e-Myco - Bulldog Bio
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1. Mycoplasma Detection limit e K562 cell M fermentans infected small cell numbers such as 12 cells e K562 gDNA M fermentans infected small quantities such as 3 25 pg e M fermentans small copy numbers such as 20 cfu ml 1 Result for the various concentration of template DNA 1 dilution eee M12 3 4 5 6 7 8 9 10 11 12 13 1415 IC Internal control Fig 1 Mycoplasma detection was performed for genomic DNA Genomic DNA was isolated from M fermentans infected K562 using the i genomic CTB DNA Extraction Mini Kit 17341 The isolated gDNA was serially diluted for PCR of mycoplasma detection These results show that it can be applied to mycoplasma detection with small quantities such as 3 25 pg of gDNA Lane M 100bp DNA Marker lane IC Internal control lane 1 50 ng lane 2 25 ng lane 3 12 5 ng lane 4 6 3 ng lane 5 3 2 ng lane 6 1 6 ng lane 7 800 pg lane 8 400 pg lane 9 200 pg lane 10 100 pg lane 11 50 pg lane 12 25 pg lane 13 12 5 pg lane 14 6 3 pg lane 15 3 25 pg 2 Result for the various cell number dilution M 12 3 45 6 7 8 9 10 11 12 13 14 15 IC z tt 1 1 44 44 7 7 e2ererr rer ee Fig 2 Mycoplasma detection was performed using the e Myco Mycoplasma PCR Detection Kit ver 2 0 method Mycoplasma detection from cell lysates of M fermentans infected K562 using the e Myco Mycoplasma Detection Kit ver 2 0 The M fermentans infected K562 cells were serially diluted2. Designed Primer A laidiawii 61 aaggtcagtg ctgcagttaa cgcattaagt tctccgcctg agtagtacgt The target regions in this kit are divided into o oe M arginini 101 gctcgcaaga gtgaaactta aaggaattga cggggacccg cacaagcget seven M types M1 M7 and one A type for M3 16T gt C M faucium 101 tcgcaagagt gaaacttaaa ggaattgacg gggacccgca caagcggtgg detecting the bulk of the species in the genus M4 17C gt T M fermentans 101 gttcgcaaga ataaaactta aaggaattga cggggatccg cacaagcggt mycoplasma The designed primers are sufficient M5 18T gt C M hyorhinis 101 atgctcgcaa gagtgaaact taaaggaatt gacgggaacc cgcacaagcg to detect major contaminants in cell cultures such M6 18T gt C 20T gt A M orale 101 ctcgcaagag tgaaacttaa aggaattgac ggggacccgc acaagcggtg as M arginini M faucium M fermentans M M7 8T gt C A laidawii 101 acgcaagtat gaaactcaaa ggaattgacg ggaccccgca caagcggtgg hyorhinis M orale and A laidlawii as well as A A laidlawii only other broad species of mycoplasma Not revealed primer sequences M arginini 151 geagcatete etttaattte aagatacecge gagaacctta cccactctte M faucium 151 agcatgtggt ttaatttgaa gatacgcgga gaaccttacc cactcttgac i M fermentans 151 ggagcatgtg gtttaatttg aagatacgcg tagaacctta cccactcttg gt POR Product size Type PCR Size M hyorhinis 151 gtggagcatg tggtttaatt tgaagatacg cgtagaacct tacccactct The size of DNA fragments that are amplified 268 bp M orale 151 gagcatgtgg tttaatttga agatacgcgg agaaccttac ccactcttga by the specific primers in this kit
3. is about 270 ll 269 bp A laidlawii 151 atcatgttgt ttaattcgaa gatacacgaa aaaccttacc aggtcttgac bp However the sizes of PCR product differ Iii 270 bp slightly from species to species 268 bp 277 4 A Ai M arginini 201 acatccttcg caatgctata This sequences are bp You can confirm by sequencing analysis VI 277 ai M faucium 201 atcctttgca aagctataga the partial sequence after T A vector cloning and other cloning p M fermentans 201 acatcttctg caaagctatg of PCR products methods M hyorhinis 201 tgacatcttc tgcaaagcta M orale 201 catcccctgc aaagctatag A laidlawii 201 atactctgca aagttcggag Table 1 Mycoplasma paas Detected by e Myco Kit ver 2 0 A laidlawii M adleri M agalactiae M alkalescenns M anseris M arginini M arthritidis M columbinasale M columbinum M equirhinis M falconis M faucium M felifaucium M fermentans M meleagridis M moatsii M mustelae M M M opalescens orale oxoniensis M penetrans M primatum M pulmonis M salivarium M spermatophilum M sualvi M subdolum M synoviae M verecundum M auris M bovigenitalium M bovirhinis M bovis M buccale M californicum M canadense M caviae M citelli M cloacale M gallinarum M gateae M hominis M hyorhinis M hyosynoviae M iguanae M indiense M iners M leopharyngis M maculosum zz mmommmnrxsxmn VDOO lt VESVOLOPOQOO NRO By E 7 V Biotechnology TECHN
4. ICAL INFORMATION e This e Myco Mycoplasma PCR Detection Kit ver 2 0 will provide a sensitive means to detect mycoplasma contamination in cell lines Under optimal conditions templates derived from supernatants of an infected cell culture will yield a maximum signal in the PCR reaction whereas an uninfected cell line will yield no PCR products Undoubtedly there will be variations in cell numbers infection amount and templates that may contribute to signal differences in your experiments e It is recommended that you use cultured cells that have cultivated for 3 6 days after subculturing as a sample for mycoplasma detection You may not detect mycoplasma infection efficiently when you use cells that are not or shortly cultivated e The PCR amplification efficiency varies by mycoplasma infection range Strong mycoplasma infections are detected in as little as 10 100 cell equivalents while weak infections require cell equivalents from the 5000 50 000 range So we recommend that you plan various cell numbers in preparing PCR templates from the cultured cells by using the boiling method Please refer to Fig 2 e If you perform genetic analysis for determining more detailed species please extract the DNA and apply it to the PCR process We recommend that you use our i genomic CTB DNA Extraction Mini Kit Cat No 17341 PHYLOGENETIC ANALYSIS TABLE e The following phylogenetic analysis table shows the classification based on the se
5. activates 8 MOP if you want to determine the detailed species of mycoplasma by DNA sequencing analysis e If you want to do genotyping excise the target band from the agarose gel then isolate the DNA fragment using a gel extraction kit eg MEGA spin Agarose Gel DNA Exttaction iNtRON Cat No 17181 MEGAquick spin PCR amp Agarose Gel DNA Extraction Kit iNtRON Cat No 17281 PROTOCOL A Using the genomic DNA extraction kit 1 Prepare cell suspensions from the test cell culture in a 1 5 ml tube Then count cell numbers by general counting methods You need at least 5x104 cells per test Note 1 Harvest adherent cells with trypsin EDTA solution using standard techniques Pipette 1 ml of TE treated adherent cells Generally with suspension cells such as K562 you need not treat with TE solution We recommend that you count the cells You should prepare at least 5x104 cells per test see Technical Guide gt 50 000 cells are needed to complete this protocol Note 2 Strong mycoplasma infections are detected in as little as 20 100 cells while weak infections require cells over 50 000 cells You can dilute the template according to the infection rates you suspect We recommend that you perform the PCR reaction after preparing serial dilutions of the straight supernatant to obtain optimal results 2 Transfer the counted cells over 5x10 cells to a 1 5 ml tube Spin the tube in a microcentrifuge for 10 15 seconds Carefully de
6. cant the supernatant 3 Resuspend the cells in 1 ml of sterile PBS or DPBS solution for washing 4 Spin the tube in a microcentrifuge for 10 15 seconds Carefully decant the supernatant Option Repeat this wash step once more 5 Resuspend the cell pellets in 100 ul of sterile PBS or DPBS solution Note If you want the best result use of PBS solution is better than Tris 10 mM pH 8 5 TE 10 mM Tris 0 1 mM EDTA or autoclaved DW 6 Heat the samples for 10 min and vortex for 5 10 sec Then centrifuge for 2 min at 13 000 rpm with a tabletop centrifuge at RT l 7 Transfer an aliquot of the heated supernatant to a fresh tube This supernatant will be used as the template in the PCR 8 Add 10 ul of the template to each tube of e Myco Mycoplasma PCR Detection Kit ver 2 0 and then resuspend after adding 10 ul of sterile water for a 20 yl PCR reaction volume 9 Perform PCR reaction as in the following table Note We recommend that you perform one negative control reaction by adding 20 ul of sterile water PCR Condition Temp Time ee Initial denaturation 94C Imin Denaturation 94 C 30 sec ee Annealing 60 C 20 sec cycles oo Benson in Final extension 120 5 min PROTOCOL B Using genomic DNA as a template 4 Add purified genomic DNA as a template using the i genomic CTB DNA Extraction Mini Kit Cat No 17431 to each tube of e Myco Mycoplasma PCR Detection Kit ver 2 0 and then resuspe
7. control app 570 bp length can function as a internal control e Check the quality of template possibility of contamination with PCR inhibitors If the PCR reaction is inhibited by impurities included in DNA preparation the use of diluted DNA as a template may be helpful If there is no internal control band please inquire with our technical support staff e Check the storage condition of product 3 Presence of amplified product in the negative control e Check contamination of D W D W can be contaminated Perform PCR again with fresh sterile water e Check contamination of lab instruments and other environments We recommend that you use filter tips to reduce contamination and that you use a pipette after sterilization All procedures should be done in sterilized conditions 4 Poor resolution on agarose gel e We recommend to use a 1 5 2 agarose gel e We recommend that electrophoresis is performed for 40 min at 100 V 14 cm using a 6 cm long 2 agarose gel YY Biotechnology TECHS PRINCIPLE OF MYCOPLASMA DETECTION SPECIES DETERMINATION BY SEQUENCING ANALYSIS e The newly developed e Myco Mycoplasma PCR Detection Kit ver 2 0 is a highly e The sequence of amplified PCR products differs slightly from species to species You sensitive PCR assay that detects various mycoplasma species that may contaminate can determine approximately the mycoplasma species by sequencing analysis with cell culture sampl
8. e Myco Mycoplasma PCR The Instruction Manual for Mycoplasma Detection Using Gene Specific Primer rere US Pat No 5 851 767 and its foreign counterparts ver 2 0 for 20 pl rxn R h l DESCRIPTION RUO esearch Use Only 25235 w 25236 V s e Mycoplasma are common and serious contaminants of cell cultures It has been shown that 30 to 87 of cell cultures are infected with mycoplasma Many mycoplasma contaminations particularly in continuous cell lines grow slowly and do not destroy host cells but are still able to affect various parameters leading to unreliable or false results These effects include changes in metabolism growth viability DNA RNA and protein synthesis and morphology Testing for mycoplasma is an essential quality control tool to assure accurate research and reliable biotechnological products The e Myco ver 2 0 product is a set of primers designed to detect the presence of mycoplasma that might contaminate biological materials such as cultured cells Conventional techniques that are used to detect mycoplasma involve culturing samples on selective media which needs at least 1 week to obtain results whereas by performing PCR with this primer set which is based on conserved 16S rRNA detection results are obtained in a few hours Because the presence of contaminant mycoplasma can be easily detected by only verifying the bands of amplified DNA fragments using electrophoresis there is no need to prepare probes that ar
9. e labeled with radioisotope etc You can determine the species groups of mycoplasma by sequencing analysis using the sequencing primers suggested in this manual Furthermore if you want to know the detailed species you may perform PCR and sequencing from your designed primers The adopted 8 methoxypsoralen 8 MOP is used to extinguish the template activity of contaminated DNAs 8 MOP is known to intercalate into double stranded nucleic acids and form a covalent interstrand crosslink after photo activation by incident light at wavelength 320 400 nm An internal control of this product was constructed to identify false negative results in each reaction The internal control was designed in such a way that the sample primer pair was used to amplify the internal control and target DNA which were differentiated by size Each tube of the e Myco Mycoplasma PCR Detection Kit ver 2 0 contains all the components for PCR except for template i StarTaq DNA Polymerase dNTPs 10x Buffer primers 8 MOP and internal control for mycoplasma partial gene amplifications So you can just add your templates and perform the PCR reaction CHARACTERISTICS Overview of Mycoplasma Detection e Premix Type This e Myco Mycoplasma PCR Detection Kit ver 2 0 contains all the components for the PCR reaction You just add template DNA or samples gt e Broad Species Detection You can detect five common cell culture infecting species of mycoplasma and al
10. es The primer sets primarily allow for detection of major the following primers Please refer to the phylogenetic table on the next page For mycoplasma species in cell culture contaminations M arginini M faucium M more detailed species analysis you should perform additional PCR reactions with fermentans M hyorhinis M orale as well as Acholeplasma laidlawii Furthermore your designed primers you can detect various mycoplasma species with this kit see below It is a quick simple reliable and cost effective method for regularly monitoring cells for e We list only the Forward primer sequences Please synthesize the primer and mycoplasma detection then analyze by general sequencing methods The primer sets used in the kit are derived from a highly conserved region within the e Sequencing primer sequences 16S rRNA gene and can detect very low levels of contamination The rRNA gene M species Forward 5 GAT TAG ATA CCC TGG TAG TC 3 20 mer sequences of prokaryotes including mycoplasma are well conserved whereas the A laidlawii Forward 5 GAT ACC CTG GTA GTC CAC GC 3 20 mer lengths and sequences of the spacer region in the rRNA operon differ from species to species So you can determine the species by sequencing analysis Note The PCR primers used in this kit differ from the sequencing primers We do not list the PCR primer sequences contained in this kit ANALYTIC INFORMAION so N The followi
11. for PCR of mycoplasma detection and then PCR was performed per the e Myco Kit s protocol These results show that it can be applied to the mycoplasma detection with small cell numbers such as 12 cells Lane M 100bp DNA Marker lane IC Internal control lane1 2x105 lane 2 1x105 lane 3 5x104 lane 4 2 5x10 lane 5 1 25x10 lane 6 6 25x102 lane 7 3 125x10 lane 8 1 56x10 lane 9 7 8x102 lane 10 3 9x102 lane 11 1 9x102 lane 12 96 lane 13 48 lane 14 24 lane 15 12 Internal control 3 Result for Mycoplasma copy number dilution erereeeo w oxwl M123 4 5 6 7 8 9 10 1112 13 14 15 16 Myco plasma Internal control Fig 3 Mycoplasma detection was performed using the e Myco Mycoplasma PCR Detection Kit ver 2 0 method Mycoplasma detection from fermentans using the e Myco Mycoplasma Detection Kit M fermentans were serially diluted for PCR of mycoplasma detection These results show that it can be applied to mycoplasma detection with small copy numbers such as 20 cfu ml Lane M 100bp DNA Marker lane1 6 6x105 lane 2 3 3x105 lane 3 1 65x10 lane 4 8 25x10 lane 5 4 12x10 lane 6 2 06x10 lane 7 1 0x10 lane 8 5 1x102 lane 9 2 5x10 lane 10 1 28x10 lane 11 6 4x102 lane 12 3 22x102 lane13 1 61x102 lane 14 80 lane 15 40 lane 16 20 Elimination of Carryover Contamination dilution 1M 2 3 45 1 1 PCR Myco Fig 4 Mycoplasma detection was perf
12. mix PCR Reaction type This product has a stable shelf life of a minimum of 1 year when stored at 20 C and unopened container Detection on agarose gel UV irradiation 10 min Discard PCR product tube REACTION TUBE COMPONENT e PCR Reaction volume 20 uL reaction e Myco Mycoplasma PCR Detection Kit ver 2 0 DNA Polymerase 2 5U Chemical Stabilizer 1x Loading Buffer 1x dNTPs 250 mM each Tris HCI pH 8 3 10 mM KCI 50 mM MgCl 1 5 mM Mycoplasma Primers Set Internal Control 8 MOP dissolved in DMSO Dried under iNtRON s instruction PROTOCOLS You can use this protocol just for detecting the contamination of mycoplasma However if you want to perform genotyping for the detailed determination of species please purify the genomic DNA of suspected mycoplasma infected cells using our i genomic CTB DNA Extraction Mini Kit Cat No 17341 You may use simply this protocol or your other general boiling methods TECHNICAL TIP 1 Use clean disposable gloves when performing the assay and make sure that the work area is clean prior to starting the assay setup 2 Keep your reagents and PCR mixture tubes on a cold block during reaction setup 3 Use positive displacement pipettes 4 The amplification and detection areas should be physically separated i e do not use the same bench area to set up the PCR reactions and run your gels CAUTIONS e DO NOT expose to UV irradiation which
13. nd after adding sterile water for a 20 ul PCR reaction volume Note Appropriate amounts of DNA template sample genomic DNA 50 ng 100 ng 2 Follow protocol A from step 9 Note Recommend to perform one negative control reaction by adding 20 ul of Sterile water We recommend to add 0 1 1ul of control DNA for positive control reaction TROUBLESHOOTING GUIDE No Target band in positive reaction gt Check internal control band If internal control band is seen PCR has been performed properly it is not a problem of the product Check the quality or concentration of template If the PCR reaction is inhibited by impurities included in DNA preparation the use of diluted DNA as a template may be helpful Whereas the signals of sample control app 570 bp length and internal control app 160 bp length are shown if the target band is not shown it indicates that the sample is not infected by mycoplasma e Check a PCR machine The problem can be caused by the PCR machine Please check the temperature and make sure to check that the machine is working properly 2 No internal control band e Check template concentration Competition can occur by using high concentrated DNA template Please repeat the PCR with a diluted template If the concentration of template is above 50 ng the signal of internal control may be disappeared by competition with the template It does not cause any problem because the signal of sample
14. ng sequences are partial sequences of major contaminants in Tab 1i SNOWS Wie broad species Ol Typ Origin general cell sae Vol can classify the ere by sequencing rae mycoplasma detected by this kit As A Cel culture Guinea Pig a e a ja 7 lela M arginini 1 GATTAGATAC CCTGGTAGTC cacgocgtaa acgatgatca ttagtoggtg sensitivity The name mycoplasma x ae A y M faucium 1 GATTAGATAC CCTGGTAGTC cacgccgtaa acgatgatca ttagtcggtg comes from the Greek words mykes ORUG aua M fermentans 1 GATTAGATAC CCTGGTAGTC cacgccctaa acgatgatca ttagctgatg S adaa One ane we E Bovine j P Canine M hyorhinis 1 GATTAGATAC CCTGGTAGTC cacgccgtaa acgatgatca ttagttegte proposed in the 1950s Mycoplasma is F Ovine Q Primates M orale 1 GATTAGATAC CCTGGTAGTC cacgctgtaa acgatgatca ttagtcggtg G Equine R Lion A laidlawii 1 GATACCCTGG TAGTCCACGC cgtaaacgat gagaactaag tgttggccaa a genus of small bacteria that lack cell H Murine S Monkey walls Many PRHE WEE purified and Insect T Mink M arginini 61 gagagttcac tgacgcagct aacgcattaa atgatccgcc tgagtagtat characterized from various origins such J Goat U Hamster M faucium 61 ggagccactg acgcagctaa cgcattaaat gatccgcctg agtagtatgc ae cell culture human and cows We K Geese V Iguana M fermentans 61 gggaactcat cggcgcagct aacgcattaa atgatccgcc tgagtagtac classify them briefly by origin M hyorhinis 61 gaataatttc actaacgcag ctaacgcgtt aaatgatccg cctgaatagt M orale 61 gaaaactact gacgcagcta acgcattaaa tgatccgcct gagtagtatg Target Primers Type
15. ormed plasma for genomic DNA a Lane M 100bp DNA Marker lane 1 Internal control lane Internal 2 25pg lane 3 12 5pg lane 4 6 3pg lane 5 3 25pg control 2 2 4 PCR tee HN Fig 5 UV irradiation 10min of 1 PCR template Lane w o UV without UV irradiation Lane w UV with UV irradiation 10 min lane M 100bp DNA Marker lane 1 PCR product 1 ul used from fig 4 lane 2 lane 2 PCR product 1 ul used from fig 4 lane 3 lane 3 PCR product 1 ul used from fiq 4 lane 4 lane 4 PCR product 1 ul used from fiq 4 lane 5 lane 5 PCR product 1 ul used from fig 4 lane 2 lane 6 PCR product 1 ul used from fiq 4 lane 3 lane 7 PCR product 1 ul used from fiq 4 lane 4 lane 8 PCR product 1 ul used from fiq 4 Lane 5 JINtRON Biotechnology
16. quence variations of PCR amplified products This cluster can be changed by which sequences are based on This cluster is just a reference table e With a suggested sequencing primer you can approximately determine the species For example because the cluster between M fermentans and M gallinarum is different you can simply classify the species with just sequencing analysis However there is no difference between M agalactiae M caviae M fermentans and M opalescens In this case you can t determine the detailed species with only this kit and a suggested sequencing primer If you want to know the detailed species you have to synthesize your own PCR primers and then analyze by sequencing analysis M agalactiae M caviae M fermentans M opalescens gallinarum adleri oe M bovigenitalium __ M californicum M phocirhinis hl verecundum citelli MONS M oxoniensis M synoviae W mustelae M bovirhinis moatsii sualvi M subdolum M auris M alkalescens M anseris hominis M cloacale M buccale M equirhinis canadense M arginini M gateae M hyosynoviae M arthritidis M faucium M salivarium M indiense M orale M falconis M iguanae M hyorhinis M pulmonis lean M spermatophilum M felifaucium M maculosum M leopharyngis M meleagridis M iners M bovis M columbinum M columbinasale M primatum lipofaciens EXPERIMENTAL INFORMATION
17. so other various species of mycoplasma See Technical Guide Species Determination You can determine the species of mycoplasma by sequencing the amplified PCR products PROTOCOL A PROTOCOL B Sample culture Internal Control Cell boiling method Genomic DNA Internal control embedded in the product prevents misjudgment that possibly method arises from an erroneous PCR test i 5 e Elimination of Carryover Contamination System J 8 MOP solution prevents carryover contamination by PCR products 2 gt APPLICATIONS 7 e e The kit is used for the detection of mycoplasma species that are most commonly i genomic CTB DNA Extraction Mini Kit encountered in cell culture including M arginini M fermentans M hyorhinis M 95 C 10min Cat No 17341 orale and Acholeplasma laidlawii Furthermore this kit can detect other various species of mycoplasma See Technical Guide N e Myco Mycoplasma KIT CONTENTS a PCR Detection Kit ver 2 0 4 2 Label Contain e Myco Mycoplasma PCR Detection Kit 48T 8T 1 tube 20 ul D W as negative control 1 tube 20 ul D W as negative control 2 tube 10 ul supernatant 10ul D W 2 tube 1 ul DNA 19 ul D W Control DNA Recombinant DNA included partial 16S sequence of M hyorhinis 20 pl 5 yl DNase RNase free Distilled Water 1ml 0 2 ml Store at 20 C e Myco Mycoplasma PCR Detection Kit is a novel vacuum dried pre
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