In order to clarify the microscopic process of Metarhizium rileyi strain CDTLJ1 infestation in the Spodoptera frugiperda larvae and to elucidate the pathogenic mechanism of the strain, scanning electron microscope, fluorescence microscope and light microscope were used to observe the process of conidia attachment on the surface of the larvae after inoculation of the 3rd instar larvae with the strain CDTLJ1, germination and penetration, and proliferation in the larvae body cavity. The results showed that the germination rate of conidia of strain CDTLJ1 was 80.36% in strumae surface topography, which was significantly higher than 72.44% in gentle surface topography (P < 0.01). Spores could form germ tubes and appressoria after germination, but most of them used germ tubes as infestation structure. Conidia germinated within 8 h after inoculation, with peak penetration into the integument of larvae at 16~24 h. Larvae moulted at 24~32 h after inoculation, and the moulted epidermis was covered with spores adhering to the surface of the protozoan. The earliest appearance of hyphae body observed in the larvae's body cavity was 48 h after inoculation. The insects died after 96 h of inoculation, Hyphae were visible in the electron microscopic field of view as it penetrated the integument and grew outward from the host at 2 h after death. The germination time of strain CDTLJ1 conidia on the isolated epidermis was 16 h, which was later than that on the epidermis of live larvae significantly.
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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.
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