How to Test for Mycoplasma in Cell Culture: The Progression from Culture-Based Methods to Rapid Molecular Detection
ARTICLE
How mycoplasma testing in cell culture has evolved through four eras of detection technology, from microbiological culture to rapid molecular methods, and what each is useful for in research, bioprocess, and regulated settings.
Mycoplasma contamination is one of the longest-standing and most consequential quality issues in cell culture. Mycoplasma species are very small (typically 0.1–0.5 µm), lack a cell wall, and replicate slowly without producing the visible turbidity that signals other bacterial contamination. An infected culture can continue to grow and yield apparently normal experimental data while the contamination remains undetected — often for weeks or months. Estimates of contamination prevalence in cell cultures used in research and biopharmaceutical work range from approximately 15% to 35%, depending on the study population and the detection method employed.
Six Mollicutes species account for approximately 95% of all detected cell-culture contaminations: M. orale, M. fermentans, and M. hominis (human origin); M. arginini and A. laidlawii (bovine origin); and M. hyorhinis (porcine origin). Even in labs with best-practice procedures, inadvertent contamination from human operators is commonplace, as Mycoplasma cells can reside in the human oral cavity as commensal organisms, or in widely used materials such as fetal bovine serum or trypsin.
This article focuses on the testing methods used to monitor and prevent mycoplasma contamination in routine research and bioprocess laboratory work — the volume application that drives the great majority of mycoplasma testing performed worldwide. An important subset of mycoplasma testing exists for pharmacopoeia-compliant batch release of biological drug products; that context is referenced in each section where it has shaped the method's development or adoption, but it is not the primary focus.
Detection methods for cell-culture mycoplasma have evolved through four broad eras: microbiological methods; biochemical / enzymatic detection; polymerase chain reaction (PCR) including quantitative PCR (qPCR); and isothermal amplification with lateral flow or visual readout. Each generation has added capability, but trade-offs between sensitivity, time-to-result, equipment dependence, throughput and operational complexity have meant that no single method has displaced the others entirely. The sections below describe each era and the products that represent it, and conclude by highlighting an important recent development in the field with the launch of Flip Mycoplasma.
Microbiological Methods
The historic gold standard for mycoplasma detection involved microbiological methods. Such techniques were the only available approach when mycoplasma was first identified as a cell-culture contaminant in the mid-twentieth century, and remained the primary approach for decades. The culture method involves inoculation of test samples into mycoplasma-permissive broth, followed by subculture onto agar plates and incubation under specified conditions. Suspected colonies are then assessed for the characteristic "fried egg" colony morphology. The indicator method, where suspect samples are grown with mammalian host cells before confirmatory DNA staining (Hoechst 33258, DAPI), was a complementary technique used to expand coverage to poorly culturable Mycoplasma species. These methods were subsequently codified in the pharmacopoeia — the European Pharmacopoeia chapter 2.6.7, the US Pharmacopoeia chapter <63>, and the Japanese Pharmacopoeia G3 — and became the compendial reference method for pharmacopoeia-compliant release testing.
The strengths of microbiological testing are that it detects only viable organisms (the relevant question when assessing infectivity risk in a finished biologic drug) and that, being based on growth rather than amplification, it sidesteps the assay-design considerations that introduce performance variability into molecular methods.
The weakness is timing. A complete mycoplasma culture assay takes approximately 28 days. As such they have never been widely adopted for routine cell-culture applications in the research laboratory. In these settings where experiments can be concluded within days and increasing numbers of valuable engineered cell lines are maintained in conventional or even 3D culture, a 28-day turnaround is increasingly incompatible with modern cell biology workflows.
DNA staining methods such as Hoechst 33258 fluorescence — once used as a faster supplementary check alongside or in place of culture — have largely fallen out of routine use. They lack the sensitivity to detect low-level contamination and depend heavily on operator interpretation under microscopy.
Biochemical Detection
Biochemical or enzymatic detection, of which Lonza's MycoAlert assay is the most widely used commercial example, operates on a different principle from culture or amplification. The assay exploits the accumulation of mycoplasma-specific metabolic enzymes that convert ADP to ATP in the culture media; the released ATP is detected via luciferase-driven luminescence. A pre-substrate / post-substrate luminescence ratio distinguishes mycoplasma-derived signal from background ATP in the sample.
The assay completes in 20–30 minutes, does not require nucleic acid extraction, and detects a broad range of mycoplasma species through shared enzymatic activity. Its limitations are sensitivity, which is generally lower than well-designed PCR assays; the indirect nature of detection (an enzyme-activity readout rather than direct molecular specificity for the mycoplasma genome); equipment dependence, since a luminometer is required and is less ubiquitous in cell-culture laboratories than a thermal cycler; reagent storage at −20 °C, which complicates use in laboratories without dedicated freezer capacity at the bench; and dependence on signal-to-noise discrimination from background sample ATP, which can be unstable in some sample types.
Biochemical methods have not, to our knowledge, been specified or used as the primary detection method in pharmacopoeia-compliant batch release testing. The combination of lower analytical sensitivity and indirect, enzyme-activity-based detection has kept the method confined to routine screening rather than regulated release. Biochemical detection remains in use in some labs as a screening tool in routine work due to its speed, but has been largely overtaken in volume by more sensitive PCR-based testing as PCR equipment and reagents have become more widely accessible.
PCR and qPCR
The introduction of PCR into routine mycoplasma testing in the 1990s and 2000s was a step-change in capability. PCR amplifies a defined nucleic acid target by repeated cycles of denaturation, primer annealing, and extension; well-designed assays can achieve analytical sensitivities below 10 CFU/mL. Real-time quantitative PCR (qPCR) integrates a fluorescent readout that enables quantification and removes the agarose-gel electrophoresis step required by endpoint PCR. PCR-based detection is currently the most widely used method for routine mycoplasma testing.
"PCR-based detection" is a chemistry family, not a quality designation. Commercial mycoplasma PCR kits span a wide analytical range: reported sensitivities run from below 10 CFU/mL for rigorously validated probe-based methods to substantially higher figures for less well-characterised products. Performance varies with primer design, target region, control strategy, and — significantly — the extent of upstream sample preparation. Two products in the same chemistry family can differ in real-world sensitivity by more than an order of magnitude as a result of these factors.
Target choice within the family also varies. Most commercial mycoplasma PCR kits target conserved regions of the rRNA operon — the 16S rRNA gene, the 23S rRNA gene, or the 16S–23S intergenic spacer region (ISR). The ISR offers higher inter-species discriminatory power than the 16S coding region alone; the 16S region is more deeply conserved across mollicute species. Different manufacturers have resolved this trade-off differently. Examples in routine use include Minerva Biolabs' Venor®GeM Classic (conventional endpoint PCR with a 16S rRNA gene target) and Roche's MycoTOOL Real-Time PCR Kit (qPCR, optimised for CHO release testing).
Higher analytical sensitivity within the PCR family is achieved by several routes, often in combination. The first is rigorous sample preparation — extraction, purification, and concentration steps designed to remove PCR inhibitors and enrich target nucleic acid. The second is optimised primer and probe chemistry. The third is the addition of a reverse-transcription step prior to amplification, which makes mycoplasma ribosomal RNA accessible to amplification alongside genomic DNA.
PCR has also become the focus of an important regulatory shift. The European Pharmacopoeia 2.6.7 chapter, updated in Issue 12.2 and effective from 1 April 2026, explicitly accepts nucleic acid amplification techniques — qPCR, RT-qPCR, dPCR — as primary methods for mycoplasma testing. This is a substantive change from the previous convention, under which culture was the primary method and PCR was an accepted alternative. The revised chapter also brings tighter definition to PCR-based testing: it retains the limit-of-detection requirement of ≤10 CFU/mL when the nucleic acid amplification technique replaces culture; introduces standardised reference materials at defined sensitivity points (10 CFU/mL and 100 CFU/mL) to support consistent validation across assays and laboratories; and reaffirms that any test deployed for pharmacopoeia-compliant testing must be validated in process by the drug manufacturer — the manufacturer cannot rely on the test supplier's published performance claims. Some DNA-only conventional PCR assays validated against earlier sensitivity bars are no longer suitable for compliant testing under the revised chapter. Suppliers are responding by retiring older kits: Minerva, for example, is phasing out Venor®GeM Classic in favour of a new Venor® Mycoplasma qPCR product that incorporates reverse transcription and is priced at approximately five times the kit it replaces.
Equivalent changes are in preparation in the USA, with the new USP chapter <77> (Mycoplasma Nucleic Acid Amplification Test) scheduled to become official on 1 October 2026. This chapter formalises the use of NAT/PCR-based methods as an alternative to traditional culture methods when appropriately validated.
For routine (non-regulated) use, the practical disadvantage of PCR-based detection is operational rather than analytical. PCR typically requires upstream nucleic acid extraction or purification of the sample, a thermal cycler, calibrated pipetting, refrigerated or frozen reagent storage, separate amplification and detection areas to prevent carry-over contamination, and trained operators. Total time from sample to result is typically two to five hours when sample preparation and gel analysis is included. These factors make PCR well-suited to centralised quality-control laboratories running batches of samples, but less well-suited to bench-side screening during routine passaging in the research laboratory. Whilst qPCR represents a faster solution and overcomes the cumbersome requirements for gel analysis of PCR assays, these tests are typically more expensive and further require a dedicated fluorescence instrument.
Isothermal Amplification with Lateral Flow or Visual Readout
Isothermal amplification chemistries — including loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), and a range of other methods — achieve molecular amplification at a single constant temperature, without the thermal cycling required by PCR. Amplification proceeds in a basic heat block, water bath, or PCR machine used as a heat block. When the amplification step is paired with a lateral flow strip or with a colorimetric tube readout, the entire workflow becomes equipment-light: heat source, pipette, and visual interpretation.
Such isothermal amplification tests for Mycoplasma contamination have started to become available over the last few years and there are now multiple cell-culture mycoplasma products available:
- InvivoGen's MycoStrip® uses LAMP-based amplification at 65 °C with a lateral flow readout, targeting the 16S rRNA gene DNA. Sample preparation plus assay totals approximately 70 minutes.
- AssayGenie's MycoGenie uses isothermal amplification with a colorimetric tube readout. Reported detection limit is 5 × 105 CFU/mL; total assay time approximately one hour.
- Vazyme's MycoBlue and Mycolor One-Step kits use isothermal amplification with colorimetric tube readout at 65 °C for one hour. Reported sensitivity of approximately 5 × 105 CFU/mL.
- MP Biomedicals' Myco-Visible uses LAMP with a visual colorimetric readout.
Despite the workflow promise of the category, adoption has remained modest relative to PCR. The reason is visible in the published performance characteristics across these products. The colorimetric implementations report analytical sensitivities four to five orders of magnitude below the 10 CFU/mL achievable with the best PCR assays; in practical terms, they detect high copy number contamination reliably but cannot match PCR for early or low-level detection. Whilst the simplicity of a heat-block and visual read-out ostensibly allows isothermal tests to overcome important limitations of PCR assays, the fact that such tests still take over 1 hour to perform and have inconsistent performance has hampered their adoption to date.
Isothermal methods have not been used for pharmacopoeia-compliant batch release testing to date. Two factors explain this: the category is relatively new and lacks the regulatory pedigree of culture and PCR; and the key advantages of isothermal amplification — speed, low equipment requirement, and bench-side workflow — are of less relevance in regulated batch release, where the operational cycle is measured in days or weeks and where centralised laboratory infrastructure is already in place. The natural market for isothermal mycoplasma testing is routine screening in research and bioprocess laboratories, where speed and equipment-independence are the binding constraints.
Until recently, no commercial product in the routine-testing isothermal category combined PCR-equivalent analytical sensitivity with a true direct-from-sample workflow at the bench in under 15 minutes. That gap is the context for the recent launch of Fuse Diagnostics' Flip Mycoplasma.
Flip Mycoplasma
Flip Mycoplasma, launched commercially in September 2025, is Fuse Diagnostics' isothermal-amplification and lateral-flow assay for cell-culture mycoplasma testing. The assay incorporates a reverse-transcriptase enzyme to generate cDNA from mycoplasma 16S rRNA, which is then amplified and detected on an integrated lateral flow strip by Fuse's proprietary chemistry. The amplification runs on a standard 0.2 mL heat block in just 10 minutes at 50 °C, without thermal cycling. The lateral flow read-out then takes just another two minutes, overcoming the need for gel electrophoresis, and the visual read-out means no fluorescence detection equipment.
The published performance characteristics of Flip Mycoplasma are summarised below. Full details are reported in the Flip Mycoplasma performance characteristics study.
Table 1 — Flip Mycoplasma Published Performance Characteristics
| Specification | Value |
|---|---|
| Analytical sensitivity (LOD) | 10 CFU/mL (M. orale) |
| Time to result | 12 minutes total (10 min amplification + 2 min readout) |
| Hands-on time | <1 minute |
| Sample volume | 30 µL, direct from culture supernatant or FBS |
| Equipment required | 0.2 mL-compatible heat block at 50 °C (or PCR machine used as heat block); optional Flip Nest USB-C heat block |
| Reagent storage | Ambient temperature (lyophilised) |
| Species detection (wet-validated) | Six species responsible for >95% of cell-culture contaminations: M. arginini, M. orale, M. fermentans, M. hominis, M. hyorhinis, A. laidlawii |
| Inclusivity (in-silico) | Sequence conservation analysis across 111+ Mycoplasma species |
| Cost per test | Approximately £10 |
| Regulatory status | Research use only (RUO) |
The 10 CFU/mL published sensitivity is comparable to the sensitivity threshold set by the European Pharmacopoeia 2.6.7 (Edition 12.2) for nucleic acid amplification methods, although Flip Mycoplasma has not been validated against the chapter's requirements. A future validation pathway is not implausible given the published performance, but the regulatory mechanics are worth noting: any test deployed for pharmacopoeia-compliant batch release testing must be validated in process by the drug manufacturer in their specific sample matrix, and the manufacturer cannot rely on the test supplier's published claims. Flip is currently positioned for routine research and bioprocess laboratory work, not for compliant release testing.
High sensitivity in Flip arises from a combination of optimised primer and probe design, the proprietary isothermal chemistry, and reverse-transcription-based amplification of ribosomal RNA. Reverse transcription is one of several routes to high analytical sensitivity rather than a unique advantage of any particular product; the relevant observation is that Flip achieves PCR-class sensitivity in a workflow that does not require thermal cycling or extensive sample preparation.
The sample workflow requires no upstream DNA extraction, nucleic acid purification, or culture passage. 30 µL of cell-culture supernatant is pipetted directly into the assay tube, and the reaction proceeds in a single closed device. The sealed device avoids release of amplicon, which is a significant problem with other combination lateral flow test designs. This workflow contrasts with compendial culture, which depends on passage through liquid and solid media; with most commercial PCR kits, which require an upstream extraction step supplied either as part of the kit or as a separate consumable; and with several other isothermal products in the category, which include sample-preparation steps prior to amplification.
The reagent format is lyophilised, allowing ambient-temperature storage. This removes the cold-chain and dedicated freezer requirements common to enzymatic assays — which typically require −20 °C storage of the substrate and reagent components — and to PCR assays, where most kits require refrigerated or frozen reagent storage.
As is typical of other tests in the field, the species inclusivity claim is reported separately for wet-validated and in silico coverage. Six species have been experimentally tested and have generated analytical performance data; these are the species responsible for the great majority of cell-culture contaminations recorded in the literature. Beyond these six, sequence conservation analysis across more than 111 additional Mycoplasma species has been confirmed.
Flip is designed for rapid bench-side testing with low throughput. Laboratories running large sample batches in a single run may find plate-based qPCR more efficient at scale, where the setup overhead is amortised across many samples; Flip is intended for routine screening rather than batch QC, and its workflow is optimised accordingly. Its ease of use and speed mean that more regular testing can now be done in settings where previously testing has been unsuitable or undesirable.
Choosing the Right Method for Your Laboratory
Method choice depends on the question being asked. The table below summarises a practical mapping between use case and method category for routine research and bioprocess laboratory work, with pharmacopoeia-compliant release testing called out separately.
Table 2 — Method Selection by Use Case
| Use Case | Recommended Approach |
|---|---|
| Routine screening in research, low-to-moderate throughput | Isothermal amplification with lateral flow readout, where the assay achieves PCR-class analytical sensitivity in a direct-from-sample workflow. Flip Mycoplasma is the current commercial example of this combination. |
| High-throughput screening (large sample batches in one run) | Plate-based qPCR (96-well or higher). Setup overhead is amortised across many samples; bench-format isothermal assays are not designed for batched workflows. |
| Pharmacopoeia-compliant batch release | Compendial culture per EP 2.6.7 remains accepted in all jurisdictions. Validated NAT methods (qPCR, RT-qPCR, dPCR) are now permitted as primary methods under EP 2.6.7 Edition 12.2 from 1 April 2026 onwards and USP chapter <77> from 1 October 2026, subject to in-process validation by the drug manufacturer. Isothermal amplification methods have not been used in this context to date. |
For routine screening — the application that drives most of a cell-culture laboratory's mycoplasma testing volume — isothermal amplification with lateral flow readout offers the best fit between sensitivity, workflow, and laboratory practicalities, provided the chosen product achieves the required analytical sensitivity together with the potential benefits of speed and ease-of-use. The workflow is bench-side rather than central-lab, the equipment requirement is minimal, time-to-result is short enough that the answer is available before the cells are next handled, and with certain tests such as Flip Mycoplasma reagent storage is at ambient temperature.
For pharmacopoeia-compliant release testing, the position remains conservative. Culture remains the historic reference and is still accepted everywhere. The EP 2.6.7 Edition 12.2 and USP chapter <77> updates broaden the acceptance of nucleic acid amplification methods substantially and are the most important recent change in the regulatory landscape, but in-process validation by the drug manufacturer remains required regardless of the test supplier's published claims.
Evaluate Flip Mycoplasma in your laboratory. Fuse Diagnostics supplies free evaluation kits to research groups. The full analytical performance summary is available on the Flip Mycoplasma product page, contact us at info@fuse-dx.com if you would like further information or to request a quote.
Flip Mycoplasma is supplied for research use only and is not intended for diagnostic use. Performance characteristics quoted are taken from the published Flip Mycoplasma validation study (FPR003-REC-MAR-00204).
