Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation

Cai, Hui; Gu, Xuelin; Scanlan, Mary S.; Lively, Chris R.

doi:10.1016/j.jpba.2011.01.010

Cited by 15 publications

(5 citation statements)

References 7 publications

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“…First, the loss of AMDV DNA occurring during the DNA extraction process is inevitable. DNA losses during extraction by commercial kits vary widely and is influenced by the type of extraction kit and the source of tissue (Fredricks Cai et al 2011). The amount of DNA lost is often large, for instance 21.75% to 60.56% , 45.1% to 99.5% , and 57.5% to 100% (Mumy and Findlay 2004).…”

Section: Discussionmentioning

confidence: 99%

A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink

Farid

Rupasinghe

2017

Can. J. Microbiol.

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this article has been changed to the CC BY 4.0 license. The PDF and HTML versions of the article have been modified accordingly. Abstract:The objective of this study was to assess the sensitivity of the Omni Klentaq-LA DNA polymerase for detecting Aleutian mink disease virus (AMDV) in mink blood and tissues by PCR without DNA extraction. The presence of AMDV DNA was directly tested by Klentaq in the plasma, serum, whole blood, and spleen homogenates of 188 mink 4 and 16 months after inoculation with the virus. Samples from bone marrow, small intestine, liver, lungs, kidneys, and lymph nodes of 20 of the same mink were also tested by Klentaq. DNA was extracted from paired samples of plasma and the aforesaid tissues by a commercial nucleic acid extraction kit (Dynabeads Silane) and tested by PCR. Compared with the extracted DNA, Klentaq detected a significantly greater number of samples in the whole blood, serum, plasma, spleen, and small intestine. It was concluded that Klentaq is a preferred system for directly detecting AMDV DNA in mink blood and tissues. The lower success rate of extracted DNA compared with Klentaq could be the result of DNA losses during the extraction process. This is an important factor in chronically infected mink, which have a low AMDV copy number in the bloodstream. Direct AMDV detection also reduces the cost of PCR amplification and lowers the risk of sample contamination.

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Section: Discussionmentioning

confidence: 99%

A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink

Farid

Rupasinghe

2017

Can. J. Microbiol.

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“…This is particularly important for monoclonal antibodies, which are known to inhibit qPCR 12, 13. Some protocols also include an extra step of DNA purification 8, 14. The same method with proteinase digestion was applied to rAAV-based drugs.…”

Section: Introductionmentioning

confidence: 99%

A Digestion-free Method for Quantification of Residual Host Cell DNA in rAAV Gene Therapy Products

Wang

Cooper

Kiladjian

et al. 2019

Molecular Therapy - Methods & Clinical Development

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Recombinant adeno-associated virus (rAAV) is a vector with increasing popularity in the field of gene therapy. Like other drug substances manufactured in cell lines, rAAV vectors are commonly contaminated with host cell DNA, and the levels must be carefully monitored. The current method for residual DNA quantification in rAAV was adapted from protein programs and required sample digestion by proteinase prior to qPCR analysis. While the method worked effectively, it was unclear if proteinase digestion was essential for releasing DNA from rAAV capsids and improving qPCR efficiency. In this study, we systematically investigated the role of each component and treatment with the goal to simplify and streamline the method. It was determined that the proteinase digestion step was dispensable, while the addition of Tween 20 to rAAV samples was essential for accurate quantification of residual DNA. Based on this finding, a digestion-free method has been established that requires only a one-step sample preparation—addition of Tween 20. The method has been tested extensively with an rAAV9-based drug substance and process intermediates and verified with other rAAV serotypes. This significantly simplified and faster assay can be easily automated for high-throughput applications.

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“…Three methods(DNA hybridization assay, Threshold® assay, and quantitative PCR)have been recommended by regulatory agencies for residual host cell DNA quantitation [14, 15]. Among these methods, qPCR is considered to be the most practical for residual DNA quantitation due to their sensitivity, accuracy, precision, and time-saving features.…”

Section: Introductionmentioning

confidence: 99%

Development and Validation of Quantitative Real-Time PCR for the Detection of Residual CHO Host Cell DNA and Optimization of Sample Pretreatment Method in Biopharmaceutical Products

et al. 2019

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Background The presence of residual DNA carried by biological products in the body may lead to an increased oncogenicity, infectivity, and immunomodulatory risk. Therefore, current agencies including WHO, EU, and the FDA limited the accepted amounts of residual DNA (less than 10 ng or 100 pg/dose). Among the methods of detecting residual DNA, qPCR is considered to be the most practical for residual DNA quantitation due to its sensitivity, accuracy, precision, and time-saving. Results In this study, the detection capacity of this method was determined by comparing the detected concentration of the commercial kit and the self-designed primer/probe set after the same treatment of the extraction method. Then, a universal sample pretreatment method based on a co-precipitant was optimized. The validation results demonstrated that the method has appropriate specificity, sensitivity, accuracy, and precision according to ICH guidelines. The limit of detection and quantitation reached 3 fg/ul and 0.3 pg/reaction respectively, which satisfies the requirement of limit of residual DNA detection in biologics. Spike recovery (82.3–105.7%) showed that the proposed qPCR assay was accurate and has good extraction efficiency. Moreover, the precision of the method based on intra- and inter-assay was 0.065–0.452% and 0.471–1.312%, respectively. Conclusions These results all indicated that the method for determination of residual DNA in biological products expressed from CHO cells is sensitive, accurate and robust. Electronic supplementary material The online version of this article (10.1186/s12575-019-0105-1) contains supplementary material, which is available to authorized users.

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Development of a quantitative PCR assay for residual mouse DNA and comparison of four sample purification methods for DNA isolation

Cited by 15 publications

References 7 publications

A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink

A fast and accurate method of detecting Aleutian mink disease virus in blood and tissues of chronically infected mink

A Digestion-free Method for Quantification of Residual Host Cell DNA in rAAV Gene Therapy Products

Development and Validation of Quantitative Real-Time PCR for the Detection of Residual CHO Host Cell DNA and Optimization of Sample Pretreatment Method in Biopharmaceutical Products

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