Development of a highly sensitive PCR/DNA chip method to detect mycoplasmas in a veterinary modified live vaccine

Area of Science:

• Microbiology
• Vaccinology
• Biotechnology

Background:

• Mycoplasmas are common contaminants in mammalian cell cultures, impacting biologics manufacturing.
• Current European Pharmacopoeia (Ph. Eur.) requires a lengthy 28-day culture assay for mycoplasma purity testing.
• The traditional culture assay has limitations in sensitivity, particularly for non-culturable mycoplasma species.

Purpose of the Study:

• To evaluate the sensitivity and specificity of a commercial nucleic acid amplification technique (NAT) for mycoplasma detection.
• To assess the suitability of NAT for testing veterinary modified live vaccines.
• To determine if NAT can replace the conventional culture method in vaccine production.

Main Methods:

• A commercially available NAT assay was used to detect mycoplasma DNA.
• The assay was tested using five Ph. Eur.-recommended reference strains.
• The NAT method was applied to a veterinary modified live vaccine spiked with mycoplasma.

Main Results:

• The NAT-based method demonstrated high sensitivity with a limit of detection of 10 CFU/mL, meeting Ph. Eur. requirements.
• No interference was observed from vaccine components when detecting mycoplasma DNA.
• The specificity of the NAT method was confirmed by the absence of detection in non-spiked vaccine samples.

Conclusions:

• The evaluated NAT-based method is a sensitive and specific tool for detecting mycoplasma in veterinary live vaccines.
• This NAT method can be reliably used without interference from vaccine components.
• The NAT assay is a promising replacement for the conventional culture method in veterinary live vaccine production.