VDS AIV H7 - Hai Kang Life
Contents
1. IRS reading below 10 000 Equipment not calibrated Contact your bioM rieux representative 7 TECHNICAL ASSISTANCE Our technical staff will provide technical assistance you may need in using this kit Simply call 852 2111 2123 during office hours Monday Friday Saturday 9 00am to 5 30pm 9 00am to 1 00pm A recorded message in English Cantonese or Putonghua may be left outside office hours Alternatively you may contact our technical staff by fax or email Fax Email 852 2111 9762 technical haikanglife com 8 WARRANTIES AND LIABILITIES Hai Kang Life Corporation Limited warrants the products manufactured by it are free of defects in materials and workmanship when used in accordance with the applicable instructions for a period equal or shorter of one year from the date of shipment of the product s or the expiration date marked on the product packaging under the storage conditions recommended in the instructions and or on the package Application protocols published by Hai Kang Life Corporation Limited are intended to be only guidelines for the buyers of the products Buyers are expected to validate the kit to their individual application Hai Kang Life Corporation Limited makes no other warranty expressed or implied There is no warranty of merchantability or fitness for a particular purpose The sole obligation of Hai Kang Life Corporation Limited with regard to the foregoing warranties shall be at i2. 1 Test 5 known negative samples and 5 known weak positive samples i e dilutions of known positives NASBA reaction generally produces strong positive ECL signals with very few intermediate ECL signals 2 The cut off can be defined as a fraction of the Instrument Reference Solution IRS which is run at the beginning of every experiment Typical values for IRS are 15 000 to 60 000 units 3 Define a cut off limit that can correctly differentiate the negative and weak positive samples tested above The cut off limit will be greater than the highest ECL signal obtained for a known negative sample and lower than the lowest ECL signal obtained for a known weak positive Generally a factor between 0 01 to 0 15 x IRS is appropriate i e 1 to 15 of the IRS 4 Further known negative and weak positive samples can then be tested to further qualify the appropriateness of the selected cut off value 5 The chosen cut off value will be valid for the current set of experiments only and must be defined again when the assay is run in the future or if other parameters are altered e g buffer composition extraction method length of incubation etc Table 2 Summary of cut off limit determination data Negative sample ECL signal 1 137 2 303 3 275 4 375 5 632 Negative control 289 Assay negative 127 IRS 36 345 Cut off limit 0 01 x IRS 363 0 015 x IRS 545 0 02 x IRS 727 0 025 x
3. Wear gloves throughout the procedure to protect your RNA samples from nucleases All procedures should be carried out at room temperature unless otherwise stated DO NOT vortex any solutions containing enzymes Nucleic Acid Amplification 1 For each amplification reaction pipette 5 ul of the nucleic acid extract into a fresh microfuge tube Add 10 ul of amplification solution see section 2 Incubate microfuge tubes for 5 min at 65 C in a heat block Cool microfuge tubes for 5 min at 41 C in a heat block Add 5 ul of enzyme solution see section 2 and mix well by flicking the microfuge tube with your finger Immediately return microfuge tubes to 41 C for 5 min 7 Briefly centrifuge microfuge tubes and incubate for 90 min at 41 C in a water bath 8 Perform the detection or store the RNA amplicons at 20 C for up to 1 month Nucleic Acid Detection 1 Take N 2 5 ml polypropylene tubes Label all tubes Keep Tube 1 for the Instrument Reference Solution IRS provided in the kit and Tube 2 for the blank control 2 Vortex the hybridisation solution until opaque see section 2 and add 20 ul solution to each of the tubes except Tube 1 3 For blank control add 5 uI NASBA water to tube 2 4 For the sample reactions add 5 ul RNA amplicons to tube 3 4 etc 5 Cover all tubes with parafilm and gently vortex all tubes until an opaque solution has formed 6 Incubate all tubes for 30 min at 41 C Gently vortex the
4. IRS 909 0 15 x IRS 5452 As the highest known negative sample has an ECL signal of 632 the most appropriate cut off limit to select on this occasion would be 0 025 x IRS 6 TROUBLESHOOTING GUIDE Symptoms Possible Causes Problem solving Low ECL signal RNase contamination The reagents provided are all RNase free All other solutions and apparatus used pipettes tips and microfuge tubes etc should be treated to ensure they are also RNase free Insufficient template RNA preparations should be stored properly to prevent Degradation of nucleic acid degradation extracts Nucleic acid samples can be stored long term at 70 C up to 7 days at 2 C to 8 C up to 48 hours at 25 C Poor amplification efficiency Add enzyme solution to the microfuge tubes by taking the microfuge tubes out of the heat block one at a time and returning them immediately after addition of enzyme Maintain the amplification temperature at 41 C 41 C Enzymes denatured DO NOT vortex the enzyme solution Enzymes are quite sensitive to physical influences and lose their activity when vortex mixed DO NOT incubate the enzymes above 42 C This will also denature enzyme activity Poor hybridisation between target RNA and probe Vortex the capture probe and generic probe tube until an opaque suspension is formed before preparing the hybridisation solution Vortex the hybridisation solution and sample mixture regularly
5. VDS AIV H7 User Manual Cat No V01 01 1112 NASBA for the detection of avian influenza virus H7 subtype in veterinary and environmental samples To be used in conjunction with the NucliSens Electrochemiluminescent Reader Includes main components for 50 reactions SRE BER a Rev 0 July 2008 HAI KANG LIFE CORPORATION LIMITED PLEASE READ THROUGH THE ENTIRE PROTOCOL BEFORE STARTING 1 KIT COMPONENTS Storage Conditions Components should be stored at 20 C with the exception of the small inner box which should be stored at 4 C Expiry dates shown on the box indicate the date beyond which reagents should no longer be used Nucleic Acid Amplification 5x6 5mg Enzyme spheres red cap contained in a foil pack with silica gel desiccant 1x0 5ml Enzyme sphere diluent red cap 5x10mg Reagent spheres blue cap contained in a foil pack with silica gel desiccant 1x0 6ml Reagent sphere diluent blue cap 1x0 5ml KCI stock solution yellow cap 1x60 ul H7 primer mix 1x25 l H7 positive control 2x1 5ml NASBA water white cap Nucleic Acid Detection Small Inner Box Contents store at 4 C 1x0 9ml Generic ECL detection probe red cap 1 x 0 65 ml H7 capture probe 2x1 7ml Instrument Reference Solution clear cap 2 PREPARATION OF REAGENTS A Nucleic Acid Amplification Re use of reagents The reconstituted reagent sphere enzyme sphere can be re used once within two weeks of opening provid
6. ed they have been stored at 70 C Re use of all other amplification reagents is possible if the remainder of the opened reagents has been stored at 20 C Bring all reagents to room temperature before use Preparation of Amplification Solution Add 80 ul reagent sphere diluent to a lyophilised reagent sphere and immediately vortex well Add 14 ul NASBA water and 16 ul KCI stock solution and vortex Add 10 ul H7 primer mix and vortex DO NOT centrifuge Use within 30 min Preparation of the Enzyme Solution Add 55 ul enzyme diluent to a lyophilised enzyme sphere Leave this enzyme solution to stand for at least 20 min at room temperature Mix gently by flicking the microfuge tube with your finger D NOT vortex any solution containing enzymes Centrifuge briefly before use Use within one hour B Nucleic Acid Detection Re use of detection reagents is possible if the remainder of opened reagents has been stored at 2 8 C Bring all reagents to room temperature before use Spin down all microfuge tubes to bring liquid to the bottom of the tube C Preparation of Hybridisation Solution Vortex Generic ECL detection probe and H7 capture probe until an opaque solution has formed For N detection reactions mix N 2 x 10 ul H7 capture probe with N 2 x 10 ul Generic ECL detection probe Vortex hybridisation solution briefly before use Use within one hour 3 PROCEDURES Remarks
7. ill show Check the worklist and click ok 13 Ensure your samples are placed on the carousel according to the sample ID order input to the computer 14 Select Routine at the top left of the screen and select Run Worklist 15 A check fluids screen will appear Click Proceed 16 The detection will start each tube will take about 1 5 min 17 After the detection stops select Routine at the top left of screen and select Assay result to obtain results Results and Interpretation The NucliSens Reader automatically measures the ECL signal of the hybridised samples and the user software calculates the corresponding result For example the results of detection will be displayed similar to the following Note The experimental data shown in the following table is for example only your results will be different Table 1 Typical NucliSens results summary Qualitative Signal Sample ID Qualification Tube g Analysis EN counts ms f vaa foa fo os ee ee so Hinnete void fa meene f neee Loo a _ vaia 3 Freetube Positive e p o a reie rene O oa e wa 6 Freewe Postve 10000001 Negaiveconiot vaio o Freewee necare w 5 DETERMINING THE CUT OFF LIMIT The calculation of the most appropriate cut off limit to adequately discriminate between genuine positive and negative samples is a relatively straightforward procedure An example is provided here as a reference
8. ts option to either replace or refund the purchase price of the product s or part thereof that proves defective in materials or workmanship within the warranty period provided the customer notifies Hai Kang Life Corporation Limited promptly of any such defect Hai Kang Life Corporation Limited shall not be liable for any defect indirect or consequential damages resulting from economic loss or property damages sustained by the buyer or any customer from the user of the product s Rev 0 July 2008 VO1 01 1112 English
9. tubes to mix every 10 min 7 Add 0 3 ml assay buffer provided with NucliSens Reader to each tube except Tube 1 8 Vortex the IRS until opaque and add 0 25 ml IRS to Tube 1 9 Place the tubes on the appropriate positions of the instrument carousel 10 Create a new run worksheet and input sample number see section 4 IPN D 4 DATA COLLECTION ANALYSIS AND INTERPRETATION Detailed explanations of the basic and advanced operating procedures and equipment maintenance are provided in the NucliSens Reader Operator Manual Create a New Run 1 Switch on the NucliSens Reader computer 2 On the start up screen click Prime to perform instrument calibration 3 When instrument is ready click start The calibration will take approximately 3 min Rev 0 July 2008 V01 01 1112 English 4 Under user name on the og in screen select service engineer and click log in There is no need to enter a password 5 Select Routine at the top left of the screen and select New Run 6 On the flush the worklist screen click yes 7 Enter a filename up to 8 characters and click ok 8 A worklist sheet will open Under selected assay select Free tube 9 Enter sample ID and click Add to list Note do not change the sample volume from the preset 100 ul 10 Repeat sample ID input for all samples 11 When finished click Close 12 The sample worklist details w
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