Compositions for use in identification of adventitious viruses

a technology for adventitious viruses and compositions, applied in the field of compositions for use in identification of adventitious viruses, can solve the problems of not being specific, adventitious viruses represent a major risk associated with the use of cell-substitute derived biologicals, including vaccines and antibodies, and the risk of containing oncogenic viruses is higher

Inactive Publication Date: 2006-09-14
IBIS BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0041] In some embodiments, the present invention provides methods for detecting or quantifying adventitious contaminant virus by combining a nucleic acid amplification process with a mass determination process. In some embodiments, such methods identify or otherwise analyze the adventitious contaminant virus by comparing mass information from an amplification product with a calibration or control product

Problems solved by technology

Adventitious viruses represent a major risk associated with the use of cell-substrate derived biologicals, including vaccines and antibodies, for human use.
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.
However, the above techniques are not specific and do not provide any information regarding the source of the RT activity.
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.
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.
DNA sequencing has significant disadvantages as an analysis method for routine use a clinical laboratory setting.
It is still relatively expensive and labor intensive, a

Method used

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  • Compositions for use in identification of adventitious viruses
  • Compositions for use in identification of adventitious viruses
  • Compositions for use in identification of adventitious viruses

Examples

Experimental program
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Effect test

example 1

Design and Validation of Primers that Define Bioagent Identifying Amplicons for Adventitious Contaminant Viruses

A. General Process of Primer Design

[0203] For design of primers that define adventitious contaminant virus identifying amplicons, a series of adventitious contaminant virus genome segment sequences were obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 150 nucleotides in length and distinguish species and / or individual strains from each other by their molecular masses or base compositions. A typical process shown in FIG. 1 is employed for this type of analysis.

[0204] A database of expected base compositions for each primer region was generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hyb...

example 2

Sample Preparation and PCR

[0216] Samples were processed to obtain viral genomic material using a Qiagen QIAamp Virus BioRobot MDx Kit. Resulting genomic material was amplified using an Eppendorf thermal cycler and the amplicons were characterized on a Bruker Daltonics MicroTOF instrument. The resulting data was analyzed using GenX software (SAIC, San Diego, Calif. and Ibis, Carlsbad, Calif.).

[0217] All PCR reactions were assembled in 50 μL reaction volumes in a 96-well microtiter plate format using a Packard MPII liquid handling robotic platform and M.J. Dyad thermocyclers (MJ research, Waltham, Mass.). The PCR reaction mixture consisted of 4 units of Amplitaq Gold, 1× buffer II (Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl2, 0.4 M betaine, 800 μM dNTP mixture and 250 nM of each primer. The following typical PCR conditions were used: 95° C. for 10 min followed by 8 cycles of 95° C. for 30 seconds, 48° C. for 30 seconds, and 72° C. 30 seconds with the 48° C. annealing temp...

example 3

Solution Capture Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads

[0218] For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, 25 μl of a 2.5 mg / mL suspension of BioClone amine terminated superparamagnetic beads were added to 25 to 50 μl of a PCR (or RT-PCR) reaction containing approximately 10 pM of a typical PCR amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed three times with 50 mM ammonium bicarbonate / 50% MeOH or 100 mM ammonium bicarbonate / 50% MeOH, followed by three more washes with 50% MeOH. The bound PCR amplicon was eluted with a solution of 25 mM piperidine, 25 mM imidazole, 35% MeOH which included peptide calibration standards.

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Abstract

The present invention provides compositions, kits and methods for rapid identification and quantification of adventitious contaminant viruses by molecular mass and base composition analysis.

Description

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.STATEMENT OF GOVERNMENT SUPPORT [0002] This invention was made with United States Government support under NIH contract N01 AI40100. Th...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12P19/34
CPCC12Q1/6851C12Q1/701
Inventor SAMPATH, RANGARAJAN
Owner IBIS BIOSCI
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