Manual - Trimgen
Contents
1. human and mammalian diseases and can cause serious illness especially in infants and the immunocompromised Enteroviruses are the most common cause of aseptic meningitis and can also result in encephalitis The diagnosis of EnV meningitis normally requires virus culture and the turnaround times are about 2 5 days A rapid protocol for EnV identification can significantly reduce unnecessary diagnostic tests and improve patient management Built upon real time PCR technology TrimGen s eQ PCR EnV Detection Kit adopts a one step RT PCR method to detect EnV in human tissue samples specifically in CSF There is no need for culturing viruses before testing eQ PCR technology improves detection sensitivity and the assay can detect as low as 2 copies of viral RNA in the tube The design covers 70 species of the EnV family Technical Support 1 888 825 6005 support trimgen com 3 TrimGen eQ PCR EnV Detection Kit User Manual Materials Provided The eQ PCR EnV Detection Kit contains reagents for 32 reactions Tube Label Cap Color Quantity 32 Rx PCR Mix 2 Purple 400 ul Enzyme Mix Red 40 ul EnV TP White 50 ul EnV CTL Yellow 40 ul NF Water Clear 500 ul Note The identity of the reagents is printed on each tube The PCR Oligo Mix EnV TP is light sensitive Keep this reagent protected from direct light PCR Mix 2 Purple cap Pre mixed reagents for reverse transcription and PCR RT PCR amplifica2. TrimGen eQ PCR EnV Detection Kit User Manual eQ PCR EnV Detection Kit User Manual CONTENTS Product Warranty Notice to Purchaser Product Use and Limitations Product Safety and Liabilities Storage Introduction Test Principle Materials Provided Equipment Required Sample Preparation Procedure 0O aO Aa WO DY YO NY NY ND Interpretation of Results Storage Upon receipt of the kit store the kit at 20 C until use At this condition the reagents are stable for 1 year After first time use store all of reagents at 2 8 C and keep the reagents protected from direct light At this condition the reagents are stable for 3 months For research use only not for use in diagnostic procedure Technical Support 1 888 825 6005 support trimgen com TrimGen eQ PCR EnV Detection Kit User Manual Limited Product Warranty It is imperative that users strictly adhere to this manual Failure to do so will void our guarantee of this product TrimGen Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose Notice to Purchaser The purchase of eQ PCR products includes a limited nonexclusive license to use the reagents and systems This license does not grant rights to use the reagents and systems for the reproduction of the eQ PCR reagents and systems to modify the eQ PCR reagents and systems f
3. ater from the_NF Water tube into the 2 In the New Detector dialogue box enter EnV into the positive control tube Detector Name Click the Reporter and select FAM dye from the down list Click Quencher and select none A 7 Add 10 ul of the DNA samples into each sample tube A 8 Cap the tubes and mix samples well by tapping the tubes New Document Wizard A 9 Centrifuge the tubes to remove air bubbles trapped in the Select Detectors ahi a Select the detectors you will be using in the document solution A 10 Load the strips directly onto the Applied Biosystems Real 3 xe ey eg BS Pan S Time PCR System Detector lame Description Reporter Quencher Detectors in Document D Env FAM none En n lt lt Ri B Programming ABI 7500 and PCR Set up B3 ira B 1 Enter the ABI 7500 SDS Software and select File gt New In the B3 New Document Wizard window click the Assay box choose the Standard Curve Absolute Quantification in the drop down list Click Next lt gt New Detector New Document Wizard iced ieee lene and template for the document and enter the operator name and comments lt Back Next gt Finish Cancel Assay ETEREE Ried Cesena on a Container 96 Well Clear z B 3 Once confirmed the target names and fluorescence options in Template Blank Document Browse the New Document Wizard window select EnV Click th
4. e Run Mode Standard 7500 z Add gt gt button to add them to the Detectors in Document dialogue box Select Next Operator hdoong Comments SDS v1 4 B 4 In the Setup Sample Plate window program the wells to be analyzed according to the following instructions 1 Select the wells to be used in the plate map 2 Select ENV detectors by clicking the check box of EnV Finish Cancel 3 Select Task to assign the detector task for each sample with Unknown Standard or NTC blank 4 Select Finish Plate Name Plate1 Technical Support 1 888 825 6005 support trimgen com Technical Support 1 888 825 6005 support trimgen com 7 8 TrimGen eQ PCR EnV Detection Kit User Manual New Document Wizard Set Up Sample Plate Setup the sample plate with tasks quantities and detectors none Unknown lt Back N Cancel B 5 In the Plate Setup window enter sample name by clicking a well A Well Inspector dialogue box will then appear Enter appropriate sample name for each well Select the next well and enter sample name until finishing naming all the wells Lastly click on the Instrument tag to set up the PCR conditions E Well Inspector Saneas Posnet tt Technical Support 1 888 825 6005 support trimgen com 9 llizdg 19 L LOZEO AL TrimGen eQ PCR EnV Detection Kit User Manual B 6 Set up the PCR conditions a
5. ector drop down list select a detector E Under Analysis Settings select Manual Ct and Auto Baseline F Click on Analyze button G At the next screen open Graph Settings in the Tools drop down list In the Post Run Settings dialog box click on the Linear button to re draw the plot H Click on each sample position from lower window to highlight the sample that you would like to analyze Select EnV in the Detector window to analyze samples Technical Support 1 888 825 6005 support trimgen com 11 TrimGen eQ PCR EnV Detection Kit User Manual Interpretation of Results Positive sample If the fluorescent curve of a sample is greater than the fluorescent curve of negative control curve the sample is considered positive The Ct value of a positive sample should be less than 40 Negative sample No Ct value will be given Select EnV in the Detector window and use the following table to determine whether EnV is presented in the samples Negative control Positive control Test Samples Interpretation Negative Positive Positive EnV positive Negative Positive Negative EnV negative Positive Positive Positive Test Fail Negative Negative Positive Test Fail Technical Support 1 888 825 6005 support trimgen com 12
6. ed FFPE tissues Ot eee A Fresh or frozen tissues Cy Sample Fine Needle Aspiration tissues C lt t Sample 3 Cells OC lt Sample 4 C lt Sample 5 Kit Name Cat No Sample 6 WaxFree RNA WR 50 50 extractions A 2 Collect PCR Mix 2 Purple cap Enzyme Mix Red cap EnV TP White cap and EnV CTL Yellow cap tubes from The eQ PCR EnV Detection Kit can detect EnV in RNA samples OX extracted from human tissues CSF and whole blood Any A 3 Using the table below prepare the PCR Master Mix in one commercially available kits are acceptable tube Reagents Formulation Volume PCR Mix 2 12 5 ul x 2 x 1 1 Sample number Enzyme Mix 1 ul x 2 x 1 1 Sample number EnV TP 1 5 pl x 2 x 1 1 Sample number Total Volume For the blank and positive controls Recommended volume correction factor for pipetting error Technical Support 1 888 825 6005 support trimgen com Technical Support 1 888 825 6005 support trimgen com 5 6 TrimGen eQ PCR EnV Detection Kit User Manual TrimGen eQ PCR EnV Detection Kit User Manual A 4 Aliquot 15 ul of the Master Mix to each sample tube B 2 For the first time user create the EnV detector file as follows If the EnV detector file has been created skip to the next A 5 Add 10 ul of NF Water into the blank control tube step A 6 Add 2 ul of EnV Control DNA from the EnV CTL tube and 8 ul 1 Select New Detector of nuclease free w
7. or resale or to use the eQ PCR reagents and systems to manufacture commercial products without written approval of TrimGen Corporation No other license expressed implied or by estoppels is granted Product Use and Limitations The eQ PCR Kit is designed as a For Research Use Only RUO kit For all of other applications user should follow the instructions provided by the appropriate regulatory authorities Product Safety and Liabilities Some reagents included in this product may cause harmful conditions The user should read the instructions on the bottle or tube and in the User Manual carefully before starting to use the reagents Handle the chemicals with caution When working with the reagents always wear lab coat gloves and protective goggles TrimGen Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the misuse the results of use or the inability to use this product Technical Support 1 888 825 6005 support trimgen com 2 TrimGen eQ PCR EnV Detection Kit User Manual Introduction The eQ PCR EnV Detection Kit is a real time PCR kit used to detect enterovirus EnV present in samples The EnV pathogens are detected by TrimGen s proprietary eQ PCR enhanced Quantitative PCR technology US patent pending This detection kit is applied to Applied Biosystems Real Time PCR System model 7500 Enteroviruseses are RNA viruses associated with a variety of
8. s follows Stage 1 Stage 2 Stage 3 Reps 1 1 40 Temperature 50 0 C 95 0 C 95 0 C 60 0 C Time 10 00 10 00 0 15 0 45 e In the Thermal Cycler Protocol window enter the values for the PCR conditions from above table into the corresponding E boxes e Enter 25 for the Sample Volume e Select Stage3 Step2 60 0 0 45 for Data collection Setup Y Instrument r Instrument Control Temperature ger FY Estimated Time Remaining hh mm Sample Heat Sink Cover Block e f Cycle Status Stage Rep Time mm ss State Thermal Cycler Protocol Thermal Profile Auto Increment Ramp Rate Stage 1 Stage 2 Stage 3 Reps 1 Reps 1 Reps 40 Add Cycle Add Hold Add Step Add Dissociation Stage Help Settings Sample Volume pL 125 Run Mode Standard 7500 v Data Collection Stage 3 Step 2 60 0 0 45 ba Technical Support 1 888 825 6005 support trimgen com 10 TrimGen eQ PCR EnV Detection Kit User Manual Data acquisition The following section is for ABI instruments for other type of thermal cyclers refer to the user manual for data acquisition A After the PCR program has finished locate the data file and open it B Click Results tab Go to the Amplification Plot C Highlight the wells to be analyzed at the bottom of the screen Then select Delta Rn vs Cycle in the Data drop down list D Inthe Det
9. tion Enzyme Mix Red cap Mix of enzymes for RT PCR EnV TP White cap Mix of PCR primers and probes for detection of EnV EnV CTL Yellow cap Positive Control DNA for EnV identification NF Water Clear cap Nuclease free water for the blank control Technical Support 1 888 825 6005 support trimgen com 4 TrimGen eQ PCR EnV Detection Kit User Manual TrimGen eQ PCR EnV Detection Kit User Manual equipment Required eQ PCR EnV Detection Kit Procedure e Applied Biosystems Real Time PCR System Model 7500 vaio pn e Nucleic acid extraction kit for isolation of EnV viral RNA This kit is applied to Applied Biosystems Real Time PCR System 7500 e Pipettes A Reaction Preparation e Sterile filter tips 2 C 8 C wh K Il Tabletop centrit ge Thaw all reagents to 2 C 8 C when ready to use Keep a reagents at 2 C to 8 C during preparations Once thawed do NOT refreeze remaining reagents Sample Preparation A 1 Prepare Optical 8 tube strips Applied Biosystems Catalog Number 4316567 not provided by the kit or MicroAmp 96 well Reaction Plate Applied Biosystems Catalog Number 4306737 not provided by the kit for the PCR reaction Label the tubes as follows Reagents for nucleic acid extraction are not provided with the eQ PCR EnV Detection Kit TrimGen provides high efficiency RNA extraction kits for the following samples Q lt Blank control aa OC lt Positive control Paraffin embedd
Download Pdf Manuals
Related Search