Development and validation of a Vero cell DNA standard for residual DNA measurement suitable for quantitative PCR method.

INTRODUCTION

The fluorescence quantitative PCR method for the detection of Vero cell residual DNA in human rabies vaccine urgently needs a national standard for Vero cell DNA quantification.

METHODS

Briefly, the genomic DNA of Vero cells was extracted, diluted to 100 μg/ml, and divided into 0.1-ml aliquots in sterile screw cap tubes. After DNA preparation, concentration calibration, applicability, and stability verification were carried out by multiple laboratories.

RESULTS

The concentration of the national standard for Vero cell DNA quantification (limited qPCR method) determined from this collaborative study was 107 ± 6 μg/mL, and the DNA purity (OD260/OD280) was 1.83. This standard is only suitable for the qPCR method, which has an average amplification efficiency of 92%. After accelerated degradation at 37 °C from 1 to 4 weeks, the standard showed amplification curves similar to those stored at -70 °C, with no significant difference in amplification efficiency, showing good stability.

CONCLUSION

This study successfully developed a national standard for Vero cell DNA quantification using the quantitative fluorescence PCR method, which has significant application potential for standardizing Vero cell DNA residue detection in human rabies vaccines.