PRRS diagnostic protocole including automated platform

PROTOCOLEAutomate metrologyTroubleshooting - Automate

Rev 1.57 / 2017-08-01


TARGET : PRRS North America / Chinese Strains


MATERIAL :

  • Tecan 8 channels + RoMA.
  • qPCR Agilent Mx3005

Purification system :  silica microbeads


BIOLOGIST CONTACT :   Dr Yannick LEQUETTE y.lequette@adnucleis.com
COMPUTING & ROBOTICS CONTACT : Pascal FRANCK, p.franck@adnucleis.com
ADD IN COPY :   support@adnucleis.com


Samples preparation - Semen
Rev 1.0 (29/06/2017)
Boar semen samples should be kept between 1 and 4 °C or on ice until use. Raw semens are first diluted 1/10 in a buffer (Elixir buffer -genePro). 100 µl of the diluted semen are used for the nucleic acid extraction.

RNA Extraction - purification - Robotic method

Rev 1.0 (29/06/2017)

Reagents and Materials included in the ADNucleis kit:

  • ADNUCLEIS Viral RNA Extraction-Purification kit (Extraction kits and Purification kits are sold separately)
    • RNA extraction kit: PVG working solution must be prepared before starting the extraction step)
      • PVG (Please reconstitute the solution in this bottle)
      • PE
      • TME
      • RNA carrier (Keep at 4°C)
(Please use immediately after preparation).
  • Nucleic acid Purification kit
    • Magnetic beads suspension (µMB)
    • Precipitation buffer SP
    • Wash buffer 1
    • Wash buffer 2
    • Wash buffer 3
    • Elution buffer (ELU)
    • Decontamination buffer for the robot needles: D1, D2 et D3

Reagents and Materials not provided in the kit

  • Consumables
    • Deep-well 96 wells plate (for the robotic method,  please use the following plate: Riplate plus PP 1mL ref. n°43001-0016, Ritter brand).
    • Aluminium foil film for 96 wells plate.
    • 96 wells PCR plate
    • QPCR film for 96 wells PCR plate
The material above  is not included in the kit, but can be purchased at ADNucleis. Please contact ADNucleis to check the compatibility of your own consumables.
  • Routine laboratory reagents and materials (to be used according to the Good Laboratory Practices): tips, micropipettes (1mL, 200µL, 20 µl, 10 µl), non-powdered gloves …
  • Ultrapure water (sterile, distilled and nuclease free).
  • RNA Standard.

RNA extraction is achieved using enzymatic activities: An enzymatic and thermal lysis releases the RNA from all microorganisms and cells present in the sample. . Preparation of the PVG working solution :
  • Resuspend PE with the appropriate volume of TME (ME) when first opening the PE vial. Homogenize
Reagents  TME volume
PE 25 mg 440 µl
PE 100 mg 1760 µl
Store at -20°C until use.
  • Prepare enough master working solution of PVG + ME (100µL of PVG for 4µL of ME) extemporaneously for X number of samples to be extracted +3 (e.g. for 20 samples, prepare a volume of master mix for the equivalent of 23 samples).
  • BE CAREFUL TO HOMOGENIZE CORRECTLY THE PVG in order to have NO volute in the end (10 times).
  • RNA carrier should be added in the mix PVG + ME at this step if extracting RNA. Add 1 µl of RNA carrier per sample (e.g. 23 µl)
Reagents 1 reaction 24 reactions 48 reactions 72 reactions 96 reactions
PVG 100 µl 2700 µl 5100 µl 7500 µl 9900 µl
ME 4 µl 108 µl 204 µl 300 µl 396 µl
RNA carrier 1µl 27 µl 51 µl 73 µl 99 µl
Volumes in the table above for 24, 48, 72, 96 reactions take into account the loss of volume due to pipetting. ADN_SOFT takes into account the loss of volume due to pipetting automatically.
  • Distribute 100µl of Extraction buffer PVG + ME (+RNA carrier if added) in each well of the deep-well plate.
  • For PIPETTOR ROBOT automated procedure, this distribution is done column by column beginning with the first column (A1, B1…H1, A2, B2…H2 etc.)
     It is possible to have the first sample placed in any well (Offset option). However, the filling of the following samples (2,3,4,...) must follow the same rule as above: samples are distributed column by column from top to bottom and no empty well is allowed between the first and last sample. The number of empty wells before the first sample is given in the "plate Layout" sheet.
  • Homogenize samples carefully, then pipette 100µl and add to a well of the deep well plate.
  • Vortex or homogenize by pipetting the sample with the PVG.
  • Put the deepwell plate with samples on the heating block (Thermoshaker)
  • Start ADN_SOFT.exe ("START A NEW RUN"), and click immediately on "Save As.." and save the archive file in the default folder.
  • fill the plate layout with sample names (each name has to be different), then click on "Lock Layout".
  • Specify the volume of elution buffer used for the RNA elution step.
  • The table below indicates reagent volumes to be filled in the different containers of the robot.
  • The program used for the Extraction-Purification method is "µMB Extract-Purif" :
  • Check that the volume in the Liquid System jar is sufficient (2% D3 in demineralized water). Empty the "Waste" jar.
  • Start Gemini software (Software controlling the Tecan Robot), then open the extraction purification program : ADNucleis_Extract_Purif.gem
  • Fill the different containers with reagents according to the positions showed on the Layout in Genimi software (Required volumes are given by ADN_SOFT).
  • Put an empty deepwell plate in "DW-Empty" position, This plate will be used to collect eluted RNA.
  • Start the Inheco Multitec heating block. The Gemini program controls the temperatures of the different blocks.
  • Click "flush" on Gemini software
  • Initialize the robot
  • Click "Play" in Gemini software to launch the extraction-purification program. A prompt window will ask the number of sample, fill the number of sample.
NB : the robot will start by the thermic lysis step. This steps takes about 30 min. The robot does not show any visual activity.
  • At the end of the program (30 min + 1min per sample), the RNA is eluted and the plate is kept at 4°C.
  • Go back to ADN_SOFT to start the setup of the PCR plate
 

qPCR plate preparation - Robotic method

Rev 1.0 (29/06/2017)

Reagents and Materials included in ADNucleis kit :

  • The RT-PCR kits are distributed in bulks of 48 reactions under the form of a 2X concentrated premix.
  • Premix: The dilution of the 2x premix is managed by the robot using ELU buffer.  The 2X concentrated PCR premix contains all the ingredients necessary for the detection of the target (s) of the PRRS virus (primers, dNTPs…) and the IPC primer/probes, except enzymes.
  • Taq (Taq polymerase Nucleis used to amplify the DNA)
  • IPC / IAC _ARN  (RNA inhibition control)
  • RT (Reverse transcriptase Nucleis used to transcribe RNA into cDNA)
  • Elution buffer ELU
  • RNA positive control  (200µL/ tube of positive control is provided for each vial of 48 reactions)

Reagents and Materials not included in ADNucleis kit :

  • Recommended consumables
    • 96 well PCR Plate PCR (Be careful to use a plate suitable for both the Inheco device and your qPCR thermocycler)
    • QPCR Sealing film (e.g. ThermalSeal RT2RR)

  • Upon reception, ADNucleis amplification kits must be stored at -20°C until use.
  • DO NOT freeze back or reuse a reconstituted PCR 1x mix.
  • The reverse transcription step to generate cDNA from RNA is performed separately from the PCR. The RT step is carried out first by adding the RT enzyme in the premix manually. When the RT step is done, the Taq polymerase Nucleis is added t the mix by the robot in each well.
  • ADNucleis recommends to avoid freeze/thaw cycles (Not >3) of the amplification kits.
  • Vials of PCR premix  can be aliquoted to avoid excessive thawing and freezing when premix are thawed for the first time (8, 16 reactions). Aliquoted premix must be kept at -20°C.
  • If the component of the kits are not aliquoted, please write on the original  vials the number of thaws and volumes pipetted.
Example of annoted vials Numbers indicate  the number of reactions  used  16, 12 and 4 puits; indicating 3 different thaws.

PROCEDURE

Generate a PCR layout and the worklist using ADN_SOFT

 
  • Choose the desired target (s) (SELECT_TARGET), and write an "x" in each well to be analyzed with the corresponding target.
    • For detection mode, the genomic quantification can be left blank. The bottom of the table shows the fluorescent channel used for each target.
  • Create the layout code for the robot by clicking "Create PCR plate(s)"
  • Fill the cell plate number (If there are several PCR plates to run all the samples extracted, the PCR plate can be generate successively by indicate the plate number). When 94 samples are tested with one target, only one PCR plate will be created (94 + 1 negative control and 1 positive control = 96 used wells). Fill the cell with "1".
  • Generate the worklist by clicking "Generate Code"

Amplification mix reconstitution

  • Work with ice or cold bloc. Inheco blocks can be used; To use the inheco device manually: Click "Temp" if the temperature is already set to 4°C. Otherwise, click "set", then "4" and "0", then click on the left top button when the temperature is written "004.0°C". Then click "Temp". The button "Set" should indicate 4°C.
  • If extracted samples have been stored at 4°C or frozen before the purification step, the deepwell containing samples should warm on the thermoshaker "Inheco DW 96 Ext" of the robot set to 4.0°C and thaw for 10 min at this temperature.
  • Thaw HQS_xxx_PRE-mix premix vials, IPC vials and the RT Nucleis vials using the 24 well Inheco block of the robot. Put the vials in each slot according to the map given by the ADN_soft. Put an empty vials to reconstitute the amplification mix.
  • The mix reconstitution is performed according to the table below
 Component Volume per well (µl) Volume per Y well (µl)
HQS_xxx_pre-mix (with IPC) 18 18 x Y
RT Nucleis 1 1x Y
IPC 3 3 x Y
reconstituted Mix(µl) 22 22 x Y
NB: ADN_SOFT will show the different volumes for the mix reconstitution. The software  takes into account the number of reactions and loss of reagent due to pipetting. Volumes of reagents provided by ADNUCLEIS in each kits take into account this loss of reagents due to pipetting. The 48 reactions kits contain enough volume for 58 reactions).

BE CAREFUL :

  1. As well as for PVG, NO VOLUTE should remain after homogenization. Pipet 10 times in order to homogenize correctly Premix with RT and then with IPC.
  2. No bubbles should be seen inside tubes (reconstituted mastermix , positive rna, Taq)

Starting the robot and QPCR

  • Put a  1.5 ml vial into the slot indicated by ADN_SOFT, add the volume of Taq given by ADN_Soft.
  • Add 10 ml of ELU buffer and 20 ml of D1 buffer into the indicated container.
  • Check if the liquid system container contains enough volume of 2% D3. Fill it if necessary according to the volume indicated by ADN_SOFT. Be careful when filling the container. Add the water first, then the adequate volume of concentrated D3 to avoid foaming.
  • Empty the waste container
  • Verify the reconstituted mix, Taq, RNA positive control vials are correctly placed according to the 24 well Inheco device map on ADN_SOFT.
  • Put a new PCR plate on the PCR Inheco device.
  • Click "flush" in Gemini
  • Then, click "initialize"
  • To run the program click file and choose the program "ADNucleis_qPCR_plate_prep", then click "Play"
  • Follow the program
  • At the end the run (around 30 min + 1 min per sample), seal the PCR plate and put it immediately in the QPCR apparatus and launch the QPCR program. If you do not run the QPCR immediatly, then freeze immediatly the PCR plate at -20°C until use.

Starting the QPCR program - template TaqMan

Rev 1.0 (29/06/2017)

How to run the QPCR Mx pro device:
  • Click the MxPro icon
  • A new window opens. Select "Quantitative PCR (multiple standards), then click OK. If the software is already opened, then click File -> New -> Quantitative PCR.
  • A new window opens -> do you wish to use Quantitative PCR plate setup adnucleis , click YES
  • The following windw opens -> do you wish to use Quantitative PCR Thermal Profile Setup, click YES.
  • Start the halogen lamp (for Mxpro QPCR device)
  • The lamp takes 20 min to warm up.
  • You can check the thermal profile by clicking on the tab Thermal Profile Setup. The thermal profile should be as follows: 95°C for 5:00 min, 42 cycles at 95°C during 0:10 sec / 60°C during 0:40 sec, then a step at 25°C for 2:00 min.
  • When the lamp is warmed up, click the RUN button.
  • On the RUN window, click "Start"  and name the file
  • Click “Save”.
The QPCR program starts (1h15 min)
  • When the run is done, save the data and export the data in excel file to be uploaded to ADN_SOFT :
  • Click on “Analysis” button
  • Click File->Export Instrument Data -> …To Excel ->Format 1…
  • Save the file and close it before to upload it into ADN_SOFT.

Results analysis using ADN_SOFT
Rev 1.0 (07/07/2017) After opening ADN_SOFT
  • Click the "Import" button and follow the instructions.
  • Results will be displayed in two tabs. On on tab, results are displayed in a PCR plate view. On the other tab, results are displayed as a list.
  • Check in the "controls" tab that the positive, negative, IPC controls are ok.
  • Save the file as archive (Save As...)
  • Data can be exported in readable excel file (.xlsx)
NB : In case of a sample inhibition ("INHIB" ), follow the procedure below : Dilute manually the RNA from the sample plate using the elution buffer. Add this diluted RNA in the next plate of extracted sample you want to test with the amplification mix. Please write this new sample in the plate layout of ADN_SOFT for the preparation of the nex PCR plate.

 

Every Day

  Wash the 100 mL trays :
  • For the µMB (micro Magnetic Beads) tray, if microbeads remain in the bottom, rince them with isopropanol 70% first.
  • For all tray, wash at 55°C max
  Autoclave all the following components: I. The washing station and pipes II. The bottle « liquid system » and the bottle « waste ».    Liquide system                                                     waste  

Every month

  The thermoshaker has a cooling liquid inside that have to be filled every month :
  • unscrew the 2 screws
  • Mont the needle with intermediate holder :
 
  • Aspirate Inheco liquid from THERMOSHAKE COOLING LIQUID and fill it inside the Thermoshake until you can see it in the filling nozzle.
 
In case of error, initialization error, please check that there was no collision done by a bad positioning of tube, rack, plates… As the robot initialize its position on each start, it is possible to clear problem by switching off the robot (wait 3 sec) and re switching on. Restart Gemini and initialization.
« Device is not implemented », happening during initialization Cause : Can be caused by a wrong initial position which causes an mechanical problem.   Corrective Task : Turn off Pipetting Robot, move the Robotic Arm on any another position of X, Y and rotation, then re launch Pipetting Robot and Gemini software.
Diluter error “tip is broken” Cause : This problem can occur when a wrong dispense operation has been done. The robot or the operator “Desactivated “ the tip. Corrective Task :  To repair this, Setup -> Configuration -> LiHa -> correct the check box where the tip is marked as broken.
Diluter error “Plunger overload” The robot stops until operator click on “initialize”. Once re initialized (click on Initialize), the robot can continue the run. If the robot doesn’t succeed in initialization procedure, switch off the robot, check if needle is not clogged, move diluter (the screw under syringe) up and down and retest. Cause :  This error can mean that needle is blocked or diluter is damaged.   Corrective Task : If the problem occurs more than 1 time each 5 runs on the same channel (which would be not plugged), diluter has to be changed. Procedure is as follows : 0- Switch off the robot, set the robotics arms in the middle in order to be able to lift the hood. 1- Unscrew bottom syringe screw 2- Put down the arm 3- Unscrew the syringe, set it on the other new diluter 4- Unscrew the 2 top tubes – BE CAREFULL OF WATER WHICH COULD DROP DOWN FROM THE 2 TUBES TO ELECTRONICS COMPONENTS OF THE ROBOT/DILUTER (you can put tubes on a wipe to absorb water.) 5- Check below the diluter if any screw retains diluter, and if there is one, unscrew it too. 6- Remove the old diluter 7- Check the address behind it, and report the same on the new one (1st diluter is “0”, 2nd is “1”, etc.) 8- Set the new one, - BE CAREFULL OF WATER WHICH COULD DROP DOWN FROM THE 2 TUBES TO ELECTRONICS COMPONENTS OF THE ROBOT/DILUTER 9- Screw firmly tubes, then syringe, then arm syringe. 10- Start robot, start Gemini and initialize. If error occurs, recheck address increment is correctly set.  
Detection error “no liquid”   Cause :   can be caused by : -  Not enough liquid in the tube/troughs -  Deionized liquid in the tube/trough so the robot cannot detect it. -  ADN_SOFT underestimated the needed volume (in this case, please contact support@adnucleis.com ), -  Evaporation of the liquid because too much time occurred between filling and program run.   Corrective Task Please refill the liquid, and click on “Retry Detection”. NB : If the volume inside looks “OK” for the estimated needed aspiration but the volume is so low that the robot can’t detect it, you can click on “Move tips to Zmax”, the robot will aspirate to the bottom of the tube / trough.