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. 2017 Jun 30:11:66-80.
doi: 10.2174/1874104501711010066. eCollection 2017.

Standardization of DNA Residual Quantification Method of Vero Cell Rabies Vaccine for Human Use

Janeth Del Carmen Arias Palacios  1 , Carlos Alberto Barrero Barreto  2 , José Salvador Montaña Lara  3 , Ángela María Londoño Navas  4
Affiliations

Standardization of DNA Residual Quantification Method of Vero Cell Rabies Vaccine for Human Use

Janeth Del Carmen Arias Palacios et al. Open Med Chem J. .

Abstract

Objectives: Normalize the quantification of residual DNA from Vero cells in the rabies vaccine for use in human VAHV I, by quantitative PCR in real time and the design of primers that amplified, highly repetitive sequences of Cercopithecus aethiops and a constitutive gene according to sequences reported in the GenBank and quantifying the residual DNA in the vaccine VAHV I in three consecutive batches according to the standard set by the World Health Organization.

Methods: A real time quantitative method based on SYBR Green chemistry has been applied for the quantification of residual DNA (resDNA) using highly repetitive DNA (Alu) and a housekeeping gene (B-actin) as target sequences.

Results: The sensitivity achieved with this white sequence is within the reported limits and who are between 5 and 50 pg. For real time PCR optimization with Alu-p53, different concentrations of MgCl2 (0.5, 0.75, 1.0, 1.25 and 1.5 mm) in combination with three different concentrations of primers (75, 100 and 150nM) were used. pDNA in concentration of 1x107 copies / ul was used as template. Optimal concentrations were 1.25 mM MgCl2 and 100nM primers. To level of detection of 1.53 ng/ul was found for p53-Alu and Alu-Glob and 0.39 ng/ul for B-actin with gDNA curves.

Conclusion: Quantification of resDNA of vaccine VAHV I with close-ups of B-actin was normalized. Reached a sensitivity of 30 pg of resDNA/dose VAHV I, with close-ups of B-actin. Found, in three consecutive batches, an amount less than 10 ng/dose, these results suggest that the production process ensures vaccine resDNA removal, meeting international requirements for biological products for use in humans that use continuous cell lines for production.

Keywords: Antirrabic vaccine; Betapropiolactone; Real time PCR; Residual DNA; Vero cells.

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Figures

Fig. (1)
Fig. (1)
PCR from genomic Vero cell’s DNA. Top gel. Lane 1, MPM 25bp (Promega). Lanes 2 and 3, amplification product with Alu-p53 primers with 400 and 40ng DNA. Lane 4, negative control. Lanes 5 and 6, amplification product with Alu-Glob primers with 400 and 40ng DNA. Lane 7, negative control. Bottom gel. Lane 1, MPM 25bp (Promega). Lanes 2 and 3, amplification product with B-actin primers with 400 y 40ng DNA. Lane 4, negative control. Lane 5, amplification product with MSP-1 region 3 primers with Plasmodium vivax
Fig. (2)
Fig. (2)
Colony PCR A) Lane 5, clone Alu-p53 1-1. B) Lane 10, clone Alu-Glob 2-9. C) Lane 12, clone B-actin 3-11. Lane 1, A, B and C. MPM 25bp. (Promega) 2% agarose gel
Fig. (3)
Fig. (3)
Plasmidic DNA extraction. Lane 1, MPM 1kb (Ladder Promega); Lane 2-4, plasmidic DNA Alu-p53 1-1, Alu-Glob 2-9 and B-actin 3-11. 1% agarose gel.
Fig. (4)
Fig. (4)
Alignment of sequenced plasmids. In A, B and C. Blue, results from sequencing of Alu-p52, Alu-Glob and B-actin plasmids. In red, partial sequence reported in GenBank.
Fig. (5)
Fig. (5)
Dissociation curves of amplification products with Alu-p53 primers. Red, pDNA as template; green, gDNA as template; blue, NTC.
Fig. (6)
Fig. (6)
Dissociation curves of amplification products with Alu-Glob primers. Red, pDNA as template; green, gDNA as template; blue, NTC.
Fig. (7)
Fig. (7)
Dissociation curves of amplification products with B-actin primers. Red, pDNA as template; green, gDNA as template; blue, NTC.
Fig. (8)
Fig. (8)
Standard curves using pDNA and gDNA as template and Alu-p53 primers. A) pDNA) B) gDNA.
Fig. (9)
Fig. (9)
Standard curves using pDNA and gDNA as template and Alu-Glob primers. A) pDNA B) gDNA.
Fig. (10)
Fig. (10)
Standard curves using pDNA and gDNA as template and B-actin primers. A) pDNA B) gDNA.
Fig. (11)
Fig. (11)
RT-PCR reaction with 10µl of VAHV I as template.
See this image and copyright information in PMC

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