Comparison Mycoplasma test kits for cell culture (research to GMP)

If you want to purchase a mycoplasma test kit, you may use this overview and comparison of manufacturers and kits as decision aid. Such a comparison is highly recommended as the prices and detection limits as well as specificity tremendously vary. There are many kits on the market and some clearly are much better than others.

In addition, we also shortly outline the principles of the PCR and DAPI test methods for mycoplasma.

Zellkultur mit Mycoplasmen-Kontamination

The following topics are covered on this page:

Mycoplasma test methods

Mycoplasma detection and test kits

EuPharm listed species

Species found in cell cultures

Mycoplasma detection methods

Mycoplasma detection can be achieved by several different test methods:

Microbiological culture method (only industry or GMP due to complexity)
Indicator cell method using DAPI (research and GMP)
PCR detection (research and GMP)
PCR hybridization (only GMP due to price)
ELISA (low sensitivity only research)
Luminescence (low sensitivity only research)

Among these methods are two that are most recommendable due to price, ease, specificity and sensitivity and these are the DAPI stain and PCR. A DAPI or Hoechst stain is easy and fast even though it is harder to read-out. The PCR test is the state-of-the-art technique as it combines - in the best case - ease of performance, speed, price, sensitivity and specificity. Below we have crudely outlined how - in a research lab - you can perform mycoplasma tests using one of these two methods.

PCR detection

In the PCR example, we show how to test an FCS batch that was newly purchased. First you prepare a known clean cell culture. To this, you add medium containing the new FCS batch. Now you grow and passage the cells (best without antibiotics) for at least a week to give the potential mycoplasma contamination time to grow. Longer culture periods would be safer. Then, you take a media sample from this culture. After boiling for approximately, 5min the sample is ready for testing using a PCR kit like the ones listed below. After 3-4 hours you get the result as bands on a gel.

Zellkultur Mycoplasmen-Nachweis PCR

When performing a PCR detection of mycoplasmas in a GMP production surrounding, this very crude and basic protocol has to be changed in several aspects and controls have to be included. This is why research level kits have a true sensitivity of roughly 1.000 CFU/mL while GMP final product testing uses tests validated to have a sensitivity of 10 CFU/mL for all relevant species.

Detection by DAPI stain

The second example outlines the DAPI stain. You start off as described above with adding the test sample to a clean culture and culturing for at least 7 days. Then. you discard the medium, you wash and fix the cells with formaldehyde and stain them with DAPI or Hoechst solution. Now you can read out the result using a fluorescence microscope under the UV setting. As this method is much less sensitive it is better to prolong the culture period to 14-21 days in order to be really sure.

Zellkultur Mycoplasmen-Nachweis DAPI

To use this test in a GMP production surrounding not much more than the cultivation period has to be changed. Of course controls have to be included.

Mycoplasma detection and test kits

Supplier	Method/DL/GMP	Species / Test result type	Price	Remarks: Research kits
Example video	PCR-Gel <1000-2500 CFU GMP: -	28 (yes/no answer) = M. orale, M. arginini, M. hyorhinis, A. laidlawii, M. fermentans, M. pneumoniae, M. synoviae, (EuPharm 7 of 7-9) M. hominis, M. penetrans, M. pirum, U. urealyticum, M. pulmonis, M. falconis, M. arthritidis, M. spermatophilum, M. opalescens, M. primatum, M. maculosum, M. bovis, M. cloacale, M. hyosynoviae, M. salivarium, M. faucium, M. genitalium, M. bovigenitalium, M. sp. ovine/caprine, M. agalactica, M. timone,	<6,63	Positive control: yes Internal control: yes This is a strongly simplified and therefore, very fast and safe PCR kit. The tubes contain most reagents in lyophilized form. Only the master mix (which already includes the loading buffer), polymerase and sample have to be pipetted. This drastically reduces the number of pipetting steps. In addition, the positive control is already added to the tubes in red which helps to avoid false positive results and cross contamination. 3 pipetting steps per sample. This kit is not available any more, but shows the principle of the method. In our MOOC, we have used this kit to demonstrate the method of PCR in a Video .
Minerva	PCR-Gel <1000-2500 CFU GMP: -	28 (yes/no answer) = M. orale, M. arginini, M. hyorhinis, A. laidlawii, M. fermentans, M. pneumoniae, M. synoviae, (EuPharm 7 of 7-9) M. hominis, M. penetrans, M. pirum, U. urealyticum, M. pulmonis, M. falconis, M. arthritidis, M. spermatophilum, M. opalescens, M. primatum, M. maculosum, M. bovis, M. cloacale, M. hyosynoviae, M. salivarium, M. faucium, M. genitalium, M. bovigenitalium, M. sp. ovine/caprine, M. agalactica, M. timone,	<10,34	Positive control: yes Internal control: yes Simplified kit as described above with lyophilized reagents in tubes. VenorGeM Advance
Sigma	PCR-Gel <1000-2500 CFU GMP: -	19 (yes/no answer) = M. arginini, A. laidlawii, M. orale, M. fermentans, M. hyorhinis, (EuPharm 5 of 7-9) M. salivarium, M. hominis, M. agalactiae, M. arthritidis, M. bovis, M. cloacale, M. falconis, M. faucium, M. hyosynoviae, M. opalescens, M. primatum, M. pulmonis, M. spermatophilum, M. timone	<9,96	Positive control: yes Internal control: yes Simplified kit as described above with lyophilized reagents in tubes. LookOut^®
Supplier	Method/DL/GMP	Species / Test result type	Price	Remarks: No internal control
R&D Systems	Hybridization- ELISA ~15 - 2500 CFU GMP: -	8 (yes/no answer) = A. laidlawii, M. orale, M. arginini,* M. hyorhinis, M. fermentans (EuPharm 5 of 7-9)* M. hominis M. pirum M. salivarium	~4	Positive control: yes Internal control: no With this kit you detect mycoplasma rRNA in a colorimetric ELISA. The assay cross reacts with Lactobacillus and therefore, is not mycoplasma-specific. In addition the sensitivity is lower. MycoProbe^®
Agilent	PCR-Gel n.a. GMP: -	Description states "most" (yes/no answer, 5 identified)	~9,8	Positive control: yes Internal control: no This kit only contains the primers. No polymerase or master mix are included. After a PCR, 5 species can be identified in a restriction digest run on a gel. The product information does not state what is meant with "most" species. Inquire and compare to the lists below! The internal control provided is a mycoplasma DNA and therefore only serves as a PCR control. Mycoplasma Plus Primer set
Supplier	Method/DL/GMP	Species / Test result type	Price	Remarks: Research kits
Greiner BioOne	PCR- Mikroarray <10 CFU GMP: +	all known species (yes/no answer), (EuPharm 9 of 7-9) 40 (identifiziert)	>560	Positive control: yes Internal control: yes Pharma-Kit developed and used as a final product release test. It is fully validated by the supplier and contains all necessary and several additional controls. Due to the double method principle, all known mycoplasma species can be detected with a yes/no answer and additionally 40 species are identified on the species level. In addition to the PCR equipment, other machinery has to be purchased to run the test. Widely used in pharma industry. Cytoinspect^TM
Roche	PCR-Gel <10 CFU GMP: +	all known species (yes/no answer), (EuPharm ? of 7-9)	>500	Positive control: yes Internal control: yes This regular PCR Pharma-Kit uses universal PCR primers that should react with all known mycoplasma species. It gives out only yes/no answers but on the other hand only PCR equipment is used. This kit was developed by Genentech for testing Roche products and has then been launched as a test kit. The validation of this kit has some flaws but it was approved for Roche production by the EMA. Revalidation is strongly recommended. MycoTool

* Price is price per sample for a kit size ~ 25 samples.

Mycoplasma species listed in the EuPharm.:

The European Pharmacopoeia precisely lists how to test for mycoplasma contamination in sterile products and which species to use for validation and verification. Depending on what you test, the detection limit has to be 10-100 CFU per mL. Depending on the product, one of these control species is run as a positive control in all batch tests. Purified mycoplasma DNA should never be used as a GMP positive control as it does not realistically reflect the results obtained with intact mycoplasma as a positive control.

A. laidlawii
M. fermentans
M. hyorhinis (where cell-culture enrichment is used, a fastidious strain such as ATCC 29052 is included)
M. orale
M. pneumoniae or M. gallisepticum
M. synoviae (where there is use of or exposure to avian material during production)
Mycoplasma arginini
Spiroplasma citri (where there is use of or exposure to insect or plant material during production).

The most important and relevant species for culture

There are several test systems and test kits on the market. Several of these detect only a limited number of species. Make sure that the species below are in the detection range (specificity and sensitivity) of the kit you purchase. There are several kits that are cheap and have a wide range of detection.

M. orale
M. hyorhinis
M. arginini
M. fermentans
M. salivarium
M. hominis