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Compositions for use in identification of adventitious virusesRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Micro-organism, Tissue Cell Culture Or Enzyme Using Process To Synthesize A Desired Chemical Compound Or Composition, Preparing Compound Containing Saccharide Radical, N-glycoside, , Nucleotide, Polynucleotide (e.g., Nucleic Acid, Oligonucleotide, Etc.)Compositions for use in identification of adventitious viruses description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060205040, Compositions for use in identification of adventitious viruses. Brief Patent Description - Full Patent Description - Patent Application Claims
RELATED APPLICATIONS [0001] The present application 1) claims the benefit of priority to U.S. Provisional Application Ser. No. 60/658,248, filed Mar. 3, 2005; 2) claims the benefit of priority to U.S. Provisional Application Ser. No. 60/705,631, filed Aug. 3, 2005; 3) claims the benefit of priority to U.S. Provisional Application Ser. No. 60/732,539, filed Nov. 1, 2005 and 4) claims the benefit of priority to U.S. Provisional Application Ser. No. 60/740,617, filed Nov. 28, 2005. Each of the above listed U.S. Provisional Applications is incorporated herein by reference in entirety. Methods disclosed in U.S. application Ser. Nos. 10/156,608, 09/891,793, 10/418,514, 10/660,997, 10/660,122, 10,660,996, 10/660,998, 10/728,486, 10/405,756, 11/060,135, and 11/073,362, are commonly owned and incorporated herein by reference in their entirety for any purpose. FIELD OF THE INVENTION [0003] The present invention provides compositions, kits and methods for rapid identification and quantification of adventitious contaminant viruses by molecular mass and base composition analysis. BACKGROUND OF THE INVENTION A. Adventitious Viruses [0004] Adventitious viruses represent a major risk associated with the use of cell-substrate derived biologicals, including vaccines and antibodies, for human use. The possibility for viral contamination exists in primary cultures and established cultures, as well as Master Cell Banks, end-of-production cells, and bulk harvest fluids. This is a major obstacle to the use of neoplastic-immortalized cells for which the mechanism of transformation is unknown is that these could have a higher risk of containing oncogenic viruses. Extensive testing for the presence of potential extraneous agents is therefore required to ensure the safety of the vaccines. Among the methods used for this purpose are animal inoculations, electron microscopy and in vitro molecular and antibody assays that provide a screen for viral agents. Another critical consideration for assessing the safety concerns associated with viral vaccines is the detection of endogenous retroviral sequences while using avian, murine, non-human primate, and human cell lines. Endogenous retroviral sequences are an integral part of eukaryotic genomes, and while the majority of these sequences are defective, a few can produce infectious virus, either spontaneously upon long-term culture. These can also be induced upon treatment with various chemical or other agents that may be part of the normal production system. The activation of an endogenous, infectious retrovirus in a cell substrate that is used for the production of biologics is an important safety concern, especially in the case of live, viral vaccines, where minimal purification and inactivation steps are used in order to preserve high vaccine potency. [0005] The currently established methods for measuring RT-activity include the highly sensitive, product-enhanced reverse transcriptase assays (PERT) that can detect 1-10 virions and transmission electron microscopy (TEM) to analyze infective retroviruses particles. However, the above techniques are not specific and do not provide any information regarding the source of the RT activity. PCR-based detection of retroviruses can be used in combination with other assays such as reverse transcriptase, electron microscopy infectivity or co-cultivation to increase the sensitivity of detection or to identify a particular adventitious agent present in the test sample. Further, while some studies demonstrate that a low level of RT activity is not generally associated with a replicating agent; major concerns remain regarding the consequences of the presence of such non-productive, non-replicating defective infections in the vaccine, as there is the potential for integration into the host genome. [0006] Retrovirus-induced tumorigenesis can involve the generation of a novel pathogenic virus by recombination between replication-competent and -defective sequences and/or activation of a cellular oncogene by a long terminal repeat (LTR) due to upstream or downstream insertion of retrovirus sequences. To address the possible integration of extraneous retroviral sequences in human cells by RT-containing particles, multiple PCR strategies have been used. These include direct PCR of DNase-treated inoculum using primers from the highly conserved pol region and Alu PCR using LTR primers in conjunction with Alu primers that specifically amplify viral-cellular DNA junctions of integrants. [0007] Future strategies to detect adventitious agents must address three fundamental problems. First, there are large numbers of known viral agents that are potential contaminants, each with a large number of potential strain variants. Second, history has shown that not all adventitious agents fall into anticipated families of viruses, so unanticipated virus families must also be considered. Third, the test must be practical to perform on a large number of samples in a standardized, high-throughput, quality-controlled fashion. The premise of this proposal is that we can leverage recently developed and validated methods using mass spectrometry analysis of broad-range PCR reactions for rapid, sensitive, cost-effective detection of broad ranges of adventitious agents, including previously unknown/uncharacterized viruses and endogenous retroviruses. B. Drug Resistance [0008] Drug resistance in bacteria and viruses is frequently mediated by point mutations in key genes whose gene products interact directly or indirectly with the drug. While there are several methods available for identification of single nucleotide polymorphisms (SNPs) in nucleic acid sequences, the functional unit that encodes each amino acid is the codon, where three successive nucleotides are responsible for encoding each amino acid. Mutations in any of the three nucleotides may or may not result in a mutation in the encoded amino acid, depending upon the particular amino acid and the rules of the genetic code. Because the genetic code is deciphered as a sequence, both the identity and the order of the nucleotides are important in determining the encoded amino acid. Thus, DNA sequencing has become the method of choice for analysis of mutations that result in amino acid changes. DNA sequencing has significant disadvantages as an analysis method for routine use a clinical laboratory setting. It is still relatively expensive and labor intensive, and thus is used only for very important analyses. An example of this is determination of drug resistance in viruses such as HIV and in bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). Drug resistance in HIV has now emerged as a significant problem in both untreated and drug-treated patient populations. The decision to select a particular drug-treatment regimen that the virus will respond to is critical to success of therapy. Drug resistance testing has been shown to improve the clinical outcome in HIV-infected individuals and thus is now recommended for new infections or for patients infected as long as two years or more prior to initiating therapy, in the case of antiretroviral failures and during pregnancy. Thus, despite the costs, DNA sequencing is currently being used for determination of viral drug resistance. Typically, a serum sample is analyzed by PCR amplification of the reverse transcriptase and protease genes, followed by sequencing of approximately 900 nucleotides of the reverse transcriptase gene and 300 nucleotides of the protease gene. The DNA sequence is then used to determine the optimal drug regimen. A drawback of sequencing is that DNA sequencing technology for identification of drug-resistant viruses is that it is not easily able to identify the components present in a mixed sample, particularly in a scenario where a fraction of the virus population has mutated. DNA sequencing was developed on the assumption that the sample being analyzed is homogeneous. However, the HIV populations that infect humans are not homogeneous, and RNA viruses such as HIV are known to rapidly mutate, creating a population of mixed sequences in each infected individual. In the presence of drug selection, mutations that mediate drug resistance that occur at low frequency grow with a selective advantage and eventually can dominate the population, causing treatment failure. In this scenario, the mutant virus starts out as an undetectable fraction of the population which increases to a higher percentage over time. It would be valuable to identify drug resistant virus populations early, before they have a chance to increase the viral load. DNA sequencing methods can identify mixed populations, but do so poorly. In a recent publication using the ABI PRISM 3100 genetic analyzer, it was reported that a viral mixture containing approximately 40% of the mutant viral population can be detected with 95% confidence. However, 40% of a typical viral load (1,800 to 10,500 HIV copies/ml) means a blood burden (assuming 5 liters of blood) of up to 21 million drug-resistant viral copies. Other analytical methods are capable of identifying mutations with more sensitivity than sequencing, but these methods are time consuming, laborious and not amenable to high throughput processes. [0009] Thus, there is a need for rapid and cost effective methods that can be applied as alternatives to sequencing in genomic analysis for variations that mediate amino acid changes. The present invention satisfies this need. The present invention provides, inter alia, methods of identifying adventitious contaminant viruses. Also provided are oligonucleotide primers, compositions and kits containing the oligonucleotide primers, which produce amplification products whose molecular masses provide the means to identify adventitious contaminant viruses at the sub-species level. SUMMARY OF THE INVENTION [0010] The present invention provides compositions, kits and methods for rapid identification and quantification of adventitious contaminant viruses by molecular mass and base composition analysis. [0011] One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 47. [0012] Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 286. [0013] Another embodiment is a composition of is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 47 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 286. [0014] One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 70. [0015] Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 286. [0016] Another embodiment is a composition of is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 70 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 286. [0017] One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 165. [0018] Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 286. [0019] Another embodiment is a composition of is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 165 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 286. Continue reading about Compositions for use in identification of adventitious viruses... 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