Microbial Detection and Elimination

Transcription

1

2 138 Contents Chapter 8 8 Microbial Detection and 8.1 Mycoplasma Test Kits Mycoplasma Solutions Bacteria Test Kit Disinfectants

3 Mycoplasma Test Kits Mycoplasma detection in cell culture, cell/virus stocks, and medium Conventional and real-time PCR Highly sensitive (detects mycoplasmas per sample volume) Fast, reproducible, and reliable results Applicable for all cell/tissue culturerelevant mycoplasma species Positive and internal controls included Convenient protocol and easy sample preparation Mycoplasmas are parasitic bacteria that frequently contaminate tissue cultures and consequently also virus stocks, vaccines, and other biological materials produced in cells. The contamination can originate from serum, laboratory staff, other contaminated cultures, or the donors from which the cells have been harvested, and it often remains unnoticed. Although mycoplasmas do not cause visible cell damage, they seriously affect cell growth and metabolism, transfection efficiency, protein and monoclonal antibody synthesis, cytokine secretion, immunological properties, signal transduction, virus proliferation and can even cause DNA and RNA damage. Since mycoplasmas are very small and lack cell walls, detection using a conventional light microscope is nearly impossible. In addition, cloudiness in the medium, typical for bacterial or yeast contaminations, does not occur even at high mycoplasma concentrations. PCR on the other hand is a highly sensitive, specific, and rapid method for the detection of mycoplasma contamination in culture supernatant, cell/virus stocks, and medium. All cell/ tissue culture relevant mycoplasma species can be detected in one assay including M. fermentans, M. hyorhinis, M. arginini, M. orale, M. salivarium, M. hominis, M. pulmonis, and M. pirum. In addition, M. pneumoniae, Acholeplasma laidlawii, M. synoviae, and Ureaplasma species can be detected. PCR Mycoplasma Test Kit I/C The PCR Mycoplasma Test Kit I/C utilizes conventional PCR technology for convenient and specific detection of more than 25 contaminating mycoplasma species. The primer set is specific for the highly conserved 16S-rRNA coding region in the mycoplasma genome. The test can be performed in 3 hours and includes internal control DNA (for verifying a successful PCR run) as well as positive control DNA. All components required for the PCR (primers, nucleotides, internal control DNA) come optimally premixed and lyophilized in PCR tubes (except the Taq polymerase). For setting up the PCR, simply cut off the number of tubes required, rehydrate with rehydration buffer (containing the Hot-Start Taq polymerase), and add the sample to be analyzed. Samples can be easily prepared by boiling the cell culture supernatant or other sample material. PCR Mycoplasma Test Kit I/RT The PCR Mycoplasma Test Kit I/RT utilizes real-time PCR technology for convenient and reproducible detection of contaminating mycoplasmas. It shows excellent sensitivity and specificity and enables reliable, quantitative PCR results extremely fast, particularly for routine testing for mycoplasma contamination. The primer and probe set is specific for the highly conserved 16S-rRNA coding region in the mycoplasma genome. It contains FRET probes emitting fluorescent light at 520 nm and is compatible with most common Real-Time PCR cyclers (e.g. Roche Light Cycler 1 and 2, ABI Prism, Cepheid Smart Cycler, and Eppendorf Mastercycler ep realplex). The test includes internal control DNA (for verifying a successful PCR run) as well as positive control DNA. All components required for real time PCR (primers, probes, nucleotides, and internal control DNA) come lyophilized in PCR tubes, except the Hot-Start Taq polymerase which is dissolved in reaction buffer. Two kit variants (kit A for e.g. Light Cycler, kit B for e.g. ABI Prism) are provided which have been optimized for different Real Time PCR instruments. For recommended kit variant, please see where you can download the manual. PCR 100 µl cell culture supernatant Boil for 10 minutes and centrifuge briefly 2 µl supernatant Analyze 5 µl of the PCR sample Fig. 1: Mycoplasma detection with the PromoKine PCR Mycoplasma Test Kit I/C is easy and convenient: Boil 100 µl cell culture supernatant or other sample material for 10 minutes; centrifuge briefly and add 2 µl supernatant to the reaction cup with the rehydrated PCR components and Taq polymerase; start PCR run; analyze PCR products by agarose gel electrophoresis. When using the PCR Mycoplasma Test Kit II, the sample is directly added to the ready-to-use, complete reaction mix to set up the PCR.

4 140 PCR Mycoplasma Test Kit III The PCR Mycoplasma Test Kit III utilizes conventional PCR technology for convenient and highly sensitive detection of mycoplasma. The primer set is specific for the highly conserved 16S-rRNA coding region in the mycoplasma genome. Thus, the kit reliably detects all mycoplasma commonly found in cell culture, including the Acholeplasma and Spiroplasma genera. The kit has been positively tested against all mycoplasma strains listed in the European Pharmacopoeia, but may detect even more mycoplasma species (~ species). It does not detect microorganisms such as Streptococcus sp., Clostridium sp. and Lactobacillus sp. which are phylogenetically closely related. The kit includes all components required for PCR (nucleotides, primers, a highperformance Taq polymerase, and an agarose gel loading dye), all premixed at optimal concentrations and in a ready-touse and ready-to-load 1x master mix. The test takes approximately 3 hours to run and obtain clear results. The kit also contains positive control DNA for easy result verification as well as an internal control DNA to verify a successful PCR run. M M 479 bp 270 bp 260 bp 160 bp Fig. 2 Top: Detection of mycoplasmas with PCR Mycoplasma Test Kit I/C. M: 100 bp DNA ladder 1: Positive control 2-6: 10; 100; 1,000; 10,000; 100,000 mycoplasmas/sample volume Internal control DNA: 479 bp band Positive sample/control: 270 bp band Bottom: Detection of mycoplasmas with PCR Mycoplasma Test Kit III. 1-4: A. laidlawii, S. citri, M. synoviae, and M. orale (50 cfu/ml each) 5: Cell culture supernatant, not infected 6: Negative control 7: Positive control M: DNA size standard Internal control DNA: 160 bp band Positive sample/control: bp band Product Size Catalog Number PCR Mycoplasma Test Kit I/C 24 tests 48 tests 96 tests PK-CA PK-CA PK-CA PCR Mycoplasma Test Kit III 100 assays PK-CA PCR Mycoplasma Test Kit I/RT; Variant A 25 assays PK-CA A PCR Mycoplasma Test Kit I/RT; Variant B 25 assays PK-CA B RTU DNA Ladder I (100 bp) 0.5 ml PK-MB N RTU DNA Ladder II (1000 bp) 0.5 ml PK-MB N For prices please see our online shop at Please see Chapter 1.11 for our ready-to-use DNA Ladders. PromoKine also provides fluorescent stains for mycoplasma detection, please see Chapter Mycoplasma Solutions Mycoplasma contamination seriously af- are disinfected (see chapter 8.4 for dis- not affect mycoplasmas. Therefore, fects cell growth and cell physiology lead- infectant sprays). However, if the af- PromoKine offers two highly effective ing to biased and unreliable experimental fected cells are unique or the result of methods to get rid of mycoplasma con- results. When mycoplasma contamination is detected, the contaminated cells are ideally discarded and all equipment, e.g. work benches, CO 2 incubators, etc., extensive work, thorough elimination of the contaminating mycoplasmas is necessary. Standard antibiotics commonly used in cell culture (e.g. Pen/Strep) do tamination: Mycoplasma-EX, a combination of non-antibiotic and antibiotic reagents, and BIOMYC 1-3, three highly effective antibiotics.

5 141 Mycoplasma-EX Mycoplasma elimination in cell/tissue culture Mycoplasma elimination in virus stocks Highly effective against mycoplasma species commonly found in cell cultures Very short treatment time Very low cytotoxicity Ready to use Easy handling Mycoplasma-EX combines a non-antibiotic and a thoroughly adjusted antibiotic treatment for the effective elimination of mycoplasmas and other microorganisms in cell/tissue culture and virus stocks. It eliminates mycoplasmas by directly killing them and not just by inhibiting their growth, while showing extremely low toxicity for the cells in culture. Mycoplasma-EX is effective with only one treatment, destroying mycoplasmas rapidly and permanently. It is applicable for most cell lines (e.g. Vero, BHK21, GBK, ML, Hep2, 293, CRFK, H9, Molt4, MT-4, Jurkat) as well as virus stocks (e.g. SHV-1, BHV-1, HSV-1, VSV, SFV, FCV, MEV) and highly effective against M. orale, M. hyorhinis, M. hominis, Acholeplasma laidlawii, M. arginini, M. fermentans, and other mycoplasma species usually encountered as contaminants in cell cultures. BIOMYC Antibiotic Solutions Mycoplasma elimination in cell/tissue culture Highly effective against mycoplasma species commonly found in cell cultures Low cytotoxicity Ready to use Easy handling Antibiotics that affect bacterial cell walls (e.g. penicillin) are ineffective against mycoplasmas, which lack a cell wall. Other antibiotics such as tylosin, neomycin, tetracycline, or gentamicin are only effective against a few mycoplasma species commonly found in cell cultures. In addition, they frequently just reduce the concentration of mycoplasmas rather than completely eliminating them. As soon as treatment is discontinued, contamination will recur. BIOMYC-1 and 2 BIOMYC-1 and 2 are two antibiotics, which eliminate mycoplasmas very efficiently when used in combination. After a four-day treatment with BIOMYC-1, a three-day treatment with BIOMYC-2 follows. This is repeated 2 to 3 times. Following this protocol, the mycoplasmas do not develop resistance to these antibiotics - a common occurrence with other antibiotic treatment methods. BIOMYC-1 is based on the antibiotic tiamutin which is produced by the fungus Pleurotus mutilus. BIOMYC-2 is based on the antibiotic minocycline, an improved derivative of tetracycline. BIOMYC-3 BIOMYC-3 is based on the antibiotic ciprofloxacin, which is a member of the fluoroquinolone group and inhibits synthesis of DNA gyrase, an enzyme necessary for bacterial DNA supercoiling. Many mycoplasma species are sensitive to BIOMYC-3, including M. orale, M. hyorhinis, M. fermentans, and M. arginini, but also Acheloplasma laidlawii. These species are responsible for most mycoplasma contaminations in cell cultures. At the concentrations recommended for use, no cytotoxic effects on the cells in culture have been found and the treatment can be performed within 12 days. Fig. 3: Electron microscope image of cells contaminated with mycoplasmas. Picture kindly provided by M. Ebert, Forschungszentrum für Medizintechnik und Biotechnologie, Erfurt, Germany. Product Size Catalog Number Mycoplasma-EX 3 treatments PK-CC BIOMYC-1 10 ml 20 ml 100 ml PK-CC D PK-CC C PK-CC B BIOMYC-2 10 ml 20 ml 100 ml PK-CC D PK-CC C PK-CC B BIOMYC-3 For prices please see our online shop at 10 ml 20 ml 100 ml PK-CC D PK-CC C PK-CC B

6 Bacteria Test Kit Eubacteria detection in cell culture Eubacteria detection in cell/virus stocks Eubacteria detection in medium Highly sensitive (detects 12 eubacteria genomes per sample volume) Reproducible and reliable results Applicable for 45 eubacteria species Positive and internal controls included Convenient protocol and easy sample preparation Bacteria are frequently found as contaminants in cell cultures. However, cell cultures sometimes lack visual signs of bacterial contamination, e.g. fast decoloration of the medium. Moreover, it has been demonstrated that standard antibiotics are mostly ineffective against resistant bacterial infection but have a strong impact on the metabolism, growth, and differentiation of the cells in culture. Frequent screening of cell cultures for eubacteria contamination is indispensable to obtain reliable results. Therefore, PromoKine offers a PCR-based Bacteria Test Kit that has been developed for accurate and reliable in vitro diagnosis of 45 eubacteria species, many of them found as usual contaminants in cell cultures (e.g. Actinomyces, Bacillus, Enterococcus, Escherichia coli, Fusobacterium, Klebsiella, Lactobacillus, Micrococcus, Mycobacterium, Peptostreptococcus, Pseudomonas, Porphyromonas, Prevotella, Salmonella, Serratia, Staphylococcus, Streptococcus, etc.). The kit provides a highly sensitive, easy, and rapid method to detect eubacteria infection in various in situ biological materials, including cell cultures. Detection requires as little as 12 eubacteria genomes (~52 fg) per sample volume. The protocol can be completed within 3.5 hours with a hands-on time of less than 40 minutes. The kit contains all necessary components for setting up a PCR including a positive control DNA as well as an internal control DNA to confirm a successfully performed PCR run. The kit does not amplify eukaryotic DNA. PCR µl cell culture supernatant Extract Bacterial DNA 5 µl DNA sample Analyze 5 µl of the PCR sample Fig. 4: Eubacteria detection with the PromoKine PCR Bacteria Test Kit is easy and convenient: Purify bacterial DNA from µl cell culture supernatant by using an appropriate, commercial DNA extraction kit; add 5 µl sample DNA to the reaction cup with the premixed PCR components and Taq polymerase; start PCR run; analyze PCR products by agarose gel electrophoresis. Product Size Catalog Number PCR Bacteria Test Kit 48 tests PK-CA For prices please see our online shop at PromoKine also offers a Bacteria Live/Dead Staining Kit as well as fluorescent dyes for staining of bacteria. Please see Chapter 1.

7