e-Myco™ VALiD-Q Mycoplasma qPCR Detection Kit is a product which is able to detect Mycoplasma by Real-time PCR method using specific probe.
- • Validation of a PCR method for the detection of mycoplasmas according to European Pharmacopoeia section 2.6.7.
- • Suitable for monitoring the manufacturing process of pharmaceuticals in the field of cell therapy / monitoring of contamination of cultured cell lines
- • High level of sensitivity in Mycoplasma detection as 10 CFU/ml
- • Able to detect over 70 species of Mycoplasma
- • Only takes 3 hours from gene extraction and has high level of sensitivity in Mycoplasma detection
- • Amplifies Mycoplasma 16S ribosomal RNA with high sensitivity and specificity
Mycoplasmas are small, round or filamentous prokaryotic organisms which are a frequent contaminant of cell cultures. Mycoplasma depend on their hosts for many nutrients due to their limited biosynthetic capabilities. Up to 30~85% of cell cultures may be contaminated with mycoplasmas, the main contaminants being the species M.orale, A.laidlawii, M.arginini and M.hyorhinis. Although these mycoplasmas do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including changes in metabolism growth, viability and morphology, there by altering the phenotypic properties of the host cells. Many methods are available for detection of mycoplasma, including isolation in broth/agar culture, direct or indirect fluorescence staining, ELISA, immunostaining, direct or indirect PCR. Among those methods, direct PCR is the highly sensitive, specific and convenient method when the primer design is optimized.
The e-Myco™ VALiD-Q Mycoplasma qPCR Detection Kit is composed of a set of primers and probe that are specific for the highly conserved mycoplasma16S-rRNA coding region including M.pneumoniae, M.argnini, M.hyorhinis, M.fermentans, M.orale and A.laidlawii. The kit is designed to detect the presence of mycoplasma that might contaminate biological materials such as cultured cells. Also, the kit can detect mycoplasma within 90minutes sensitively up to 10CFU/ml and includes internal control for verifying a qPCR run as well as positive control DNA.
The kit is used for the detection of mycoplasma species that are most commonly encountered in cell culture, including M.peumoniae, M.arginini, M.fermentans, M.hyorhinis, M.orale, and A. laidlawii. Furthermore, this kit can detect other various species of mycoplasma.
The test using positive control (M.hyorhinis) and internal positive control showed target signal amplification and HEX channel amplification respectively (Ct value around 20) (picture a). Otherwise, only IPC signal amplification was observed when tested with genomic DNA of 6 non-Mycoplasma samples and NTC (no template control) (picutre b~h). Excellent specificity of e-MycoTM VALiD-Q Mycoplasma qPCR Detection Kit was observed through 3 times of repeated experiment.
Fig. Analytical Sensitivity of e-Myco™ VALiD-Q Mycoplasma qPCR Detection Kit
A) Mycoplasma arginini, B) Mycoplasma fermentans, C) Mycoplasma hyorhinis, D) Mycoplasma orale, E) Mycoplasma pneumoniae, F) Acholeplasma laidlawii.
e-Myco™ VALiD-Q Mycoplasma qPCR Detection Kit using 6 Mycoplasma species was carried out, and the test proved the Kit’s excellent sensitivity to detect all 6 Mycoplasma species. Besides, minimum detection sensitivity for 6 Mycoplasma species was identified as 10 CFU/ml. Accordingly, replaceability of direct plating method to e-Myco™ VALiD-Q Mycoplasma qPCR Detection Kit was proved.
Equivalence test to compare with direct plating method
Fig1. Mycoplasma Culture and Colony Assay.
Fig2. Comparability test of e-Myco™ VALiD-Q Mycoplasma qPCR Detection Kit
10㎕ of 10-6 diluted culture medium showed 11 CFU formation (standard variation ±2) (Fig 1). Consequently, CFU of initial culture medium was calculated as 0.9 ~ 1.1 x 109 CFU/ml by substituting dilution rate and spreaded volume to CFU value. Meanwhile, Mycoplasma was collected from 1ml of initial culture medium then diluted with 50㎕ PBS. The diluted culture medium was resuspended then 10-fold diluted followed by boiling to use 5㎕ of it as template DNA for Real-time PCR. The result is indicated in the Fig 2. In case of direct injection, 10 CFU/ml sensitivity was observed (Fig 2) and signal detection in 1 CFU/ml was identified by repeated test. Also, the result from NTC test proved test reliability by detection of IPC signal excluding target signal