TITLE: Salmonella detection with Kingfisher Flex/Apex extraction and 7500-FAST enrichment PCR
AUTHORS: Laura Goodman, Rebecca Franklin-Guild, Renee Anerson

[DESCRIPTION]
This procedure is used to test enrichment broth from environmental or animal specimens using the MagMAX™ CORE Nucleic Acid Purification Kit and the MicroSEQ Salmonella spp. Detection Kit.

The RVS enrichment broth should be prepared in advance to allow for quality control. The rest of the method is performed over two days:
Day 1 - sample setup and enrichment
Day 2 - DNA purification and real-time PCR

The steps for Kingfisher automated extraction were forked from "Extraction of bacterial DNA using MagMAX™ CORE Nucleic Acid Purification Kit on KingFisher™ Flex Instrument" (https://dx.doi.org/10.17504/protocols.io.81wgb781ovpk/v2)

[GUIDELINES]
Wipe down sample tubes and gloves with 10% bleach to minimize cross-contamination.

[STEPS]
SECTION: DNA Purification
24. Prepare plates for Robot
SECTION: DNA Purification
24.1. Prepare Wash Plate 1 by adding 500 μL of MagMAX™ CORE Wash Solution 1 to each well for each sample to be extracted plus controls.
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24.2. Prepare Wash Plate 2 by adding 500 μL of MagMAX™ CORE Wash Solution 2 to each well for each sample to be extracted plus controls.
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24.3. Prepare Elution Plate by adding 100 μL of MagMAX™ CORE Elution Buffer to each well for each sample to be extracted plus controls.
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24.4. Set Tip Comb in a 0.5 ml 96-Well Plate

 
       Plate setup of Processing Plates:   KingFisher™ Flex instrument                      Plate ID         Plate Type         Reagent         Volume Per Well            Wash Plate 1         Deep Well         MagMAX™ CORE Wash Solution 1          500 μL            Wash Plate 2         Deep Well         MagMAX™ CORE Wash Solution 2          500 μL            Elution Plate         Standard         MagMAX™ CORE Elution Buffer         100 μL            Tip Comb Plate         Standard         Place a tip comb in the plate      
 
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24.5. Prepare Bead/PK mix by adding 20 μL of MagMAX™ CORE Magnetic Beads and 10 μL of MagMAX™ CORE Proteinase K for the required number of samples plus 10% overage.
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24.6. Prepare Lysis/Binding solution by adding 350 μL of MagMAX™ CORE Lysis Solution and 350 μL of MagMAX™ CORE Binding Solution for for the required number of samples plus 10% overage. Mix by inverting the tube or bottle at least 10 times.
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24.7. Invert the tube of Bead/PK Mix several times to resuspend the beads, then add 30 μL of the Bead/PK Mix to the required wells in the 2.4 mL 96- deep well Sample Plate.
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24.8. Transfer 200 μL of RVS enrichment broth to a well with 30 μL Bead/PK mix in the Sample Plate. Seal the plate and shake vigorously for 2 minutes on a plate shaker at room temperature, or pipette mix them and incubate for 2 mins at room temperature.
 

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24.9. Add 700 μL of Lysis/Binding Solution to each well containing sample.
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25. Operate on KingFisher machine
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25.1. Select the appropriate script for your instrument if not already loaded:

Download the KingFisher Flex heated script: MagMAX CORE_Flex.bdz at https://www.thermofisher.com/order/catalog/product/A32700#/A32700, and then use BindIt sofware to transfer this script from a computer to the machine with the connection line.

For the KingFisher Apex, use the following script instead:
https://assets.thermofisher.com/TFS-Assets/BID/Methods-&-Protocols/MagMAX_CORE_Heated_v1.kfx

BindIt software can be requested and downloaded at the website https://www.thermofisher.com/us/en/home/global/forms/life-science/download-bindit-software-kingfisher-instruments.html
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25.2. Start the run, then load the prepared plates in the appropriate positions when prompted by the instrument. The instrument will direct the user as to which plate to load in what position.
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25.3. Load the Tip Comb Plate
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25.4. Load the Elution Plate
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25.5. Load the Wash Plate 2
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25.6. Load Wash Plate 1
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25.7. Lastly, load the Sample Plate and press Start. The program takes about 20 min.
SECTION: PCR Setup
26. Program the ABI 7500-FAST machine with the following settings (use fast mode):

     PCR   amplification conditions in ABI7500 Fast under Fast conditions, SDS v1.4        Stage      Reps      Temperature      Duration (sec)        Stage 1      1      95 ͦ C      20        Stage 2      40      95 ͦ C      03        60 ͦ C      30   
 

SECTION: Sample processing and enrichment
14. Thoroughly disinfect a biosafety cabinet (BSC) using a disinfectant spray. Place a piece of absorbent bench paper in the BSC after cleaning. Allow the BSC to run for several minutes before placing items in it.
SECTION: Sample processing and enrichment
15. Obtain the following and place into the running BSC:
Prepared aliquots of RVS (9ml per sample) to accommodate the total number of samples being tested plus one negative enrichment setup control. Positive controls are optional for this exercise. Retrieve additional aliquots of RVS if you will be adding positive controls.
Racks to hold 15 ml tubes
Lab towels soaked with 10% bleach contained within a plastic holder.
Small biohazard bag (to collect waste)
Labeled samples (organized in appropriate sized rack) 
Optional: one positive control culture each for Salmonella Typhimurium and Salmonella Dublin grown on blood agar
Sterile 10 µL loop (optional if using an in-house positive culture control)
SECTION: Sample processing and enrichment
16. For the Vet-LIRN Salmonella Interlaboratory Comparison Exercise, samples to be tested will be liquid suspensions of bovine intestinal scrapings. The test sample tubes contain 1ml of sample in a 15 ml tube. Important! The entire sample will be used; do not transfer the sample out of this tube at this time. 
SECTION: Sample processing and enrichment
17. Place the first sample in a tube rack and loosen the cap. Clean your entire gloved hand with the 10% bleach towel.
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18. Chose one 9ml RVS aliquot and pour the entire volume into the first sample tube. Use care to avoid back splashing. Recap the tube. Discard the empty aliquot tube in the biohazard waste.
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19. Clean your entire gloved hand with the 10% bleach towel. Place the inoculated tube, which now has both sample and RVS broth, back into the original rack. Clean your gloves again.
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20. Repeat steps 16-19 with the remaining samples. 
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21. Reserve one aliquot of RVS broth and label as negative setup enrichment control: this tube will remain un-inoculated. Note! After over-night incubation, the negative setup enrichment control will be extracted and tested by PCR along with the samples.
SECTION: Preparation of RVS broth and Quality Check
1. This broth is used for the enrichment of specimens for Salmonella spp. isolation. The Rappaport Vassiliadis (RVS) media has an expiration date of 6 months from the preparation date.

Determine batch size:
 
     RO   water      0.5 L       1 L      2 L      3 L      4 L      5 L        RVS   broth powder  HIMEDIA   #M1491      13.55 g      27.11 g      54.22 g      81.33 g      108.44 g      135.55 g   
 
SECTION: Preparation of RVS broth and Quality Check
2. Add half the amount of RO water needed for the batch and a sterile stir bar to a sterile
flask of appropriate size
SECTION: Preparation of RVS broth and Quality Check
3. Place the flask on a stir plate and start rotating the stir bar to “stir” the water
SECTION: Preparation of RVS broth and Quality Check
4. Add the entire amount of RVS powder to the flask while stirring
SECTION: Preparation of RVS broth and Quality Check
5. Add the remaining amount of water to the flask
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6. Heat the flask until the powder is completely dissolved 
SECTION: Preparation of RVS broth and Quality Check
7. Dispense 10 ml of the broth from the flask into sterile 20 x 125 mm screw-capped
tubes using sterile tubing and the DoseIt. Loosely cap each tube.
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8. Autoclave the tubes of broth for 15 minutes on liquid cycle @ 121oC
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9. Allow to cool to RT and then tighten caps.
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10. Label racks of tubes as “RVS Broth”, date made, initials,& expiration date (months)
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11. Perform QC using the fields below according to the manufacturer’s directions
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12. Store RVS Broth @ 2-8oC protected from light
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13. Quality Control Procedure:RVS Broth

RVS Broth/BP inoculated:Initials:Date:

BP examined:Initials:Date:
BP    :□growth    □no growth

BG/XLD inoculated:Initials:Date:

BG/XLD Subculture plates examined: Initials:Date:

Positive Control plates:
BG:    □typical colonies    □atypical colonies    □no growth    □mixed growth
XLD:□typical colonies    □atypical colonies    □no growth    □mixed growth

Final Results(check one)    Pass □        Fail/Batch Discarded □

Date:
Initials:

SECTION: PCR Setup
27. Set up reactions at ROOM TEMPERATURE (do not use cold blocks or ice). Chilled nucleic acid and lyophilized components will make resuspension of the reaction mixture difficult.

Slowly remove the rounded caps on the tube strips, one row at a time, and discard them.
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28. Add 30 µl of sample nucleic acid elution to the PCR tube containing the lyophilized master mix. Wait 3-5 seconds and then resuspend the reagent pellet by pipetting up and down 2-3 times. 

Important! It is critical to wait 3-5 seconds to avoid aspirating part of the still lyophilized PCR master mix into the pipette tip.
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29. Add 30 µl of the negative amplification control (supplied with the kit) to a PCR tube containing the lyophilized master mix. Wait for 3-5 seconds and then resuspend the reagent pellet by pipetting up and down 2-3 times.
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30. Add 30 µl of the positive amplification control to a PCR tube containing the lyophilized master mix. Wait for 3-5 seconds and then resuspend the reagent pellet by pipetting up and down 2-3 times.
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31. Cap the tubes with the flat caps provided with the kit. The cap strip must be seated level as shown in the picture. 

Place a small mark at one end of each of the strip caps in order to keep proper orientation and order of the tubes. Do not mark the sides of the tubes or place any marks over the main surface of the optical cap.

  


  
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32. Pulse spin the strip tubes in order to collect liquid in the bottom of the tubes and displace any air bubbles.
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33. Place Samples in ABI-7500 FAST thermocycler using the tube strip adapter. Balance the tubes on each side according to the manufacturer’s instructions (blank tubes may be used for this purpose if necessary). If you do not have this adapter or are using a different machine, you can transfer the reactions into a plate that is compatible with your setup if needed.

  
SECTION: PCR Setup
35. Following run, analyze the results using the automatic analysis setting. All samples should have a VIC (internal control) signal. If the VIC is >35 or undetermined, repeat the PCR using a 1:5 dilution of the sample in nuclease-free water and interpret based on the result that gives the lowest Ct for the VIC.

  

SECTION: PCR Setup
36. Save a copy of the original run file and export the data into an Excel file. Interpret the results according to the following guidelines:


 
 
     FAMa      VICb      Interpretation      Action        undetermined      detected      Not   Detected      None   – this result is final        <   35      disregard      Positive      Culture   confirmation is optional        ≥ 35      detected      Suspect      Culture confirmation is optional        undetermined      undetermined      Inconclusive      Culture confirmation is optional   

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37. Copy the results into the provided SecureSheet file. 
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34. Start the run.
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38. Optional: proceed with culture confirmation for samples with a Salmonella (FAM) Ct detected
SECTION: Sample processing and enrichment
23. Move the rack(s) to a 40-44oC incubator, with shaking preferred. Incubate overnight, at least 18-24 hours.
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22. Optional: Inoculate one positive control using a sterile loop.
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25.8. The elution plate contains the eluted DNA to be run on qPCR.