Design and development of a simple method for the detection and quantification of residual host cell DNA in recombinant rotavirus vaccine

Abstract

Rotavirus recombinant vaccine is usually produced in Vero cells . Residual host DNA may reside in the final product and is considered a source of contamination. WHO protocols indicate that biological products should be free of any type of impurity such as nucleic acids , endotoxins , and host cell intermediate materials. Therefore, all recombinant biological therapeutics should be assessed for residual host DNA. In the present study, a sensitive and specific real-time PCR method was developed to detect residual host cell DNA in the final product. The Beta-actin gene of Vero cells was selected to detect residual host cell DNA. One set of primers and a TaqMan probe were designed for the gene using AlleleID 6 software. Real-time PCR reactions were set up, and efficiency of 84% was obtained. The sensitivity and limit of detection of the assay were determined to be 0.176 Fg/μl and 0.044 Fg/μl, respectively. The intra-assay and inter-assay variations were 4.4% and 1.04%, respectively. Furthermore, the specificity and sensitivity of the assay were high enough, and the detection limit was lower than that of the FDA and WHO standards. This indicates that our assay is highly specific and sensitive to detect residual host DNA of Vero cells in the recombinant rotavirus vaccine . • A specific and sensitive TaqMan Real-time PCR can be used to quantify Residual DNA. • The assay to detect Beta actin is reproducible with a CV% ranging from 1.04 to 4.4 • The sensitivity and limit of detection of the assay were determined to be 0.176 Fg/µl and 0.044 Fg/µl, respectively.