Mycoplasma contamination poses a persistent challenge in biological research and biopharmaceutical production. Conventional detection methods, such as classical culture assays, are time-consuming and lack sensitivity, while the standard PCR is prone to inhibitor interference and incapable of quantification, failing to meet the demand for rapid and precise quality control in vaccine production or laboratory settings.

This study aimed to establish a universal, highly specific, and sensitive quantitative real-time PCR (qPCR) method for efficient screening of mycoplasma contamination in cell cultures, live viral vaccines, and biological raw materials.

The SILVA_138.1_SSURef database, encompassing 16S/18S rRNA sequences of bacteria, archaea, and fungi, was utilized to extract 181 non-redundant 16S rRNA sequences from classified mycoplasma species. The hypervariable V6-V8 region was identified as the optimal target via multiple sequence alignment (MEGA 11.0). Three primers (forward primers MF1: 5′-GCAAARCTATRGARAYATAGYVGAG-3′; MF2: 5′-GCAAAGGCTTAGAAATAAGTTCGGAG-3; reverse primer MR: 5′-CCARCTCYCATRGTKTGACGG-3′) and a dual-quenched TaqMan probe (5′-FAM-ACAGRTGGTGCATGGYTGTCGTCAGCTC-BHQ1-3′) were designed using Primer Premier 5.0, with primer-probe ratios and annealing temperatures optimized to establish the qPCR assay. Validation included: (1) primer-probe specificity testing against 11 Mycoplasma/Acholeplasma species (e.g., Mycoplasma anatis, Mycoplasma bovis, Ureaplasma urealyticum); (2) sensitivity assessment via 10-fold serial dilutions (1.0×10⁸-1.0×10¹ copies/μL) of Mycoplasma synoviae BHQ03, Mycoplasma gallisepticum, and Mycoplasma hyopneumoniae, with standard curves generated; (3) specificity evaluation against four common bacteria (Salmonella, Clostridium perfringens, Escherichia coli, Brucella) and eight animal cell lines (Marc145, Vero, CEF, etc.); (4) repeatability analysis (intra- and inter-assay variability) using M. synoviae BHQ03 dilutions (1.0×10⁸–1.0×10¹ copies/μL); (5) parallel testing of 24 live viral vaccine batches (poultry, swine, and canine), 20 cell culture samples (8 types), and 8 viral seed stocks via qPCR, conventional PCR, and classical culture methods.

The optimized qPCR protocol employed a two-step amplification program (56 ℃ annealing temperature). Specificity testing confirmed positive detection of all 11 Mycoplasma/Acholeplasma strains and no cross-reactivity with non-target bacteria or cell lines. Repeatability tests showed coefficient of variation (CV) values <2% for Ct values across replicates. Sensitivity assays demonstrated limits of detection (LOD) of 1-2 copies/μL for M. synoviae, M. gallisepticum, and M. hyopneumoniae. Comparative analysis of clinical samples revealed high concordance between qPCR, conventional PCR, and culture methods, with qPCR exhibiting superior sensitivity.

The universal qPCR method developed in this study provided an accurate, reliable, and rapid detection tool for potential mycoplasma contamination in cell cultures and live viral vaccines.