Tag: <span>PCR</span>

Adventitious Virus Contamination Testing: Massively Parallel Sequencing in a Multimodal Solution for Biopharmaceutical Safety Testing

Virus contamination of commercially valued cell cultures can be a health risk to the general population and imposes financial burdens on manufacturing and biopharmaceutical companies. We investigated the use of massively parallel sequencing/next-generation sequencing (MPS/NGS) and bioinformatics for the detection and subsequent identification of adventitious contamination. Specifically, the Illumina® HiSeq 2000 instrument, in the 2 × 100 base pair (bp) paired end (PE) run configuration, was used to determine the identity and limit of detection of a DNA virus spiked into a virus vaccine, and an RNA virus spiked into a mammalian master cell bank (MCB). This configuration provided sufficient sequence read lengths and depth of coverage to detect and identify spiked-in SV40 and measles viruses in a background of vaccine, MCB, and host nucleic acid that make up the bulk of these samples. Furthermore, the detection of 30 SV40 reads within one tested sample suggested a < 1 plaque forming unit (pfu) sensitivity in a background of 2.8 × 107 infectious adenovirus serotype 5 (Ad5) particles. Results from subsequent virus vaccine testing suggested the presence of non-viable virus DNA contamination in the sample. Therefore, we propose that a multimodal approach, in which broad-range screening for known or unknown adventitious agents by MPS/NGS is complemented by targeted virus detection assays (e.g., PCR-based and infectivity assays), should provide the most useful safety monitoring information. We suggest that MPS/NGS and the accompanying bioinformatics is a sensitive, broad-range, and long-lasting tool with the ability to improve upon existing biosafety testing within a larger testing program…

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Biologics Production

bioinformatics cell culture dna contamination host cell proteins mammalian cell bank massively parallel sequencing master cell bank mcb measles virus next generation sequencing PCR plaque forming unit SV40 vaccine production vaccines viral contaminants virus contamination virus detection assays virus spikes

PCR Turns 25: Celebrating the Past and Anticipating the Future

Beginning with the production of the very first ­recombinant deoxyribonucleic acid (DNA) molecule in 1972 and continuing through the ­following decade, DNA-focused molecular biology researchers firmly positioned themselves on the cutting edge of ­scientific inquiry. With each passing day, month and year, these researchers made ­significant leaps forward in understanding, modifying and manipulating the key roles that DNA plays in all living organisms. Some of the key milestones…

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Nucleic Acids Research

amplification gene expression genetic research genotyping molecular research PCR polymerase chain reaction

Improved Analysis of PCR Viral Detection Using Chip-based Capillary Gel Electrophoresis

Validating the safety of biological preparations requires thorough testing for contamination by adventitious agents. Utilizing mammalian cell cultures to produce recombinant proteins as biopharmaceuticals requires testing for viral contamination. The polymerase chain reaction (PCR) may be employed to specifically detect the presence of viral DNA or RNA with great sensitivity. PCR assays are particularly useful for the qualification of recombinant cell banks. Regulatory agencies recommend that mammalian cell banks be tested for a variety of possible human viral contaminants. In most cases the cells used to produce the cell bank have been previously analyzed for viral contamination. The use of PCR for the detection of viruses in the final banked cells can alleviate the need for difficult, costly, and time-consuming infectivity assays. In some cases the relevant viruses cannot be cultured, eliminating the ability to perform infectivity assays. The PCR assay can provide a sensitive and specific method for detection of viral contamination when standard infectivity assays are unsatisfactory…

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Cell & Tissue Banking

analysis electrophoresis PCR PCR- based assay safety testing viral detection

FTA Filter Applications: A PCR Format to Alleviate Technical Barriers in the Detection of Foodborne Pathogens in Complex Matrices

The safety of our food supply is a major public health concern for consumers, government regulatory agencies, and the food industry. Earlier generations may recall when fresh produce was largely domestic and seasonal. Today we live in a global marketplace, where fresh fruits and vegetables may be on the vine overseas one day and on our grocer’s shelf the next. Although this has provided more yearround variety of foods for the consumer, a lack of uniformity in established agricultural standards and practices among international trading partners (e.g., sanitary issues and inspections) may ultimately lead to deleterious health effects. This is evident in the number of food-borne illness outbreaks and associated deaths. Negative economic consequences also result through lost wages and productivity, and health care costs. The impact can be far greater in developing nations. Therefore, refining domestic and international food safety policies is at the forefront of many government agencies’ efforts toward protecting the public health. Reducing the number of such incidences has become a priority for government regulatory agencies and the food industry…

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Regulatory

food safety food-borne illness food-borne pathogen FTA filter PCR PCR- based assay standards