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The Genequence® Salmonella Microwell Assay for the Detection of Salmonella in a Variety of Foods

Laboratory Procedure MFLP-20
September 2004

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Health Products and Food Branch
Ottawa

Don Warburton and Premalatha Himawan
Evaluation Division
Bureau of Microbial Hazards, Food Directorate,
Postal Locator: 2204A1
HPFB, Ottawa, Ontario, K1A 0L2
Don_Warburton@hc-sc.gc.ca

1. APPLICATION

This method is applicable to the detection of Salmonella spp. in a wide variety of food products, including meat, poultry, fish and seafood, fruits and vegetables, eggs, nuts, flours, confectionery products, dairy products, spices, pet foods, and miscellaneous processed foods.

2. DESCRIPTION

The GENEQUENCE° Salmonella Microwell assay is a DNA probe-based diagnostic in kit format, which permits rapid and accurate detection of Salmonella spp. in foods. Results are available in approximately 48 hours. The test exhibits sensitivity equivalent to that of reference culture procedures and is capable of detecting the presence of as few as 1 Salmonella cell in a 25 g food sample when the specified enrichment protocols are used. The GENEQUENCE method is designed for use by qualified lab personnel who follow standard microbiological practices.

3. PRINCIPLE

The GENEQUENCE DNA hybridization test employs Salmonella-specific DNA probes labeled with horseradish peroxidase and a colorimetric endpoint for the detection of Salmonella spp. in food samples following broth culture enrichment. After sample pre-enrichment, selective enrichment, and post enrichment incubation periods, target bacteria are lysed enzymatically at 65°C and Salmonella -specific oligonucleotide probes are added for a 60 minute hybridization incubation at 45°C. If Salmonella ribosomal RNA (rRNA) is present in the test sample, detector probe directly labeled with horseradish peroxidase (HRP) and polydeoxyadenylic acid (poly dA)-tailed capture probe hybridize to the target organism rRNA sequences. Concurrently, base pairing between the poly dA-tailed capture probe and polydeoxythymidylic acid (poly dT) coated polystyrene microwells facilitates solid phase capture of the probe-target hybrid molecules. Unbound probe is removed by washing, and substrate-chromogen is added to each well. The reaction of HRP with substrate-chromogen produces a blue color. The reaction is stopped with the addition of sulfuric acid, which changes the color of the substrate from blue to yellow. A microwell plate or microwell strip reader (A ₄₅₀) measures absorbance; absorbance in excess of the threshold value indicates the presence of Salmonella spp. in the test sample. Positive assay results must be confirmed by standard culture methods.

4. DEFINITION OF TERMS

See Appendix A of Volume 3.

5. COLLECTION OF SAMPLES

See Appendix B of Volume 3.

6. MATERIALS AND SPECIAL EQUIPMENT

Note: The Laboratory Supervisor must ensure that the analysis described in this method is carried out in accordance with the International Standard referred to as "ISO/IEC 17025:1999 (or latest version): General Requirements for the Competence of Testing and Calibration Laboratories".

The media listed below are commercially available and are to be prepared and sterilized according to the manufacturer's instructions. See also Appendix G of Volume 3 for the formula of individual media.

Note: If the analyst uses any variations of the media listed here (either product that is commercially available or made from scratch), it is the responsibility of the analyst or Laboratory Supervisor to ensure equivalency.

1) Supplied with kit - (no. 6700; sufficient for 96 tests - Neogen Corporation, Phone: 517-372-9200, fax: 517-372-0108, web: www.neogen.com) (formerly know as Gene-Trak).

1 Microtiter plate, 96 coated wells in divisible strips
2 vials Lysis Reagent Concentrate (Reagent 1A)
1 bottle Lysis Reagent Buffer (Reagent 1B), 12 mL
1 bottle Hybridization Solution (Solution 2), 18 mL
1 bottle Salmonella Probe Solution (Solution 3), 6 mL
1 bottle Wash Solution 20X concentrate (Solution 4), 50 mL
1 bottle Substrate-Chromogen Solution (Solution 5), 15 mL
1 bottle Stop Solution (Solution 6), 5 mL
1 bottle Positive Control (5 mL)
1 bottle Negative Control (5 mL)
1 Hybridization/probe mixture chart
1 Package insert

Store microtiter plate, Positive Control, Negative Control, and Reagents 1A, 2, 3, and 5 at 2-8°C. Other reagents may be stored at 2-25°C. The entire kit may be stored at 2-8°C.
Once Reagent 1A has been reconstituted, it must be stored at -20°C and is stable for 60 days in this form.

Precautions: Stop Solution contains 4.0 N sulfuric acid. Hybridization Solution contains formamide. Avoid contact with skin and mucous membranes. Refer to Material Safety Data Sheet for more information.

2) Enrichment broths - vary by food type (see MFHPB-20)

Lactose broth (or other pre-enrichment medium as appropriate for sample type) Tetrathionate-brilliant green broth
Rappaport-Vassiliadis broth
Gram negative broth

3) Materials

Equipment

Blender or homogenizer
Incubator at 35°C
Incubator or water bath at 42°C
Water bath or heater block at 65°C
Small orbital platform shaker capable of 150 rpm (optional)
Heater block (with cover) or air incubator at 45°C
Vacuum source
Microtiter plate washing device with 8 wells per strip orientation
Micropipette to dispense 150 μL volume
Micropipette or repeater pipette to dispense 100 uL volume
8-channel pipette (recommended) or repeater pipette to dispense 50 μL, 125 μL, and 150 μL volume
Microtiter plate or strip reader at 450 nm with discrimination of at least 0.01 absorbance unit
500 mL or 1 L graduated cylinder
Test tube rack
Timer

Note: It is the responsibility of each laboratory to ensure that the temperature of the incubators or waterbaths is maintained at the recommended temperatures. Where 35°C is recommended in the text of the method, the incubator may be 35 ± 1.0°C. Similarly, lower temperatures of 30 or 25 °C may be ± 1.0°C. However, where higher temperatures are recommended, such as 43 or 45.5°C, it is imperative that the incubators or waterbaths be maintained within 0.5°C due to potential lethality of higher temperatures on the microorganism being isolated.

Supplies

Culture bottles for sample pre-enrichment
Culture tubes for 11 mL sample volume
Test tubes, glass, 12 x 75 mm
Tips for pipettes
Absorbent paper
10 mL or 25 mL pipettes

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7. PROCEDURE

7.1 Handling of Sample Units

7.1.1 Analyse samples as soon as possible, preferably within 24 h of receipt. During transport, with the exception of shelf-stable products, keep the sample units, refrigerated (0-5°C) or frozen depending on the nature of the product. Thaw frozen samples in a refrigerator, or under time and temperature conditions which prevent microbial growth or death. Foods which are partially thawed should be stored in a refrigerator.

Note: Use appropriate containers to ensure that drippings from the product do not contaminate the laboratory environment.

7.1.2 If the sample unit received for analysis is less than the recommended analytical unit, analyse the entire amount and record the weight used. Adjust the volume of enrichment broth to maintain a 1 in 10 dilution of the sample.

7.1.3 Use aseptic techniques and sterile equipment at all stages of analysis. Containment during the handling of powdered products is critical if cross-contamination of the work environment is to be avoided.

7.2 Preparation for Analysis

7.2.1 Have ready sterile enrichment broth and other media and supplies.

7.2.2 Clean the surface of the working area with a suitable disinfectant.

7.3 Preparation of Samples

Each sample unit may be analysed individually or the analytical units may be combined. Carry out the test in accordance with the following instructions:

7.3.1 To ensure a truly representative analytical unit, agitate liquids or free flowing materials until the contents are homogeneous. If the sample unit is a solid, obtain the analytical unit by taking a portion from several locations within the sample unit. To reduce the workload the analytical units may be combined for analysis. It is recommended that a composite contain no more than five analytical units.

7.4 Pre-Enrichment and Enrichment

7.4.1 Aseptically weigh 25 g or mL (the analytical unit) into a filter stomacher bag and add 225 mL of Nutrient broth. See MFHPB-20 (Volume 2) for the use of other enrichment broths. Incubate 22 +/- 2 hr. at 35°C.

Note: All enrichment media should be pre-warmed to room temperature before inoculation.

7.4.2 Remove pre-enrichment culture from incubation and mix well. Transfer 1 mL of pre-enrichment culture to 10 mL Tetrathionate Broth (TBG). Transfer 0.1 mL of pre-enrichment culture to 10 mL Rappaport Vassiliadis Broth (RV). Incubate TBG culture 18-24 h. at 35°C and RV culture 18-24 h. at 42°C.

7.4.3 Remove TBG and RV cultures from incubation and mix well. Transfer 1 mL of TBG culture to 10 mL Gram Negative broth (GN). Transfer 1 mL of RV culture to a second 10 mL of GN. Incubate GN cultures 6 hr. at 35°C. Save TBG and RV cultures in the refrigerator for possible confirmation.

Note: GN broths may be refrigerated overnight or up to 48 h prior to GENEQUENCE assay.

7.4.3 Perform GENEQUENCE assay using a pooled sample containing 0.2 mL of each GN culture. Save GN cultures in the refrigerator for possible confirmation.

7.5 Test Procedure

Note: All pipetting transfers should be made using either a disposable pipette and pipetting bulb or micropipette with disposable tips. Do not mouth pipette. This assay should be performed in a normal laboratory environment with respect to humidity, lighting, etc. Steps requiring room temperature incubation should be performed at 18-30oC. Kit reagents that have been refrigerated should be equilibrated to room temperature before use but should not be left out unrefrigerated for long periods of time.

7.5.1 Prior to starting the assay:

7.5.1.1 Turn on the water bath or heater block and adjust to 65°C. The water bath should be filled to a level of approximately 1.5 inches. The heater block wells should be filled about 1/3 with deionized water.

7.5.1.2 To Reagent bottle 1A (Lysis Reagent Concentrate), add 6 mL Solution 1B. (Lysis Reagent Buffer). Dissolve contents by gentle swirling and place the bottle on ice.

Note: Lysis Reagent is stable in the reconstituted form for 60 days when stored at -20°C. To thaw, place bottle at room temperature. When thawed, place on ice (freeze-thawing does not adversely affect the reconstituted reagent).

7.5.1.3 For each sample to be tested, label a 12 x 75 mm glass test tube with the appropriate samples designation and place in a rack. Include tubes for 1 Positive Control and 1 Negative Control per experimental run.

7.5.1.4 Prepare 1X Wash Solution. Dilute Solution 4 (20X Wash Solution Concentrate) by mixing contents of bottle (50 mL) with 950 mL distilled or deionized water. Fill buffer reservoir of plate washing device (see manufacturer's instructions for set-up and use).

Note: 1X Wash Solution can be stored in a closed bottle at room temperature for up to 60 days.

7.5.1.5 Prepare a mixture of Solution 2 and Solution 3 in an appropriate size plastic or glass container in a 4:1 proportion to achieve a sufficient amount for the number of samples to be tested. Use the formula below or refer to the mixing chart included with the test kit.

Volume Solution 2 = [(N x 0.1) + 1.6] mL
Volume Solution 3 = [(N x 0.025) + 0.4] mL

where N is the number of samples to be tested including controls

Note: The above formula provides for an excess volume of 2.0 mL; a different excess volume may be appropriate depending on the type of container used to prepare the mixture.

7.5.1.6 Place the appropriate number of coated microtiter wells in the plate frame, filling the frame left to right and front to back in rows of 8. Include wells for the reagent blank, Negative Control, and Positive Control. Avoid touching the bottoms of the wells. If the last row has fewer than 8 wells, fill in the row as necessary (special colored wells are available from Neogen Corp. for this purpose).

7.5.2. Assay Procedure

7.5.2.1 Mix the sample cultures (GN cultures). Add 0.2 mL of each of the two GN cultures for each sample to the appropriate tubes (0.2 mL of each of the two GN cultures for each sample pooled into one tube). Shake the Positive Control and Negative Control solutions and add 0.4 mL of each control to the appropriate tubes.

7.5.2.2 Add 0.1 mL Solution 1 (reconstituted Lysis Reagent) to each tube. Shake the rack of tubes by hand for 5 seconds. The resulting solution should be blue in color. If any tubes are not blue, check for proper reagent addition. Incubate the tubes in the 65°C water bath or heater block for 5 minutes.

7.5.2.3 Remove the tubes from the 65°C water bath or heater block. Transfer 0.150 mL of each lysed sample, including the controls, to the designated microtiter well. The first well should be reserved for the reagent blank and receives no sample. The second well should be used for the Negative Control, and the third well for the Positive Control.

7.5.2.4 Vigorously mix the Solution 2/3 (Hybridization/Probe Solution) prepared in step 7.5.1.5 above. Add 0.125 mL to each well except the reagent blank well. If using a multi-channel pipette, mix the contents of the wells by drawing up and dispensing back into the wells at least 5 times. Otherwise, shake the plate on an orbital shaker at room temperature for 2 minutes at 150 rpm.

7.5.2.5 Incubate the plate in a covered heater block or air incubator at 45°C for 60 minutes.

7.5.2.6 Wash the wells 5 times at room temperature using a microwell strip washing device. For each wash cycle, process one 8-well strip at a time, aspirating the liquid, filling the wells, and then proceeding to the next strip. After the last wash, aspirate the liquid from the wells, then remove residual liquid by inverting the plate and tapping it onto absorbent paper. Hold the plate by gently squeezing on the sides of the frame to keep the strips in place.

7.5.2.7 Add 0.15 mL Solution 5 (Substrate-Chromogen Solution) to each well, including the reagent blank well. Incubate the plate at room temperature for 20 minutes.

7.5.2.8 Add 0.5 mL Solution 6 (Stop Solution) to each well, including the blank well. Gently tap the side of the plate frame a few times to ensure complete mixing.

7.5.2.9 Read absorbance at 450 nm using a plate or strip reader according to the manufacturer's instructions. Blank using the first well containing substrate-chromogen and stop solution (do not blank with air).

7.6 Interpretation of Results

7.6.1 Control Values

The absorbance value for the Negative Control must be < 0.15. Otherwise, the assay is invalid and should be repeated.

The absorbance value for the Positive Control must be > 1.00. Otherwise, the assay is invalid and should be repeated.

7.6.2 Negative Criterion

Assays producing an absorbance value < 0.10 are indicative of the absence of Salmonella spp. in the test sample.

7.6.3 Positive Criterion

Assays producing an absorbance value > 0.10 are indicative of the presence of Salmonella spp. in the test sample. These samples must be confirmed by standard culture procedures.

7.7 Confirm Positive Results

Positive samples must be confirmed culturally as described in MFHPB-20. Spread plate to specified selective agars and confirm biochemically and serologically.

7.8 Limitations

The DNA probes used in this kit are not reactive with serovars of the species Salmonella bongori.

8. REFERENCES

8.1 D'Aoust and Purvis. 1998. Isolation and Identification of Salmonella from Foods. In Volume 2. The Compendium of Analytical Methods. Health Canada. http://www.hc-sc.gc.ca/food-aliment/mh-dm/mhe-dme/ compendium/volume_2/e_mfhpb2001.html

8.2 Technical Committee 34. Microbiology of food and animal feeding stuffs -Horizontal method for the detection of Salmonella spp. International Organization for Standardization. (ISO 6579:2002, 4^th ed). International Organization of Standards: Geneva. Available from http://www.iso.ch/iso/en/CatalogueDetailPage.CatalogueDetail?
CSNUMBER=29315&ICS1=7&IC S2=100&ICS3=30

8.3 US Department of Agriculture, Food Safety and Inspection Service, Office of Public Health and Science. Microbiology Laboratory Guidebook [Internet]. Washington: The Dept; c1998 [rev 2001 January 10; cited 2003 June]. Chapter 4, Isolation and Identification of Salmonella from Meat, Poultry, and Egg Products [about 14 screens]. Available from http://www.fsis.usda.gov/OPHS/microlab/.mlgchp4.pdf

8.4 US Food & Drug Administration, Center for Food Safety & Applied Nutrition.
Bacteriological Analytical Manual Online [Internet]. Washington: The Admin; c1998 [rev 2001 October; cited 2003 June]. Chapter 5, Salmonella [about 12 screens]. Available from
http://vm.cfsan.fda.gov/~ebam/bam-5.html