Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine

Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine Publisher Pubmed

Paryan M¹ ; Khodayar M² ; Kia V³ ; Mohammadiyeganeh S^{4, 5} ; Kaghazian H¹

Show Affiliations

1. Department of Research and Development, Production and Research Complex, Pasteur Institute of Iran, P.O.Box: 3159915111, Tehran, Iran
2. Department of Cellular and Molecular Biology, Tehran East Branch, Islamic Azad University, Tehran, Iran
3. Department of Medical Biotechnology, Faculty of Medicine, Zanjan University of Medical Sciences, Zanjan, Iran
4. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
5. Department of Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Source: Applied Biochemistry and Biotechnology Published:2016

Abstract

Biological therapeutic products such as recombinant hepatitis B virus (HBV) vaccine, produced by microbial fermentation in complex media, should be evaluated for host cell DNA contamination in purification steps. Eliminating these contaminations increases the efficacy of the vaccine and decreases its side effects. The objective of the present study is to trace the residual host cell DNA (HCD) in recombinant HBV vaccine by developing a TaqMan Real-Time PCR method which is more sensitive, specific, and reproducible than traditional methods such as Picogreen analysis and Threshold DNA assay. Primers and a probe were designed for the most highly conserved regions of Pichia pastoris genome. To determine the specificity of the assay, in addition to performing a BLAST for the primers and the probe in NCBI nucleotide database, 20 different human genomes and 8 bacterial and viral genomes were used. Moreover, serial dilutions of plasmids, from 102 to 107 copies/μL (from 0.00064 to 6.4 pg/μL), were prepared to find the sensitivity and the limit of detection (LOD) of the assay. Using 28 different genome samples, the specificity of the assay was determined to be 100 %. In addition, the sensitivity and LOD of the method was 0.39 × 10−5 pg/μL. Moreover, the reproducibility of the assay based on intra- and inter-assay was 1.03 and 1.06 %, respectively. Considering the suitable specificity and sensitivity, ease of use, relatively low cost, and rapidity of the assay, it can be a reproducible and sensitive method to examine recombinant vaccines for P. pastoris residual DNA. © 2016, Springer Science+Business Media New York.

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Style	Citing Format
MLA	Paryan M, et al.. "Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine." Applied Biochemistry and Biotechnology, vol. 179, no. 3, 2016, pp. 375-382.
APA	Paryan M, Khodayar M, Kia V, Mohammadiyeganeh S, Kaghazian H (2016). Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine. Applied Biochemistry and Biotechnology, 179(3), 375-382.
Chicago	Paryan M, Khodayar M, Kia V, Mohammadiyeganeh S, Kaghazian H. "Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine." Applied Biochemistry and Biotechnology 179, no. 3 (2016): 375-382.
Harvard	Paryan M et al. (2016) 'Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine', Applied Biochemistry and Biotechnology, 179(3), pp. 375-382.
Vancouver	Paryan M, Khodayar M, Kia V, Mohammadiyeganeh S, Kaghazian H. Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine. Applied Biochemistry and Biotechnology. 2016;179(3):375-382.
BibTex	@article{ author = {Paryan M and Khodayar M and Kia V and Mohammadiyeganeh S and Kaghazian H}, title = {Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine}, journal = {Applied Biochemistry and Biotechnology}, volume = {179}, number = {3}, pages = {375-382}, year = {2016} }
RIS	TY - JOUR AU - Paryan M AU - Khodayar M AU - Kia V AU - Mohammadiyeganeh S AU - Kaghazian H TI - Development of an In-House Taqman Real-Time Pcr-Based Method to Detect Residual Host Cell Dna in Hbv Vaccine JO - Applied Biochemistry and Biotechnology VL - 179 IS - 3 SP - 375 EP - 382 PY - 2016 ER -

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