Description

The e-Myco™ VALiD-Q Real-time PCR Kit (v3.0 PRO) provides a rapid, reliable, and guideline-compliant method to detect mycoplasma contamination in cell-based manufacturing processes. The assay offers comprehensive coverage of international pharmacopeia standards (EP, USP, JP, and Korea MFDS), enabling simultaneous detection of compendial reference species within a single integrated panel. Panelized primers and probes, developed through extensive sequence alignment and in-silico validation, ensure high specificity and meet established detection limits without cross-reactivity with non-target microorganisms.

▶ Full compliance with Major 4 pharmacopeias : A performance class beyond comparison

▶ Just 53 minutes : Far beyond the limitations of culture methods

▶ Internal Control (IC) and External Control (EC) ensure process validity across all steps, from nucleic acid extraction to PCR amplification

 B, (Insect, Plant, or Avian-derived species) : In the Japanese Pharmacopoeia (JP), species of insect or plant origin (e.g., Spiroplasma citri) should also be tested in addition to the listed species. When avian cells or materials are used, avian-derived species (e.g., Mycoplasma synoviae) may also be considered for inclusion in the test panel.

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Storage

▶ Storage Temperature : Below - 20°C

▶ Shelf Life : 1 year (Stable Within 6 months after opening, within expiry date of the kit) ​

 

Kit Contents

No.	Kit Contents	Unit
1	2X qPCR mix	500 μl × 2 tubes
2	Detection solution	280 μl × 1 tube
3	Internal Control	150 μl × 2 tubes
4	External Control	25 μl × 3 tubes
5	DNase/RNase free water	1,000 μl × 1 tube
6	Quick guide	1 ea

 

Technical Data

1. Limit of Detection (LOD) test

 

● Detection limit evaluation of the e-Myco™ VALiD-Q Real-time PCR Kit (v3.0 PRO)​

To evaluate the detection sensitivity of the assay, genomic DNA from ten quantified Mycoplasma strains(A. laidlawii, M. arginini, M. gallisepticum, M. fermentans, M. hyorhinis, M. orale, M. pneumoniae, M. salivarium, M. synoviae, and S. citri) was serially diluted to 10 CFU/mL and 1 CFU/mL. Nucleic acids were extracted directly from culture media and tested using the e-Myco™ VALiD-Q Real-time PCR Kit (v3.0 PRO). All species showed positive detection at the lowest dilution level, confirming a detection limit of 1 CFU/mL for each Mycoplasma species tested.

2. Specificity test

 

● ​Analytical specificity evaluation of the e-Myco™ VALiD-Q Real-time PCR Kit (v3.0 PRO)​ 

The analytical specificity was evaluated against six non-Mycoplasma strains according to the European Pharmacopoeia (EP) 2.6.7 guidelines. No amplification signals were observed in the non-target strains, confirming high analytical specificity.

3. Robustness test

 

● ​Reliability and reproducibility of the Mycoplasma Target (FAM) assay under different instrument, sample matrix,extraction methods, PCR reagents conditions​ 

Performance was evaluated for A. laidlawii (10 CFU/mL) across four experimental variables PCR instruments, nucleic acid extraction methods, sample matrices, and PCR conditions. Under all conditions, Ct values remained within the expected range, and the coefficient of variation (CV) was ≤5%, demonstrating stable performance. 

4. Dual-Fluorescence Control Ensuring Confidence in NAT Results

 

● ​Dual-channel External Control (EC) system for identifying true positives and carry-over contamination in the e-Myco™​ VALiD-Q Real-time PCR Kit (v3.0 PRO)​ 

Amplification curves represent : (1) True Positive (Sample), FAM-only amplification, (2) External Control Positive, FAM + Cy5 amplification generated from the EC template, (3) False Positive (PC carry-over), dual FAM/Cy5 fluorescence detected in a test sample due to contamination. Dual-channel positivity in a sample indicates carry-over of EC or positive-control material; such reactions are invalid and must be repeated.​