Universal Mycoplasma Detection

Transcription

1 Universal Mycoplasma Detection

2 ATCC UNIVERSAL MYCOPLASMA DETECTION KIT Quick and sensitive PCR-based test to detect mycoplasma contaminants in cell culture Designed for routine use in research laboratories All components required for sample preparation are included All components required for the PCR reaction are provided and have been optimized for amplification Universal primers are specific to the 16S rrna coding region in the mycoplasma genome High specificity is obtained through the utilization of a proprietary mix of buffers, dntps and thermostable polymerase Touchdown PCR regimen increases sensitivity and specificity Meets European Pharmacopeia guidelines for nucleic acid detection of mycoplasma (Biologicals 38, March 2010) Competitively priced compared to other test kits 2

3 FEATURES AND BENEFITS Superior Sensitivity Detects 2.5 to 25 fg of target DNA, corresponding to 4 to 40 genome copies per assay, for the mycoplasma species most likely to infect cell culture Confirmed Specificity Does not cross-react with closely-related bacterial or mammalian genomic DNA Broad Detection Range Recognizes more than 60 mycoplasma species Fast Results in less than 3 hours Convenient Provides all components for detection in an easy-to-use format 3

4 DESCRIPTION ATCC K (40 assays) Store at -20 C $279/kit Components: Component Volume Composition Lysis Buffer 2 ml Lytic agent + digestive enzymes Sample Lysis Tubes 40 each 2-mL snap cap tubes, tight seal Universal PCR Mix 0.8 ml Proprietary mix of buffers, dntps, thermostable polymerase Universal Primers 0.1 ml Proprietary mix of universal forward and reverse primers Positive Control 50 µl 1 pg/µl puc19::m.arginini target in TE 4

5 SIMPLE PROCEDURE ATCC UNIVERSAL MYCOPLASMA DETECTION KIT FLOWCHART Grow cells Scrape and suspend cells, or count Add lysis buffer Perform PCR Run gel Works with adherent and suspension cells. Use 10 4 to 10 5 cells per assay. Transfer cell suspension to Sample Lysis Tubes. Pellet cells. Cell pellet can be stored until ready to assay. Incubate. Heat at 95. Spin. Transfer cell lysate to new microfuge tube. Cell lysate can be stored until ready to assay. Perform optimized touchdown PCR using Universal PCR + Primers Mix. Clear results in less than 3 hours. 5

6 RESULTS THAT SPEAK FOR THEMSELVES Sensitivity Wide range of species detected Optimized system Sample preparation Thermostable polymerase selection Cycling conditions Superior performance compared to other kits HIGH SENSITIVITY Titration of M. arginini DNA; 25 fg to 1.0 fg target DNA/assay 6

7 WIDE RANGE OF MYCOPLASMA SPECIES DETECTED Species Mycoplasma arginini Mycoplasma fermentans Mycoplasma hominis Mycoplasma hyorhinis Mycoplasma orale Mycoplasma pirum Mycoplasma salivarium Acholeplasma laidlawii Acheloplasma axanthum Acholeplasma granularum Acholeplasma oculi Mesoplasma coleopterae Mycoplasma anatis Mycoplasma arthritidis Mycoplasma bovigenitalium Mycoplasma bovirhinis Mycoplasma bovis Mycoplasma bovoculi Mycoplasma buccale Mycoplasma californicum Mycoplasma canadense Mycoplasma canis Mycoplasma capricolum Mycoplasma caviae Mycoplasma columbinasale Mycoplasma columbinum Mycoplasma columorale Mycoplasma cricefuli Mycoplasma cynos Mycoplasma equirhinis Mycoplasma falconis Species Mycoplasma faucium Mycoplasma felis Mycoplasma gallinaceum Mycoplasma gallisepticum Mycoplasma gateae Mycoplasma genitalium Mycoplasma glycophilium Mycoplasma hyosynoviae Mycoplasma insons Mycoplasma mobile Mycoplasma muris Mycoplasma opalescens Mycoplasma ovipneumoniae Mycoplasma penetrans Mycoplasma pneumoniae Mycoplasma preputii Mycoplasma primatum Mycoplasma pulmonis Mycoplasma spermatophilum Mycoplasma synoviae Mycoplasma tullyi Spiroplasma cantharicola Spiroplasma citri Spiroplasma floricola Spiroplasma ixodetis Spiroplasma lineolae Spiroplasma monobiae Spiroplasma platyhelix Spiroplasma tabanidicola Spiroplasma taiwanense Ureaplasma parvum Top 8 species in bold 7

8 MODEL SYSTEM TO ASSESS PERFORMANCE ATCC No. Name Infected with Cell Line-1 CRL-2891 BHK M. arginini (sequence-verified) Cell Line-2 CRL-2892 BHK M. orale (sequence-verified) Cell Line-3 CRL-2893 BHK M. fermentans (sequence-verified) Control Cell line CCL-10 BHK None, Control BHK cells infected with M. arginini, M. orale and M. fermentans were cultured to approximately 5 x 10 5 cells/ml Duplicates of 10X serial dilutions were prepared and cultured overnight in T25 flasks with 10 ml of fresh media 8

9 DETECTION EFFICIENCY AND IMPORTANCE OF SAMPLE PREPARATION Cell Line 1 Cell Line 2 P M N P M N P M N Supernatant Pellet of Supernatant Cell Suspension (ATCC) Dilution Colony Forming Units (CFU) (1 ml of Cell Suspension) Cell Line-1 (M. arginini) 2, Cell Line-2 (M. orale) 2, Cell Line-3 (M. fermentans) 1,

10 SAMPLE PREPARATION MAKES A DIFFERENCE BHK cells infected with M. arginini, M. orale and M. fermentans were cultured to approximately 5 x 10 5 cells/ml. Duplicates of 10X serial dilutions were prepared and cultured overnight in T25 flasks with 10 ml of fresh media. Samples from each infected cell line were prepared as follows: (1) Supernatant 1 ml of culture supernatant was used directly for detection, (2) Pellet of supernatant - 1 ml of culture supernatant was spun down, supernatant was discarded and pellet used for detection, and (3) Cell suspension 1 ml cells were scraped into remaining media as described in the Universal Mycoplasma Detection kit instructions. Cells counts were performed by adding trypsin to the duplicate serial dilutions and using 1 ml for counting. The total number of cells used for detection of mycoplasma ranges between 100,000 cells (lane 1) to single cells (lane 6). One ml of cells from each serial dilution was plated in solid media to determine CFU as shown in the table. Gel photos show that the cell suspension samples give the highest detection sensitivity, while the pellet derived from the supernatant shows a loss of about fold sensitivity, and detection is reduced at least 100-fold in the supernatant sample. The cell suspension preparations were the only samples detected down to 1 CFU/mL for all three infected cell lines. 10

11 AMPLIFICATION ENZYME ENHANCES SPECIFICITY Enzyme G Universal enzyme 11

12 SUPERIOR SPECIFICITY USING TOUCHDOWN PCR Touchdown PCR: the annealing temperature is decreased by 0.5 C per cycle for 20 cycles, then 20 cycles at 60 C per the kit protocol Standard PCR settings are 40 cycles at 60 C Duplicate samples of M.arginini genomic DNA at 25, 2.5, 1, and 0.25 fg are shown with positive (+) and negative (0 fg) controls Lanes 1 and 16 contain an 100 bp ladder 12

13 COMPETITIVE AUDIT ATCC VS. SUPPLIER 1 P M N P M N P M N ATCC Cell Line 1 Cell Line 2 Cell Line 3 P M N P M N P M N Supplier 1 Supernatant Cell Line 1 Cell Line 2 Cell Line 3 Pellet 13

14 COMPETITIVE AUDIT ATCC VS. SUPPLIER 2 P M N P M N P M N Cell Line 1 Cell Line 2 Cell Line 3 Dilution Cell Line Cell Line Cell Line Control Cell line Positive Negative Borderline, test after 48 hours 14

15 COMPETITIVE AUDIT ATCC VS. SUPPLIER 3 P M N P M N P M N Cell Line 1 Cell Line 2 Cell Line 3 Dilution Cell Line Cell Line Cell Line Negative Control Positive Control Positive Negative 15

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