Regular ArticleReal-time Quantitative PCR for Retrovirus-like Particle Quantification in CHO Cell Culture
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A direct RT qPCR method for quantification of retrovirus-like particles in biopharmaceutical production with CHO cells
2020, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :However, it requires large sample volume (3–6 L), is labor intensive, time consuming, expensive, and has low sensitivity and high variability [8]. Quantitative real-time PCR based methods for RVLPs are available but require RNase digestion of samples followed by total nucleic acid extraction, then digestion with DNase followed by reverse transcription and PCR [8–10]. Although the qPCR is a very sensitive method, the efficiency of nucleic acid extraction impacts the sensitivity and usefulness of the qPCR-based quantification method.
Evaluation of infectivity and reverse transcriptase real-time polymerase chain reaction assays for detection of xenotropic murine leukemia virus used in virus clearance validation
2015, BiologicalsCitation Excerpt :Because RVLPs are noninfectious, an infectivity assay cannot be used directly to detect these particles. Real-time polymerase chain reaction (RT-PCR) assay has been used for the quantification of the RVLP from CHO cells [10]. In the biopharmaceutical industry, xenotropic murine leukemia virus (X-MuLV) is commonly used as a specific model virus in evaluating the removal of RVLP by purification processes.
Growth curve of murine xenotropic leukemia virus-related virus grown in Chinese hamster ovary cells
2014, Journal of the Chinese Medical AssociationCitation Excerpt :Nowadays, the PCR assay is a popular method of detecting viruses, especially real-time PCR. Lute et al,10 Shi et al,11 and Wit et al 12 used the Taqman prober to detect XMulV particles in CHO cells for the purpose of evaluating viral clearance; the limit of detection in their experiments ranged from 1.0 to 1.5 viral RNA copies/μL. In this study, the SYBR Green fluorescence qPCR was used to detect virions, and the quantity of virions on different days was calculated using the 2−ΔΔCt method.
Robustness of virus removal by protein A chromatography is independent of media lifetime
2008, Journal of Chromatography ASpecific detection of residual CHO host cell DNA by real-time PCR
2007, BiologicalsCitation Excerpt :qRTPCR have been applied to characterize and detect numerous bacteria, fungal and viral DNA [10–13]. In the biologics industry, qRTPCR has been used to detect viral load in protein samples [14–16]. However, to date, there are no published methods to specifically monitor host cell DNA contamination using this sensitive technique although it is available as a service provided by laboratories specializing in quality assurance [17].
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