttcgtgcagc atgaatgggg aaaggaccaa ggctggcact 360 gtcatgtgct gattggaggc aaggacttta gtcaagctca aggaaaatgg tggagaaggc 420 agctaaatgt gtactggagt agatggttgg tgactgcctg taatgttcaa ctaacaccag 480 ctgaaagaat taaactaaga gaaatagcag aggacagtga atgggtcact ttgcttacct 540 ataagcataa gcacaccaag aaggactata ccaagtgtgt tctttttgga aacatgattg 600 cttattactt tctaagcaaa aagaaaatat gtaccagtcc accaagggac ggaggctatt 660 ttcttagcag tgactctggc tggaaaacta actttttgaa agagggcgag cgccatctag 720 tgagcaaact gtatactgat gagatgaaac cagaaacggt cgagaccaca gtgaccactg 780 cacaggaagc tagcgcggc agaattcaaa ctagaaagga ggtctcgatt aaaaccacac 840 900 960 agatatgtac actgactcta gcaagaacca aaacagcctt tgacttgatt ctggaaaaag 1020 ctgaaccag caaactagcc aacttttcta tggctagcac cagaacctgt agaatctttg 1080 ctgagcatgg ctggaactat attaaagtct gccatgccat ctgttgtgtg ctaatagac 1140 aaagggcaa aaagacact gtgctctttc acggaccagc cagcacaggc aaatctatca 1200 ttgcacaagc catagcacaa gcagttggta atgttggttg ttacaatgct gccaatgtga 1260 actttccatt taatgactgt accaacaaaa acttgatttg ggtggaagaa gctggtaact 1320 ttggccagca agtaaccaa ttcaaagcta tttgttctgg ccaaaccata cgcattgatc 1380 aaaaaggaaa aggcagcaaa cagattgaac caacaccagt tatcatgacc accaacgaga 1440 acattaccgt ggtcagaata ggctgtgagg aaagaccaga acacactcaa ccaatcagag 1500 acagaatgct caacattcac ctgacacgta cactgcctgg tgactttggt ctggtggata 1560 agcacgaatg gcctctgatc tgtgcttggt tggtgaagaa tggttaccaa tctaccatgg 1620 cttgttactg tgctaaatgg ggcaaagttc ctgattggtc agaagactgg gcggagccga 1680 agctagac tcctataaat tcgctaggtt caatgcgctc accatctctg actccgagaa 1740 gtacgcctct cagccagaac tacgctctta ctccacttgt atcggacctt gcggacctag 1800 ctctggagcc ttggagcaca ccaaatactc ctgttgcggg cactgcagca agccagaaca 1860 ctggggaggc tggttcgca gcctgtcaag gtgctcaacg gagcccaacc tggtccgaga 1920 tagaagcaga cttgagagct tgcttcagtc aagaacagtt ggagagcgac ttcaacgagg 1980 agctgacctt ggactaa 1997 <210> 36 <211> 2019 <212> DNA <213> Rat minute virus 2a <400> 36 atggctggaa acgcttactc cgatgaggtt ttgggagcaa ccaactggct aaaggacaaa 60 agtagccagg aggtgttctc atttgttttt aaaaatgaga acgtccaact aaatgggaag 120 gatacggtt ggaatagtta cagaaaggag ctacagatg acgagctgaa gtctctaca 180 cgaggggcgg agaccacttg ggaccaagc gaggacatgg aatgggagag cgcagtggat 240 gatagacca aaaagcaagt attcatttt gattctttgg ttaagaagtg tctgttttgaa 300 gtgctcagca caaacat agctcctagt gatgttactt ggttcgtgca gcatgaatgg 360 ggaaaggacc aaggctggca ctgtcatgtg ctgattggag gcaaggactt ggtcaagct 420 caggaaaat ggtggagaag gcagctaaat gtgtactgga gtagatggtt ggtgactgcc 480 tgtaatgttc aactaacacc agctgaaaga attaactga gagaatagc agaggacagt 540 gatgggtca ctttgcttac ctataagcat aagcacacca agaaggacta taccaagtgt 600 gttcttttg gaaacatgat tgcttattac ttctagca aaagaaaat atgtaccagt 660 ccaccaaggg acggaggcta tttcttagc agtgactctg gctggaaac taacttctg 720 aaagagggcg agcgccatct agtgagcaaa ctgtatactg atgagatgaa accagaaacg 780 gtcgagacca cagtgaccac tgcgcaggaa gctaagcgcg gcagaattca aactagaaag 840 gaggtctcga ttaaaccac actcaaagag ttggtgcata aaagagtaac ctcaccagaa 900 gactggatga tgatgcagcc agacagttac attgaaatga tggctcaacc aggtggagaa 960 aacttgctta aaaatacact agagatatgt acactgactc tagcaagaac caaaacagcc 1020 tttgacttga ttctggaaaa agctgaaacc agcaaactag ccaacttttc catggctagc 1080 accagaacct gtagaatctt tgttgagcat ggctggaact atattaaagt ctgccatgcc 1140 atctgttgtg tactaatag acaaggaggc aaaaggaaca ctgtgctctt tcacggacca 1200 gccagcacag gcaaatctat cattgcacaa gccatagcac aagcagttgg taatgttggt 1260 tgttacaatg ctgccaatgt gaactttcca tttaatgact gtaccaacaa aaacttgatt 1320 tgggtggaag aaggctggtaa ctttggccag caagtaaacc aattcaaagc tatttgttct 1380 ggccaaacca tacgcattga tcaaaaagga aaaggcagca aacagattga accaacacca 1440 gttatcatga ccaccaacga aaacattacc gtggtcagaa taggctgtga ggaaagacca 1500 gaacacactc aaccaatcag agacagaatg ctcaacattc acctgacacg tacactgcct 1560 ggtgactttg gtctggtgga taagcacgaa tggcctctga tctgtgcttg gttggtgaag 1620 aatggttacc aatctacaat ggcttgttac tgtgctaaat ggggcaaagt tcctgattgg 1680 tcagaagact gggcggagcc gaagctagag actcctataa attcgctagg ttcaatgcgc 1740 tcaccatctc tgactccgag aagtacgcct ctcagccaga actacgctct tactccactt 1800 gcatcggacc ttgcggacct agctctggag ccttggagca caccaaatac tcctgttgcg 1860 ggcactgtag caagccagaa cactggggag gctggtttcg cagcctgcca aggtgctcaa 1920 cggagcccaa cctggtccga gatagaagca gacttgagag cttgcttcag tcaagaacag 1980 ttggagagcg acttcaacga ggaactgacc ttggactaa 2019 <210> 37 <211> 192 <212> DNA <213> Artificial <220> <223> consensus sequence <400> 37 ctarrrarga rgtytcdatt aaaacyacac tyaaagaryt rgtrcataar agagtaacct 60 caccagarga ctggatgatg atgcagccag acagttayat tgaaatgatg gctcarccag 120 gkggagaaaa cytrctdaar aatacrctag aratytgtac rctractcta gchagaacma 180 aaacagcmtt tg 192

Claims

1. (a) Below: (i) a sample of a positive amplification control (PAC) plasmid comprising a unique artificial plasmid specific sequence (UAPS) that does not recognize or anneal to any naturally occurring parvovirus sequence; a target amplification polynucleotide (TAP) sequence comprising all or a portion of an NS-1 sequence of a rodent parvovirus, wherein the NS-1 sequence of the parvovirus is at least 88% identical to any one of SEQ ID NOs:12-37 or at least 97% identical to SEQ ID NO:9; and a nucleic acid extraction control (NEC) nucleotide sequence comprising an M13 bacteriophage nucleotide sequence; (ii) Unique detection probe (UDP); (iii) TAP forward oligonucleotide primer; (iv) TAP reverse oligonucleotide primer; (v) a TAP detection probe, and (vi) a DNA polymerase, mixing the (b) Below: (i) a test sample; (ii) UDP, (iii) TAP forward oligonucleotide primer; (iv) TAP reverse oligonucleotide primer; (v) a TAP detection probe, and (vi) a DNA polymerase, mixing the (c) subjecting the mixed sample of step (a) and the mixed sample of step (b) to polymerase chain reaction (PCR); (d) monitoring the PCR in real time for production of a TAP signal resulting from binding of a TAP detection probe to a TAP sequence in the PAC plasmid or a TAP sequence in a biological contaminant, and for production of a UDP signal resulting from binding of UDP to a UAPS in the PAC plasmid; and (e) detecting the presence or absence of a biological contaminant in the test sample; Including, (i) if no TAP signal is observed for the test sample and a TAP signal is observed for the PAC plasmid sample, then the test sample does not contain a biological contaminant; (ii) if a TAP signal and a UDP signal are observed for the test sample, the test sample is cross-contaminated with a portion of the PAC plasmid sample; or (iii) if a TAP signal is observed and no UDP signal is observed for the test sample, the TAP signal is not due to cross-contamination from the PAC plasmid and the test sample contains a biological contaminant; A method for detecting the presence or absence of a biological contaminant, including a parvovirus.

2. The method of claim 1, wherein the UDP further comprises at least one fluorophore and at least one quencher.

3. 3. The method of claim 2, wherein the fluorophore is selected from the group of fluorophores having an excitation wavelength in the range of 495 nm to 680 nm and an emission wavelength in the range of 515 nm to 710 nm.

4. 4. The method of claim 1, wherein the TAP detection probe further comprises at least one fluorophore and at least one quencher.

5. 5. The method of claim 4, wherein the TAP signal is monitored at about 533 to about 580 nm.

6. The method of any one of claims 1 to 5, wherein the UAPS comprises SEQ ID NO:

10.

7. The method of any one of claims 1 to 6, wherein the parvovirus NS-1 sequence comprises SEQ ID NO:9 or any one of SEQ ID NOs:12-37.

8. The method of any one of claims 1 to 6, wherein the parvovirus NS-1 sequence comprises SEQ ID NO:

37.

9. 9. The method of claim 1, further comprising detecting oligonucleotide extraction from a reaction vessel, Steps (a) and (b) (vii) a NEC-specific forward oligonucleotide primer; (viii) a NEC-specific reverse oligonucleotide primer, and (ix) a NEC-specific oligonucleotide detection probe; wherein the NEC-specific oligonucleotide detection probe binds to a NEC sequence in the PAC plasmid sample or the test sample, said mixing Further comprising: step (d) further comprises monitoring the PCR in real time for production of a NEC signal resulting from binding of the NEC-specific oligonucleotide detection probe to a NEC nucleotide sequence in the PAC plasmid sample or the test sample; and (f) detecting extraction of the oligonucleotide from the reaction vessel; Including, (i) if an NEC signal is observed for the test sample and the PAC plasmid sample, and no UDP signal is observed for the test sample, the NEC signal indicates oligonucleotide extraction from the reaction vessel; or (ii) if an NEC signal and a UDP signal are observed for the test sample, the test sample is cross-contaminated with a portion of the PAC plasmid sample; method.

10. 10. The method of any one of claims 1 to 9, wherein the M13 bacteriophage nucleotide sequence is an M13K07 nucleotide sequence.

11. The method of claim 10, wherein the M13K07 nucleotide sequence comprises SEQ ID NO:

8.

12. 12. The method of any one of claims 9 to 11, wherein the NEC-specific oligonucleotide detection probe comprises a cyanine dye.

13. 13. The method of claim 12, wherein the cyanine dye is Cy5, which has an absorption maximum at about 650 nm and an emission maximum at about 670 nm.

14. 14. The method of any one of claims 1 to 13, further comprising detecting the presence or absence of a biological contaminant or a portion of the PAC plasmid sample in the PCR reagent, (g) Below: (i) a buffer sample; (ii) UDP, (iii) TAP forward oligonucleotide primer; (iv) TAP reverse oligonucleotide primer; (v) a TAP detection probe, and (vi) a DNA polymerase, mixing the (h) subjecting the mixed buffer sample to polymerase chain reaction (PCR); (i) monitoring the PCR in real time for production of a TAP signal resulting from binding of a TAP detection probe to a TAP sequence in the PAC plasmid or biological contaminant, and for production of a UDP signal resulting from binding of UDP to a UAPS in the PAC plasmid; and (j) detecting the presence or absence of a biological contaminant or a PAC plasmid in the PCR reagent; Including, (i) if no TAP or UDP signal is observed in the buffer sample, the PCR reagents are not contaminated with biological contaminants or the PAC plasmid; (ii) if a TAP signal and a UDP signal are observed in the buffer sample, the PCR reagents are contaminated with a portion of the PAC plasmid sample; or (iii) if a TAP signal and no UDP signal are observed in the buffer sample, the PCR reagents are contaminated with a biological contaminant; method.

15. The method of any one of claims 1 to 14, wherein the PAC plasmid comprises the nucleic acid sequence of SEQ ID NO:11.

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