Mycoplasma hominis RG detect

Transcription

1 Mycoplasma hominis RG detect Kit for detection of Mycoplasma hominis DNA using the Rotor-Gene 3000/6000/Q Cat. # P0706 Cat. # P0706s Institute of Applied Biotechnologies a.s. User manual Version 09/2011

2 TABLE OF CONTENTS 1. KIT CONTENTS STORAGE TRANSPORT OF KIT EQUIPMENT AND MATERIAL GENERAL PRECAUTIONS PATHOGEN INFORMATION PRINCIPLE OF REAL-TIME PCR TO PERFORM SAMPLING DNA ISOLATION AMPLIFICATION PROGRAMMING OF THE ROTOR-GENE ANALYZER INTERPRETATION OF RESULTS SETTING OF THE SOFTWARE FOR EVALUATION EVALUATION OF RESULTS DISPOSAL INFORMATION...16 Praha10,

3 1. Kit Contents Kit M. hominis RG detect is ready to use system for detection of Mycoplasma hominis DNA in clinical material. The kit comes in two sizes, with the catalog number P0706 for 96 reactions and P0706s for 20 reactions. Composition of kits for 96 reactions: Reagent Premix I Description colourless transparent liquid under the white wax Volume [ml] Tube volume [ml] Quanti ty 0,01 0,2 96 Premix II Violet liquid 1,1 1,5 1 Pozitivní kontrola (PK) Negativní kontrola amplifikace (NCA) Vnitřní kontrola transparent colourless liquid transparent colourless liquid transparent colourless liquid 0,2 1,5 1 0,5 1,5 1 1,0 1,5 1 Praha10,

4 Composition of kits for 20 reactions: Reagent Premix I Description colourless transparent liquid under the white wax Volume [ml] Tube volume [ml] Quanti ty 0,01 0,2 20 Premix II Violet liquid 0,22 0,5 1 Pozitivní kontrola (PK) Negativní kontrola amplifikace (NCA) Vnitřní kontrola transparent colourless liquid transparent colourless liquid transparent colorless liquid 0,035 0,5 1 0,035 0,5 1 0,2 0, Storage All elements of the RG detect M. hominis kit must be kept in the dark at about + 2 to +8 C. Under these conditions the kit is stable until the expiration date on the label. Praha10,

5 3. Transport of kit Kit must be transported as long as necessary in a box with a cooling load. The temperature during transport must be in the range of +2 to +8 C. 4. Equipment and material Necessary equipment and instrumentation which is not included in the kit. Vortex mixer DNA isolation kit Centrifuge pikofuge Disposable gloves Adjustable pipettes Sterile pipette tips with filters PCR box (Aura mini) Rotor-Gene Analyzer 3000/6000/Q Stand on the 0,5ml/1,5ml tubes Cooling block for 0,2 ml tubes Praha10,

6 5. General Precautions The user should follow following advices: Use sterile filter tips. Store and handle positive material (samples, positive control) separately from other reagents and exclusively in the PCR or flow-boxes. Before each use, add all ingredients to the reaction mix and briefly centrifuge. Because of the fluorescent dyes component, is necessary to minimize their exposure to daylight. 6. Pathogen Information Mycoplasmas are the smallest auto replicated bacteria. They have no cell wall and are parasites which attack the mucosal membrane of respiratory and urogenital tract. Morphologically are very small cocci (diameter 300 nm), but the absence of cell walls may develop fibrous form. Species invading urogenital tract caused nongonococcal urethritis. These are surface parasites who create locally high concentrations of toxic metabolites causing inflammation. Among the sexually Praha10,

7 transmitted types include: Ureaplasma urealyticum, Mycoplasma hominis and Mycoplasma genitalium. 7. Principle of Real-Time PCR Pathogen detection by PCR is based on in vitro amplification of specific genome fragments. In case of Real-Time PCR technology the amplified product is detected with fluorescent dyes usually bound on oligonucleotides. Monitoring of fluorescent signal during the experiment enable to ascertain the product without the necessity to open the reaction tube. This notably reduces the time and minimizes the possibility of crosscontamination of laboratory by amplicons. This is a qualitative test. The kit includes Internal Control, which serves as the control of both isolation and possible PCR inhibition. 8. To perform Sampling Kit is intended for analysis of DNA isolated from the urogenital swabs, prostate secretions and urine samples. Praha10,

8 Swab sampling is recommended to perform following the instructions in the Kit for the collection and transport of endocervical / urethral swabs (P0709/P0815). For determination of pathogen from urine, it is recommended to collect the first portion of morning urine into sterile tube DNA Isolation Kit M. hominis RG detect is optimized for use with isolation kits DNA express and DNA express B. For isolate DNA from urogenital swabs (endocervical and urethral above) and urine samples is determined isolation kit DNA Express kit (P0701). For isolate DNA from prostate secretion is determined isolation kit DNA express B (P0813). The negative control of isolation (NCI is a part of the isolation kits) must undergo a isolation procedure under the same conditions as the real samples. When using a different isolation method, the compatibility test is necessary to carry out in advance. Praha10,

9 8. 3. Amplification The total reaction volume is 25 ul 1. Make sure the cooling pads are cooled to a temperature of +4 C 2. Prepare the required number of 0.2 ml tubes Premix I and the number of DNA samples + Positive and Negative controls (NCI, NCA). 3. Add 10 µl of Premix II without damage of the wax layer. 4. Add 10 µl of sample (isolate DNA) and controls. PK - Positive Control NCI- negative isolation control NCA - negative control amplification 5. Close tubes tightly and place them in a Rotor-Gene instrument in order: 1. Positive control 2. Samples (1, 2, 3..) 3. Negative control isolation (NCI) 4. Negative control amplification (NCA) Praha10,

10 Final setting of profile on Rotor Gene analyzer is a follows: the total volume of reaction mixture to 25 ul if the analyzer Rotor-Gene has function "Oil layer volume",activate it Hold 1. Cyklus (5 opakování) 2. Cyklus (40 opakování) 95 C, 15 min 1. krok 95 C, 5 sec 2. krok 60 C, 20 sec 3. krok 72 C, 15 sec 1. krok 95 C, 5 sec 2. krok 60 C, 20 sec Snímání FAM(Green) a JOE (Yellow) 3. krok 72 C, 15 sec FAM (Green) JOE (Yellow) 5-10 FI 4-8 FI 9. Programming of the Rotor-Gene Analyzer Turn on the Rotor-Gene Analyzer 3000/6000/Q (the green indicator READY is shining). Place the CD, which is part of the kit, into the CD-ROM drive of your PC and launch the software template for Rotor-Gene Analyzer by double-clicking on the icon: Praha10,

11 A dialog window New Run will open automatically or click the icon New : In this dialog window select the Advanced tab and the option Open A Template in Another Folder. Choose a RUN file STD RG 3000.ret or STD RG 6000.ret from CD-ROM drive. Select the type of rotor and 36-hole course to confirm the use of tubes with flat caps. Press Next repeatedly and then Start run Choose a folder where you want to save the data and name your file. After confirming by picking Save the experiment will start. In final window you can specify names of the samples. Praha10,

12 10. Interpretation of Results Setting of the software for evaluation Data analysis can be performed after finishing of analysis (Rotor-Gene Analyzer is stopped and the light READY is on) Click the button Analysis 2. Open Quantitation tab 3. Select Cycling A.FAM/Sybr (Green)* 4. Press Show. 5. Close the window with a cross. 6. In the dialog window Quantitation Analysis Cycling A. Fam/Sybr (Green) activate Dynamic Tube Praha10,

13 7. Press More Settings (Outlier removal) and open dialog Windows Quantification Settings (Outlier removal). Set value NTC Threshold on 0 % and confirm OK. 8. Set value Threshold on 0,1 9. Set value Eliminate Cycles before on 1. This is the final step of software setting for analysis in channel Fam/Sybr (Green). Now continue with data analysis of the internal control in the JOE (Yellow) channel. Click the button Analysis (step 1) and repeat above-mentioned protocol with a difference in the steps: 3. Choose Quantitation Analysis Cycling A. JOE (Yellow) 7. In window Quantification Settings (Outlier removal) set value NTC Threshold on 5 %. Praha10,

14 Summary table of software settings for data analysis: Activated: Dynamic Tube FAM (Green) JOE (Yellow) Threshold Eliminate Cycles before Quantification Settings (Outlier removal) Slope correct 0,1 1 0 % off 0,1 1 5 % off You can print obtained analysis using icon Reports for both channel Evaluation of results 1. The results can be evaluated in a file on the CD: From the dialog box "Quant. Results - Cycling A FAM / SYBR (Green) and Quant. Results - Cycling A. Joe (Yellow) 'copy the values in columns "Ct " to the appropriate columns on the scoreboard. Then copy from the column "Name" labelling of all samples from the position 3 to the last one in the sample list. Analysis of results is automatically displayed in the Evaluation column. If the positive and negative controls Praha10,

15 are evaluated as the "bad experiment, it is necessary to perform the whole experiment again. If any of the samples is denoted "repeat", the reaction is not valid and must be repeated PCR, DNA isolation, or sampling. Internal control (Vnitřní kontrola-vk) is evaluated in the JOE channel and M. hominis in the FAM channel.. The sample is evaluated as positive if the Ct value in the FAM channel is even or below 33 and value in the JOE channel is even or below 30. The sample is evaluated as negative if there is no signal (no Ct value) in the FAM channel, and Ct value in JOE channel is even or below 30. Praha10,

16 2. Interpretation could be made manually according to following table: Experiment FAM/Sybr (Green) PK NCI NCA Valid Ct 33 neg neg Experiment JOE (Yellow) PK NCI NCA Invalid Ct 30 Ct 30 neg Reaction FAM/Sybr (Green) JOE (Yellow) Pozitive Ct 33 Ct 30 Strongly positive Ct 22 Ct 35 or none Negative No Ct value Ct 30 Non-valid (one of the values are as indicated repetition is required) Ct > 33 Ct > 30 or none 11. Disposal Information Disposal of all materials used at work must be carried out according to the internal regulations for disposal. Praha10,

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18 Manufacturer: Institute of Applied Biotechnologies a.s. Praha 10 IČO: Produced by: Institute of Applied Biotechnologies a.s. Tymiánová 619/14 Praha 10 Date of the last revision: