• Decrease font size
  • Return font size to normal
  • Increase font size
U.S. Department of Health and Human Services

Food

  • Print
  • Share
  • E-mail

FDA Survey of Domestic Fresh Produce - Domestic Produce Assignment (DFP # 05-20)

HIGH Priority ORA Concurrence # 2005052501 - Amendment to 6/10/05 Assignment

 

Memorandum 
Attachment A: Detection and Isolation of Shigella species from Produce.
Attachment B: Soak Method for the Detection of Salmonella in Basil, Cilantro, Scallions/Green Onions, Loose-Leaf Lettuce, Parsley, and Spinach
Attachment C: Soak Method for Salmonella analysis in Cantaloupe
Attachment D: Tomato Soak Method for Salmonella analysis
Attachment E: Protocol for the Detection of Viruses in Produce

 Memorandum

Date: August 3, 2005
From: Consumer Safety Officer, Division of Field Programs
Through: Chief, Compliance Programs Branch, HFS-636 /s/
Subject: Domestic Produce Assignment (DFP # 05-20) - HIGH Priority
ORA Concurrence # 2005052501--Amendment to 6/10/05 Assignment
To: District Directors: (#)
Directors, Regional Laboratory: (#)
Director, Laboratory Branch: (#)
Director, Microbiology Branch: (#)
Directors, Compliance Branch: (#)
Info: Regional Food and Drug Directors: ALL
Directors, Investigations Branch: (#)
Field Food Committee Chairperson
Field Food Program Monitors: (#)

Note: Material that is not releasable under the Freedom of Information Act (FOIA) has been redacted/deleted from this electronic version of the program. Deletions are marked as follows: (#) denotes one or more words were deleted; (&) denotes one or more paragraphs were deleted; and (%) denotes an entire attachment was deleted.

Note: Assignment has been amended to expand sample collection directions and clarify reporting of presumptive positive analytical results. In addition, a new method for Shigella is included as revised Attachment A. The assignment is being re-transmitted in its entirety for ease of reference. Modifications are noted by asterisks and bold font.

OBJECTIVES

  • To expand surveillance of fresh produce, including focused sampling of fresh produce with a history of association with illness outbreaks, and sampling of a broader range of produce items to obtain baseline data on the incidence of contamination with pathogens.
  • To take appropriate regulatory actions on lots found contaminated, including follow-up inspections of growers, packers and/or distribution centers, where warranted, to help identify conditions and practices that could lead to, or spread, contamination.

BACKGROUND

Over the past fifteen years, the number of foodborne-outbreak associated illnesses associated with fresh fruits and vegetables has increased. In the 1990s, the number of foodborne-outbreak-associated illnesses associated with fresh fruits and vegetables doubled from the previous decade. CDC estimates that, in the 1990s, at least 12 percent of all foodborne-outbreak-associated illnesses implicated fresh fruits and vegetables. Since the 1990s, FDA has focused significant resources on reducing foodborne illness from all sources. However, despite these efforts, foodborne illness associated with fresh produce has not decreased.

Fresh fruits and vegetables are grown in fields and orchards. Consequently, surfaces of produce during growth become contaminated with non-pathogenic and possibly pathogenic microorganisms. In addition to growing conditions, packaging and further processing (e.g., cutting, slicing, packaging) under insanitary conditions provides additional opportunities for contamination of harvested produce with pathogens. Fresh produce is of special concern because it is likely to be consumed without further treatment to "destroy or remove" microorganisms. The increase in foodborne illnesses associated with fresh produce may be due, in part, to increased ability to detect foodborne illness outbreaks and to an increase in the consumption of fresh produce. CFSAN has conducted a number of assignments since 1999 to gather data on the incidence and extent of pathogen contamination on selected produce, both imported and domestic. This assignment is being issued to continue this effort and is specified in the FY05 WorkPlan under Import and Domestic Micro Assignments.

The "Produce Safety from Production to Consumption: 2004 Action Plan to Minimize Foodborne Illness Associated with Fresh Produce Consumption" (the Action Plan) released in October 2004 reiterates the importance of the imported and domestic produce assignments. The overarching goal of the Action Plan is to minimize the incidence of foodborne illness associated with the consumption of fresh produce. To achieve this goal, the Action Plan has four general objectives: 1) Prevent Contamination of Fresh Produce with Pathogens; 2) Minimize the Public Health Impact When Contamination of Fresh Produce Occurs; 3) Improve Communication with Producers, Preparers, and Consumers about Fresh Produce; and 4) Facilitate and Support Research Relevant to Fresh Produce. For each objective, the Action Plan identifies steps to achieve the objective. The second and fourth objectives are applicable to the domestic and imported produce assignments.

In the second objective, "Minimize the Public Health Impact When Contamination of Fresh Produce Occurs," three of the steps to achieve this objective directly relate to the produce assignments. They are to:

  • Expand surveillance of fresh produce including focused sampling of fresh produce with a history of association with illness outbreaks and expanded sampling of a broader range of produce items to obtain baseline data on the incidence of contamination;
  • Use facility inspections and surveys to increase routine monitoring of practices and conditions in the fresh produce supply chain including farms, packing facilities, and distribution centers, to identify practices that could lead to the spread of contamination in produce; and
  • Continue to improve and refine appropriate regulatory follow up when insanitary conditions are identified or contamination of fresh produce is found.

In the fourth objective, "Facilitate and Support Research Relevant to Fresh Produce," the information collected in the domestic and imported produce assignments will be an important source of information to proceed with several of the steps included in this objective.

Produce commodities are selected for sampling based on results of previous assignments, recent foodborne illness outbreaks, and other data needs such as those outlined in the Action Plan. The elements of the assignment including pathogens targeted and analytical methods are kept as constant as possible for continuity between assignments and to facilitate completion of the assignment. Where changes from previous versions are necessary, they will be noted.

Over the past few years, there have been several studies involving the ability to recover and detect pathogen contamination of fresh produce. Most often, analyses involve rinsing the outside of the fruit or vegetable and analyzing the rinse water for pathogens. However, for some commodities and pathogens, the rinse method has a very low incidence of pathogen recovery and detection. For example, it is difficult to recover pathogens from the rough surface of cantaloupe. Pathogen recovery is significantly improved if cantaloupe is soaked rather than rinsed. If pathogens have been internalized into the product, a surface rinse may again fail to detect their presence. Internalization is a particular concern for microbiological analysis of tomatoes. Prior to the most recent assignment, a study was done within CFSAN to analyze the accuracy of two different Salmonella soak methods, one for cantaloupe and another for whole tomatoes. The study determined that the soak methods yielded more accurate results than when these two commodities were examined by the rinse method (fewer false negative results). In this assignment, the soak methods will be used for the Salmonella analyses of all commodities, with modified soak methods for cantaloupe and for tomatoes.

This assignment is the domestic counterpart to the recently issued Import Produce Assignment (DFP # 05-16).

APPROACH

This assignment is to be implemented upon receipt. Each district should collect samples as seasonally available. The assignment will continue through September 30, 2005 if needed to meet sampling goals.

SAMPLE COLLECTION AND SHIPMENT

Sample Collection

The following table shows the collecting districts, products to be collected, and the number of requested samples per commodity per district.

Commodity District
Cantaloupe  (#)
Tomatoes
Loose-leaf lettuce
Scallions/Green Onions - split sample
Spinach
Cilantro
Basil
Parsley
Totals per district

Each district should collect samples indicated as available. If product availability requires districts to substitute commodities between other commodities from the table to meet overall district sampling obligations, this will be acceptable. Districts should attempt to meet the totals per district. All samples should be field grown commodities where microbial contamination may be of concern due to poor agricultural practices on the field and/or poor manufacturing practices, such as at packing houses, i.e., districts should not knowingly collect hot house or greenhouse produce. Only produce grown in the United States is to be collected. Districts should collect locally grown produce (i.e., same or neighboring state). Districts should continue to collect samples until their obligation is fulfilled or until September 30, 2005.

Collect investigational samples. Samples must be officially sealed to ensure integrity. It is not necessary to document interstate commerce at the time of collection, although you must identify the grower.

CFSAN recommends that all collections be made at packers; however, if a district *is unable to collect samples at a packer and the district is able to: 1) identify the grower, and 2) determine that the product has not been combined with product from other growers then the district may collect samples at a re-packer or wholesaler. When collecting samples at a packer or repacker, describe on the collection report where in the process the sample was collected (e.g., incoming product, after washing, final box package). If other than incoming product, note handling steps performed on product at that facility (e.g., wash, repack, ripening room, etc.).*

If readily available, obtain either an actual label or a photograph of a label along with the codes on each container sampled.

If a dealer informs the FDA investigator that a sampled lot will be held pending receipt of FDA analytical results or specifically requests that FDA inform him or her of the results, the investigator will coordinate notification to the dealer of the *presumptive positive results once the results are reported by servicing lab*. The initial notification may be done by district or lab personnel and made by telephone.

The intent of this assignment is to collect samples from as large a number of growers as possible. Districts should not collect samples of the same product/grower combination under this assignment without prior CFSAN concurrence.

For all products, a sample will consist of ten (10) sub-samples (no 702(b) required). For all products except cantaloupe and tomato, each sub-sample will consist of 454 grams (1 pound). For cantaloupe, each sub-sample will consist of two cantaloupes. For tomatoes, each sub-sample will consist of 900 grams (2 pounds).

NOTE: When two laboratories are involved with the analysis of scallions/green onions, districts should collect a split sample. Split sample indicates the sample is to be collected in duplicate under one sample number; one portion of the sample is sent to one laboratory and another portion is sent to another laboratory. Each split sample must consist of ten sub-samples.

Coordinate shipment and analysis of samples with the analyzing laboratories before collecting to ensure that the sample will be analyzed expeditiously.

Aseptic Sampling

Collect all samples ASEPTICALLY; see IOM, Chapter 4, Section 426.

Note: It is important that each sub-sample be collected into a separate bag, and controls (i.e., open and unopened collection bags, and unopened sterile disposable gloves) be submitted with the sample. All controls must be identified and placed in the container with the sample. analysis.

Collect sub samples at random to ensure that the sample is representative of the lot.

Sampling containers for cantaloupe samples:
If whirl pack bags of sufficient size are unavailable, investigators may use unopened, new, small commercial garbage or trash bags. Previous experience indicates that the interiors of these bags do not support the growth of bacteria. If these containers are used, then an unopened intact bag must also be submitted to the laboratory as a control.

Sample Shipment

Ship samples to servicing laboratories according to the following table:

Commodity Servicing Laboratories
Cantaloupe  (#)
Tomatoes
Loose-leaf lettuce
Scallions/Green Onions - split sample
Hepatitis A
All other analyses
Spinach
Cilantro
Basil
Parsley

Because fresh produce is highly perishable, ship refrigerated samples by the fastest means possible and coordination should be made by the District to the servicing labs for this assignment. Consideration should also be made to not collect samples late in the day if they cannot be prepared and shipped the same day.

Ensure that the coolant used does not come in direct contact with the commodity.

REPORTING REQUIREMENTS

Data Reporting

Laboratories are required to report positives and monthly sample status to headquarters.

Report all analytical finding into FACTS using PAF = MIC (problem code MICROID, PFGE, SERO as appropriate); secondary PAF = SAL, ABR.

Data Reporting Requirements:

Cantaloupe: 22A [ ] B01 Raw
22A [ ] C01 Raw, Fresh, Refrigerated
Tomatoes: 24F [ ] B50 Raw
24F [ ] C50 Raw, Fresh, Refrigerated
Loose-leaf Lettuce: 24T [ ] B32 Raw
24T [ ] C32 Raw, Fresh, Refrigerated
Scallions, Green Onions: 25J [ ] B04 Raw
25J [ ] C04 Raw, Fresh, Refrigerated
Spinach: 24T [ ] B25 Raw
25J [ ] C04 Raw, Fresh, Refrigerated
Cilantro: 24T [ ] B46 Raw
24T [ ] C46 Raw, Fresh, Refrigerated
Basil: 28A [ ] B04 Raw
28A [ ] C04 Raw, Fresh, Refrigerated
Parsley: 24T [ ] B21 Raw
24T [ ] C21 Raw, Fresh, Refrigerated
PAC: Report all domestic operations accomplished under PAC 03F100.

 

ANALYTICAL

*Negative or presumptive positive test results for Salmonella and Shigella will be available within 3 days from receipt in the lab. Hepatitis A negative or presumptive positive test results will be available within 5 days from receipt in the lab. E. coli negative results will be available in 3 days and confirmed positive and MPN within 7 days from receipt in the lab. E. coli 0157:H7 negative results will be available in 3 days and presumptive positive results will be available within 5 days from receipt in the lab.

Presumptive positive findings must be reported to the CFSAN assignment contact carrie.lawlor@fda.hhs.gov and the collecting district once known. The collecting district in coordination with the servicing laboratories needs to notify the firm immediately of presumptive positive results or when FDA's test results are negative.*

ANALYTICAL RESULTS
Microorganism Presumptive Positive Positive
E. coli Not Applicable IMVIC, API20E, VITEK GNI+, modified 3.21.05
E. coli 0157:H7 Absence of Sorbitol fermentation on TC-SMAC agar and Spot Indole Test, modified 3.21.05 Serology and PCR
Salmonella VIDAS (api20E or VITEK GNI+) and Salmonella antiserum H and O Groups
Shigella First round PCR (of amplified product and/or microbiology with PCR analysis of colonies) using ipaHF/ipaHR primers Nested PCR (of amplified product and/or microbiology with PCR analysis of colonies) using ipaH3/ipaH4 primers
Hepatitis A BG7/BG8 Sequencing

*The analyzing laboratory should immediately notify the collecting district and the Center assignment and compliance contacts if any of the following pathogenic microorganisms are detected and confirmed:

  • Salmonella
  • E. coli 0157:H7
  • Shigella
  • Hepatitis A

NOTE: The Laboratory should notify the CFSAN Assignment Contact (Carrie Lawlor at carrie.lawlor@fda.hhs.gov) when levels equal to or greater than 100 MPN of E. coli are found.*

At the first of each month, each laboratory is requested to e-mail a list of samples received and/or completed during the previous month to the DFS contact (Atin Datta at atin.datta@fda.hhs.gov). The information will include the sample number, product, sampling district and analysis results (if completed). This information will be compiled and sent to CFSAN assignment contact (Carrie Lawlor at carrie.lawlor@fda.hhs.gov). The labs will immediately notify the DFS contacts and the CFSAN assignment contact of positive samples by e-mail. Laboratories should also immediately notify the collecting district.

Sample Analysis

  1. Analyzing laboratories: (#)

    Due to the perishable nature of fresh produce, it is important that analyses start immediately upon arrival of samples to the laboratories. Samples should not be frozen at any time prior to analysis.

    Use the following table to determine which commodities to sample for each pathogen.

      E. coli E. coli 0157:H7 Salmonella Shigella Hepatitis A
    Cantaloupe X   X X  
    Tomatoes X   X X  
    Loose-leaf lettuce X X X    
    Scallions/Green Onions X   X X X
    Spinach X X X    
    Cilantro X X X X  
    Basil X   X    
    Parsley X X X X  
    Salmonella cannot be enumerated when detected by the soak method.
  2. Sample splitting:

    Tomato and Cantaloupe
    Each sub sample will be split into two approximately equal portions. One portion of each sub sample will be analyzed for E. coli, and Shigella using the sub sample rinse method. The other portion of each sub sample will be used for Salmonella testing as per the specialized soak methods for the particular commodity.

    Loose-leaf lettuce and Spinach
    Each sub sample will be split into two approximately equal portions. One portion of each sub sample will be analyzed for E. coli and E. coli O157:H7 using the sub sample rinse method. The other portion of each sub sample will be used for Salmonella testing as per the specialized soak method for the particular commodity.

    Scallions/Green Onions
    Each sub sample will be split into three portions. One portion of each sub sample will consist of 50 grams and will be analyzed for Hepatitis A using the protocol for the detection of viruses in produce (Attachment E). The remaining quantity will be split into two approximately equal portions; one portion will be analyzed for E. coli and Shigella using the sub sample rinse method, and the other portion of each sub sample will be used for Salmonella testing as per the specialized soak methods for the particular commodity.

    Cilantro and Parsley
    Each sub sample will be split into two approximately equal portions. One portion of each sub sample will be analyzed for E. coli, E. coli O157:H7, and Shigella using the sub sample rinse method. The other portion of each sub sample will be used for Salmonella testing as per the specialized soak method for the particular commodity.

    Basil
    Each sub sample will be split into two approximately equal portions. One portion of each sub sample will be analyzed for E. coli using the sub sample rinse method. The other portion of each sub sample will be used for Salmonella testing as per the specialized soak method for the particular commodity.

  3. Sub-sample Rinse Preparation

    Pre-sample preparation for all products that are to be analyzed by the Rinse Method.

    To conduct meaningful bacterial analyses, the samples must be prepared for analysis in a manner that closely simulates the actions typically taken by consumers who provide minimal preparation (e.g., washing and/or trimming) prior to consumption.

    All product analyses except Salmonella: Perform a light rinse to remove visible dirt. "Light rinse" means to place produce commodity under cold running tap water for several seconds until visible dirt is removed. For Salmonella analyses only, a preliminary rinsing of the produce under running tap water is not recommended.

    For each individual sub sample, place contents into a sterile plastic bag. Add a volume of Butterfield's phosphate buffer solution that is equal to the weight of the product. For instance, if one sub sample weighs 500 g, add 500 ml Butterfield's phosphate buffer solution (1:1 dilution). Gently shake the bag with contents for 5 minutes using a shaker (e.g., orbital) at 100 rpm. This is considered to be the "sub sample rinse". Do not discard the "sub sample rinse" until the entire analysis is completed. Refrigerate the remaining rinse.

  4. Sub-Sample Rinse Method for
    1. E. coli (Cantaloupe, tomatoes, loose-leaf lettuce, scallions/green onions, spinach, cilantro, basil, and parsley)

      From each sub sample rinse (10 analyses/sample):

      Prepare decimal dilutions by removing 50 ml (of sub sample rinse) into 450 ml of Butterfield's phosphate buffer solution (1:10). Then follow methodology as outlined in the BAM for E. coli.

      NOTE: E. coli analysis: inoculation of the LST tubes will be conducted from 10-1 to 10-5 dilutions, only. It will not be necessary to prepare/use tubes for dilutions greater than 10-5for an end-point. Therefore, the maximum result that can be encountered would be > 110,000 MPN/g.
      The Laboratory should notify the CFSAN Assignment Contact (Carrie Lawlor at carrie.lawlor@fda.hhs.gov) when levels equal to or greater than 100 MPN of E. coli are found.
    2. E. coli O157:H7 (Loose-leaf lettuce, spinach, cilantro, and parsley)

      From each sub sample rinse (10 analyses/sample):

      Remove 125 ml (of sub sample rinse) and place in a sterile beaker/flask with 125 ml 2X EEB to perform the E. coli O157:H7 analysis. Then follow methodology as outlined in BAM in Chapter 4a, Sections K-Q.

      See BAM Ch. 4a, Section M.

      DO NOT USE THE OPTIONAL METHOD DESCRIBED THAT USES DYNA BEADS.

      Positive E. coli O157: H7

      Submit one (1) isolate from each E. coli O157:H7 positive sub sample to the specified laboratories per the instructions for PFGE and ribotyping. All laboratories are to send isolates for PFGE and/or ribotyping analyses within 24 hrs after completion of the analytical portion of the sample analysis.

      All cultures should be shipped by FedEx overnight and should conform to the rules and regulations regarding the shipment of infectious agents. Consult your supervisor if you have any questions.

      Ensure that the appropriate servicing laboratory is selected and identified in FACTS - MIC screen.

      Submit E. coli O157:H7 isolates for Pulse Field Gel Electrophoresis (PFGE) assay to respective servicing laboratories.

      Submit E. coli O157:H7 isolates for ribotyping only to:

      (#)
      Regional Laboratory (HFR-PA260)
      19701 Fairchild
      Irvine, CA 92612

      Submit E. coli O157:H7 isolates for antibiotic sensitivity assay to:

      (#)
      6th Avenue & Kipling Street
      DFC BLDG. 20
      Denver, Colorado 80225-0087
      (#)

    3. Shigella Only analyze cantaloupe, tomatoes, scallions/green onions, cilantro, and parsley for Shigella.

      See Attachment A: Detection and Isolation of Shigella species from Produce.

      Shigella analysis must be done on a composite basis (i.e., 2 composites per sample). Each composite for Shigella analysis will consist of 250 ml.

      If the district laboratories do not have PCR primers and the positive control strain, please notify Keith Lampel, Ph.D., the CFSAN scientific contact for Shigella.

  5. Soak Method for
    1. Salmonella - Do not use this method for tomatoes or for cantaloupe

      Salmonella analysis will be done on a composite basis (i.e., 2 composites per sample). Each composite for Salmonella analysis will consist of 375 ml.

      Prepare each composite by removing 75 grams from each of five (5) sub samples into a sterile beaker/flask. Add 3375 ml of appropriate pre-enrichment broth (see Attachment B).

      Incubate 24+2 h at 35° C. Then follow the methodology as outlined in BAM On-line, for Salmonella, Chapter 5.

      Screen all samples for Salmonella using rapid methods listed in the memo entitled, "Guidance for the Use of Rapid Methods for Food Microbiology" dated April 24, 1998. If the laboratory does not have a copy of the memo, they should request a copy from the Division of Field Science, HFC-140.

      If there is a presumptive positive based on the test kit, then perform:

      [1] Confirmation analyses as outlined in the BAM.

    2. Special Salmonella method only for use on cantaloupe:

      See Attachment C: Soak Method for Salmonella analysis in Cantaloupe.

    3. Special Salmonella method only for use on tomatoes:

      See Attachment D: Tomato Soak Method for Salmonella analysis

    4. Salmonella Positive Isolates

      One (1) isolate from each Salmonella positive composite or sub sample is to be sent to each of the specified laboratories per the instructions for serotyping (speciation) and PFGE.

      Following speciation, one slant from each serotyped isolate will be sent for antibiotic resistance testing.

      All laboratories are to send Salmonella isolates for serotyping and PFGE analyses within 24 hrs after completion of the analytical portion of the sample analysis.

      All cultures should be shipped by FedEx overnight and should conform to the rules and regulations regarding the shipment of infectious agents. Consult your supervisor if you have any questions.

      For Salmonella serotyping, all bacterial cultures should be prepared and submitted according to the directions specified in the Bacteriological Analytical Manual (BAM), in Chapter 5, E.11, "Submission of cultures for serotyping."

      Ensure that the appropriate servicing laboratory is selected and identified in FACTS - MIC screen.

      The serotyping (speciation) laboratory should batch isolates of Salmonella for antibiotic resistance testing (20 isolates per batch) and ship to the identified laboratory for antibiotic resistance testing.

      An electronic mail should be sent to the recipient before the shipment.

      Submit Salmonella isolates for serotyping to the laboratories listed below.

      Isolates from (#) will be serotyped in ARL:

      Arkansas Regional Laboratory
      3900 NCTR Road Building 26
      Jefferson AR 72079
      (#)

      Isolates from (#) will be serotyped in DEN:

      Denver District Laboratory
      6th Avenue & Kipling Street
      DFC Building 20
      Denver Colorado 80225-0087
      (#)

      Serotyping laboratories submit Salmonella isolates for antibiotic sensitivity assay to:

      (#)
      6th Avenue & Kipling Street
      DFC BLDG. 20
      Denver, Colorado 80225-0087
      (#)

      Submit Salmonella isolates for Pulse Field Gel Electrophoresis (PFGE) assay to respective servicing laboratories.

  6. Hepatitis A Only analyze scallions/green onions for Hepatitis A

    See Attachment E: Protocol for the Detection of Viruses in Produce.

REGULATORY/ADMINISTRATIVE FOLLOW-UP

*Presumptive positive findings must be reported to the CFSAN assignment contact (carrie.lawlor@fda.hhs.gov) and the collecting district once known. The collecting district in coordination with the servicing laboratories needs to notify the firm immediately of presumptive positive results or when FDA's test results are negative.* The analyzing laboratory should immediately notify the collecting district and the Center assignment and compliance contacts if any of the following pathogenic microorganisms are detected and confirmed:

  • Salmonella
  • E. coli 0157:H7
  • Shigella
  • Hepatitis A

NOTE: The Laboratory should notify the CFSAN Assignment Contact (Carrie Lawlor at carrie.lawlor@fda.hhs.gov) when levels equal to or greater than 100 MPN of E. coli are found.

In most cases, the contaminated produce will have already been distributed by the time sample results become available. Therefore, the collecting district should encourage the firm to conduct a voluntary recall of any affected produce still in the marketplace and press should be considered based on current agency guidance (see Regulatory Procedures Manual, Section 7-7-3, Public Warning). The collecting district should encourage the firm to either recondition the produce to destroy the pathogen or to destroy the product if reconditioning is not possible. If the lot is available and voluntary corrective action is rebuffed, seizure can be pursued charging adulteration within the meaning of 21 U.S.C. 342(a)(1), provided interstate commerce is documented. If interstate commerce is not documented, but the lot is available and voluntary corrective action is rebuffed, the collecting district should immediately refer the lot to their local state regulatory officials.

Once a pathogen is detected and confirmed in a produce sample, CFSAN will issue an assignment for inspection of the firms directly involved in the production and distribution of that product to determine their conditions and practices for handling the produce, coupled with identifying possible sources and routes of contamination. An inspection of the distributor, packing shed and grower should be conducted. Once CFSAN learns of a positive that is traceable to a grower, packer and/or processor, a request to the home district will be issued to conduct a follow-up inspection. The inspection at the grower should follow the guidance contained in the Guide to Produce Farm Investigations (DFI is finalizing the guide, which should issue shortly. If the guide is not available on the DFI website, and is needed for follow-up to a positive sample, please contact Barbara Marcelletti, DFI). During inspections of the growers, the investigative team should encourage the farm's management to become familiar with FDA's Guide to Minimize Microbial Food Safety Hazards for Fresh Fruits and Vegetables, which the farmer can access through FDA's website.

SUMMARY/EVALUATION

The Office of Plant and Dairy Foods will prepare a summary and evaluation of the findings within 120 days of the completion of the assignment.

CONTACTS

CFSAN Assignment Contact:

Carrie Lawlor, (301) 436-2068
Office of Compliance, Division of Field Programs
Compliance Programs Branch, HFS-636

CFSAN Domestic Regulatory Contacts:

Dennis Dignan, (301) 436-2051
Office of Compliance, Division of Enforcement
Domestic Branch, HFS-607

Don Greaves, (301) 436-2057
Office of Compliance, Division of Enforcement
Domestic Branch, HFS-607

Brandon Bridgman, (301) 436-2073
Office of Compliance, Division of Enforcement
Domestic Branch, HFS-607

Dwayne Johnson, (301) 436-1782
Office of Compliance, Division of Enforcement
Domestic Branch, HFS-607

CFSAN Program Office Contact:

Amy Green, (301) 436-2025
Office of Plant and Dairy Foods
Division of Plant Product Safety
Regulatory and Policy Branch, HFS-306

CFSAN Scientific Contacts:
E. coli and E. coli O157:H7:

Peter Feng, PhD, (301) 436-1650
Office of Plant and Dairy Foods
Division of Microbiological Studies
Microbiological Methods Development Branch, HFS-516

Shigella:

Keith A. Lampel, PhD, (301) 827-8617
Office of Applied Research and Safety Assessment
Division of Virulence Assessment
Virulence Mechanisms Branch, HFS-025

Salmonella:

Wallace H. Andrews, PhD, (301) 436-2008
Office of Plant and Dairy Foods
Division of Microbiological Studies
Microbiological Methods Development Branch, HFS-516

Hepatitis A:

Biswendu B. Goswami, PhD, (301) 827-8627
Office of Applied Research and Safety Assessment
Division of Molecular Biology
Virulence Mechanisms Branch, HFS-025

ORA Analytical Inquiries:
Atin Datta, PhD, Division of Field Sciences, HFC-141 at (301) 827-1030.

Marsha Hayden, Division of Field Sciences, HFC-141 at (301) 827-1039.

ORA Investigational Contact: Barbara Marcelletti, Division of Field Investigations, HFC- 130 at (301) 827-5635.

ATTACHMENTS

Attachment A: Detection and Isolation of Shigella species from Produce.
Attachment B: Soak Method for the Detection of Salmonella in Basil, Cilantro, Scallions/Green Onions, Loose-Leaf Lettuce, Parsley, and Spinach
Attachment C: Soak Method for Salmonella analysis in Cantaloupe
Attachment D: Tomato Soak Method for Salmonella analysis
Attachment E: Protocol for the Detection of Viruses in Produce

 

BKAloi for
Carrie Lawlor

(#)

 

 Attachment A - Revised 8/2/2005

Domestic Produce Assignment FY 2005 DFP # 05-20
Detection and Isolation of Shigella species from Produce

Notes:
Microbiological analysis and PCR are to be performed concurrently. PCR analysis should be performed on both the amplified product and suspect colonies.
Addition of a positive control spike of the rinse will run in parallel to the sample analysis (conventional and molecular) throughout the method.

Media and reagents:

For preparation of media and reagents, refer to the Bacteriological Analytical Manual (BAM). The number in parentheses following each medium refers to its listing in the media section of the BAM.

Media

  1. Shigella broth with novobiocin (M136)
  2. MacConkey agar (M91)
  3. Hektoen Enteric agar (M61)
  4. Xylose Lysine Desoxycholate agar (M179)
  5. Triple sugar iron (TSI) agar (M149)
  6. Motility test medium (semisolid) (M103)
  7. Decarboxylase basal medium (lysine, Falkow) (M44), or lysine iron agar (M89)

Reagents

  1. 1N Sodium hydroxide solution (R73)
  2. 1N Hydrochloric acid (R36)

Enrichment in broth medium

  1. Split the 250 ml, five-sub composite rinse to obtain two 125 ml rinse aliquots. One aliquot will serve as a positive control.
  2. Transfer each sub sample rinse (125 ml each) to one of two sterile 500 ml Erlenmeyer flasks containing 125 ml of 2X Shigella broth with novobiocin (final concentration should be 0.5 ug/ml). The positive control rinse aliquot will be spiked at a level of < 60 cfu/250 ml of rinse/enrichment broth.
  3. Adjust pH, if necessary, to 7.0 ± 0.2 with sterile 1 N NaOH or 1 N HCl.
  4. Incubate at 40oC +/- 2oC overnight (16 to 24 hours), aerobic conditions, with no shaking.

Bacteriological testing:

Procedure

  1. Streak a loopful of enrichment and positive control broths onto MacConkey (MAC), Hektoen (HE), and Xylose Lysine Desoxycholate (XLD) agar plates immediately after enrichment.
  2. Incubate plates overnight (16 to 24 hrs) at 37°C +/- 2°C.
  3. Identify suspect colonies.
  4. Prepare lysate from suspect colonies and test via PCR protocol (first round and nested). Include one positive control colony from each agar type.
  5. Inoculate as many as six to twelve suspicious colonies into the following media: TSI agar slant, lysine decarboxylase broth or lysine iron agar slant, and motility agar. Pick from the center of the colony without touching the agar). Include one positive control colony from each agar type.
  6. Incubate tubes at 37°C + 2°C for 20 hrs, but also examine after 48 hrs.

Examination/results

  1. Follow standard microbiological criteria for examining test results.
  2. The characteristics of Shigella are summarized as follows:
    1. MAC agar: Shigella colonies are translucent, with or without rough edges.
    2. HE agar: Shigella colonies are approx. 2-3 mm and appear green.
    3. XLD agar: Shigella colonies are 1-2 mm diameter, transparent or red, smooth colonies.
    4. TSI: K/A, H2S-, no gas
    5. Negative for motility
    6. Negative for production of lysine decarboxylase

PCR

  1. Note: If enrichment broth culture appears to contain large amounts of plant material and debris, decant approximately 50 ml of the enrichment broth cultures from sample and spiked rinse into centrifuge tubes (50 ml). Perform a slow-speed spin (2000 rpm for 2 minutes) to remove debris. Carefully transfer 1.0 ml of supernatants to fresh micro centrifuge tubes. Centrifuge at 14,000 rpm for 3 minutes to pellet bacterial cells. Remove as much of the supernatant as possible (to avoid leaving behind any inhibitory compounds; aspiration may be a better alternative than decanting the supernatant).
  2. Add 50 - 500 µl of 1 X phosphate buffer solution (PBS) (this will depend upon the size of the pellet). If the pellet is hardly visible, then add the smallest amount of 1 X PBS. PBS formulation: 11.42 grams NaCl, 1.08 grams Na2HPO4, 0.315 grams KH2PO4, 1.5 liters dH2O pH 7.4
  3. For PCR template preparation: Boil the cell suspension in a water bath for 5 minutes, cool on ice and centrifuge at 14,000 rpm for 3 minutes. Transferring the supernatant to another tube is not necessary; use the supernatant as template without disturbing the pellet.
  4. PCR setup

    The following is a typical set up for PCR methodology.

    dH20 4-6.5 µl
    Mix 12.5 µl 10 X stock (DNA polymerase buffer*)
    Primers 2.5 µl each primer (10 pmoles/ µl stock) (will be provided) ipaHF and ipaHR
    Template 1-2.5 µl (e.g., positive control or PCR template prep)
    Total volume is 25 µl

    (*HotStart Taq Master Mix Kit (Qiagen)

    Primer sequences:
    ipaHF 5'GTT CCT TGA CCG CCT TTC CGA TAC CGT C 3'
    ipaHR 5'GCC GGT CAG CCA CCC TCT GAG AGT AC 3'

    NOTE:
    A S. flexneri strain (2457M) is provided on a slant and should be streaked out onto nutrient agar plate (e.g., isolated colonies). This strain is a positive control, which can be used, with the PCR primers. However, this strain can be differentiated from any other Shigella isolate with another set of primers (607, 608), if needed, and its resistance to the antibiotic kanamycin [50 µg/ml]. All presumptive positive samples should be confirmed by nested PCR and also by primers 607 and 608 to make sure the positive samples are not due to cross contamination with the control strain. Amplification (PCR) with primers 607 and 608 will yield a 1.8 kb amplicon from the control strain (2457M) only; all other Shigella spp. will have a 1.0 kb amplicon. Control reactions can be run simultaneously (not multiplex) to ensure proper quality control of sample processing. A negative result, i.e. no PCR product from reactions using primers 607 and 608 only should not be interpreted as being negative for Shigella; the virulence plasmid could be completely or partially deleted. The ipaHF and ipaHR primers target the ipaH genes-there are 9 copies in the chromosome and virulence plasmid. Template is prepared by boiling a colony from an agar plate in 150 µl dH20 and using 1 µl in a PCR control tube. Another set of primers specific for this strain is available. It is recommended that the positive control be used to ensure that the reagents are working properly.

  5. PCR Amplification cycles and steps

    First file is set at 95°C for 15 minutes

  6. Following cycles consists of the following steps:
    1. 94°C for 1 minute (denaturation)
    2. 60°C for 1 minute (annealing)
    3. 72°C for 1 minute (extension)

      Total number of cycles is 30.

    4. 4°C for indefinite period of time (soak file)
       
  7. Agarose Gel Analysis of PCR Products

    After amplification, transfer 10 µl of the PCR products to another microcentrifuge tube containing 2 µl of tracking dye and load on 1% agarose gel.

    NOTE: Do not add dye directly to the PCR product.

    A 100 base pair ladder is used as a molecular weight standard. A 620 base pair product is expected from the positive samples. When reactions are completed, keep PCR product at 4°C or stored at -20°C.

    If a 620 bp amplicon is seen on the agarose gel (presumptive positive), proceed to the nested PCR step below for confirmation.

    NOTE: PCR primers

    PCR primers (ipaH-F and ipaH-R) are targeted to the ipaH genes; there are multiple copies of this gene residing in the chromosome and in the virulence plasmid of Shigella.

  8. Nested PCR protocol 

    Primers ipaH3 and ipaH4 are directed to internal sequences within the 620 bp amplicon generated from PCRs using primers ipaHF and ipaHR. Using primers ipaH3 and ipaH4, a 290 bp product should be amplified if the 620 bp fragment was generated from Shigella DNA. The objective of using the nested PCR assay is to confirm that the 620 bp fragment was amplified from Shigella.

  9. The PCR assay is as follows:

    1. Distilled water 6.5 µl
    2. 10 X buffer: 12.5 µl (see note I below)
    3. Primers 2.5 µl ipaH3 (stock is 10 pmol/µl)
    2.5 µl ipaH4 (stock is 10 pmol/µl)
    4. Template 1.0 µl (see explanation II below)
  10. PCR conditions (cycles and temperatures) are identical to amplification using
    PCR primers ipaHF and ipaHR.

    After reactions are complete, run 8-10 µl through a 1 % agarose gel in 0.5 X Tris-acetate EDTA buffer, pH 8.3. Each gel should have a 100 bp ladder as molecular weight marker. A positive reaction generates a 290 bp fragment. This is a confirmatory test.

    Notes:

    1. HotStartTaq Master Mix Kit (Qiagen)
    2. Usually if 1 µl of the PCR product is used directly from the reaction that amplified the 620 bp fragment, then 3 bands may be seen on the gel; the correct band at 290 bp and 2 other approximately 400 and 500 bp. The larger 2 bands are due to primers ipaHF and ipaHR being carried over from the first reaction. The larger amplified bands were generated from the combinations of ipaHF-ipaH4 and ipaHR-ipaHF. To avoid this, dilute the reactions that yielded a presumptive positive product 1:10 and 1:100 in separate tubes with dH2O. Use 1 µl of the diluted products as template. In some cases, faint bands around 400 and 500 bp may be seen on agarose gels using the 1:10 diluted product as template; this is explained above. A band at 290 bp is confirmation for the presence of Shigella.
    3. The DNA polymerase used to develop this assay was from Qiagen. Others are probably suitable.

      Primer sequence
      ipaH3: 5'-CCA CTG AGA GCT GTG AGG
      ipaH4: 5'-TGT CAC TCC CGA CAC GCC

      It is recommended that the positive control be used to ensure that the reagents are working properly.

      If a presumptive sample is analyzed by nested PCR and a 290 bp product is detected, save the PCR products from both sets of reactions (first round and nested). If necessary, send the products to:

      Dr. Keith A. Lampel
      FDA/CFSAN, HFS-515
      5100 Paint Branch Parkway
      College Park, MD 20740
      301-436-2007

Reporting/actions

Interpretation:

Bacteriological:
Lactose negative on MacConkey/lactose agar plates Negative for motility, hydrogen sulfide production, lysine decarboxylase activity, no gas production from glucose.

PCR (of amplified product and of colonies):
Presumptive positive if first round PCR yields a 620 bp product Confirmation is by nested PCR-amplification of 290 bp product

  1. Presumptive positive: first round PCR analysis of amplified product is positive AND/OR first round PCR analysis of colonies is positive.
  2. Confirmed positive: nested PCR analysis of amplified product is positive, AND/OR nested PCR analysis of colonies is positive.
  3. Negative: both PCR analysis of amplified product AND microbiology with PCR analysis of colonies are negative.

Reference:

Bacteriological Analytical Manual Online.

 

 Attachment B

Domestic Produce Assignment FY 2005 DFP # 05-20
Soak Method for the Detection of Salmonella in Basil, Cilantro, Scallions/Green Onions, Loose-Leaf Lettuce, Parsley, and Spinach

(Note: The method below is unchanged from the method cleared for the Import Produce Assignment issued March, 2005.)

Note: This sample preparation is only to be used for the detection of Salmonella in basil, cilantro, scallions/green onions, loose-leaf lettuce, parsley, and spinach. See Attachment C for cantaloupe and Attachment D for tomatoes.

  1. Pre-sample preparation.
    Do not rinse the produce, even if there is visible dirt. Examine the produce "as is".
  2. Sub-sample soak preparation:

    For basil and cilantro:

    Combine 75 g from each of 5 individual sub-samples into a sterile flask (375 g composite), or other appropriate container. Add 3375 ml tryptic soy broth and swirl, so that all of the produce is fully wetted. Loosely cap the flask. Let stand for 60 ± 5 min. Adjust pH to 6.8 ± 0.2, if necessary. Allow the samples to remain in the pre-enrichment broth during incubation.

    For scallions/green onions, loose-leaf lettuce, parsley, snow peas, and spinach:

    Combine 75 g from each of 5 individual sub-samples into a sterile flask (375 g composite), or other appropriate container. Add 3375 ml lactose broth and swirl, so that all of the produce is fully wetted. Loosely cap the flask. Let stand for 60 ± 5 min. Adjust pH to 6.8 ± 0.2, if necessary. Allow the samples to remain in the pre-enrichment broth during incubation.

  3. Sample preparation/method: Incubate composite at 35 C for 24 ± 2 h.

    After pre-enrichment, produce are to be selectively enriched as described below. The selective enrichment strategy is dependent on whether the culture method or a rapid method is to be used. Only rapid methods, for Salmonella, listed in the memo entitled, "Guidance for the Use of Rapid Methods for Food Microbiology" dated April 24, 1998, may be used. If the laboratory does not have a copy of the memo, they should request a copy from the Division of Field Science, HFC-140.

    For the Salmonella culture method (BAM, method 995.20) and rapid method 996.08, use Rappaport-Vassiliadis (RV) medium and tetrathionate (TET) broth. For the rapid methods 989.14, 990.13, 992.11, or 993.08 use selenite cystine (SC) and TET broths.

    For the Salmonella culture method, transfer 0.1 ml mixture to 10 ml RV medium and 1 ml mixture to 10 ml TET. Vortex. Treat all of the above produce as high microbial load foods. Incubate RV medium for 24 ± 2 h at 42 ± 0.2°C and incubate TET broth 24 ± 2 h at 43 ± 0.2°C. Incubate both RV and TET in air incubator. After incubation, follow the BAM Online, Salmonella, Chapter 5, section D, Isolation of Salmonella. 

    For rapid method kits, selectively enrich as instructed by the kit manufacturer.

    None of the rapid method test kits listed in the memo entitled, "Guidance for the Use of Rapid Methods for Food Microbiology", dated April 24, 1998, have been validated for the analysis of all of the different kinds of produce found in this assignment. Thus, should the analyst decide to use a rapid method which has not been validated for a particular kind of produce, it will be necessary to do a comparative seeding study.

    If a sample is a presumptive-positive based on the test kit, then perform confirmation analysis as described in the BAM Online, above.

 

 Attachment C

Domestic Produce Assignment FY 2005 DFP # 05-20
Soak Method for Salmonella analysis in Cantaloupe

(Note: The method below is unchanged from the method cleared for the Import Produce Assignment issued March, 2005.)

Note: This sample preparation is only to be used for the detection of Salmonella in Cantaloupe; when other organisms are being analyzed for, follow the soak method as stated in Attachment B.

  1. Pre-sample preparation

    Do not rinse the cantaloupes, even if there is visible dirt. Examine the cantaloupe "as is".

  2. Sub-sample soak preparation

    For each individual sub sample (e.g., one cantaloupe), place contents into a sterile plastic bag (Biopro Sample Bag, 12 x 18 inches, catalog number BP-41218, available from International Bioproducts, phone 800-729-7611). Add a volume of lactose broth that is needed to allow the cantaloupe to float. Normally this volume of lactose broth is 1.5 times the weight of the cantaloupe. For instance, a cantaloupe weighing 1200 g will need a volume of 1800 ml lactose broth. Place the plastic bag, with cantaloupe and lactose broth, into a non-sterile 5 liter beaker for support during incubation. Allow the open-end flap of the plastic bag to "fold over" so as to form a secure, but not airtight, closure during incubation.

  3. Sample preparation/method

    Incubate each, individual cantaloupe sub sample at 35 C for 24 plus or minus 2 hours.

    After incubation, manually mix the contents of the bags containing the cantaloupes and the pre-enrichments. The sub sample pre-enrichments are then to be "wet composited". From each of 5 incubated sub samples, remove 0.1 ml lactose broth pre-enrichment and place into a tube or flask containing 50 ml Rappaport-Vassiliadis (RV) medium. For the other 5 incubated sub samples, remove 0.1 ml lactose broth and place into a tube or flask containing 50 ml RV medium. Incubate the 2 RV medium composites at 42 plus or minus 0.2 C in a circulating, thermostatically controlled water bath for 24 h.

    In addition to subculturing the sub sample pre-enrichments to RV medium, these sub sample pre-enrichments are to be sub-cultured to tetrathionate (TET) broth. From each of 5 incubated sub samples, remove 1.0 ml lactose broth pre-enrichment and place into a tube containing 50 ml TET broth. For the other 5 incubated sub samples, remove 1.0 ml lactose broth and place into a tube containing 50 ml TET broth. Incubate the 2 TET broth composites at 35 C for 24 hours.

    After incubation of the RV and TET composites, continue as directed in the BAM Online.

    Note: Only the VIDAS Salmonella (SLM) Assay (Method 996.08 has been validated for use with cantaloupes.
    None of the rapid method test kits listed in the memo entitled, "Guidance for the Use of Rapid Methods for Food Microbiology", dated April 24, 1998, have been validated for the analysis of cantaloupe. Thus, should the analyst decide to use rapid methods other than the VIDAS Salmonella SLM Assay, it will be necessary to do a comparative seeding study as described in the guidance memo of April 24, 1998 and in ORA-LAB1.

If a sample is a presumptive positive based on the test kit, then perform confirmation analysis as described in the BAM Online, above. It should be noted that the use of this soak method does not allow a most probable number determination of Salmonella for those samples that are qualitatively positive for this pathogen.

Please note that this procedure involves wet compositing of incubated sample pre-enrichments as opposed to dry compositing as described in the BAM. Validation data to support the use of wet compositing may be found in the following references:

  1. Price, W. R., R. A. Olsen, and J. E. Hunter. 1972. Salmonella testing of pooled pre-enrichment broth cultures for screening multiple food samples. Applied Microbiology. 23:679-682.
  2. Silliker, J. H. and D. A. Gabis. 1973. ICMSF methods studies. I. Comparison of analytical schemes for detection of Salmonella in dried foods. Can. J. Microbiology. 19:475-479.

 

 Attachment D

Domestic Produce Assignment FY 2005 DFP # 05-20
Tomato Soak Method for Salmonella analysis

(Note: The method below is unchanged from the method cleared for the Import Produce Assignment issued March, 2005.)

Note: This sample preparation is only to be used for the detection of Salmonella in tomatoes; when other organisms are being analyzed for, follow the soak method as stated in Attachment B.

  1. Pre-sample preparation: Do not rinse the tomatoes, even if there is visible dirt. Examine the tomatoes "as is".
  2. Sub-sample soak preparation: For each individual sub-sample (e.g., approximately 454 g tomatoes), place contents into a sterile plastic bag (Biopro Sample Bag, 12 x 18 inches, catalog number BP-41218, available from International Bioproducts, phone 800-729-7611). Add enough lactose broth to allow the tomatoes to float. This volume of lactose broth may be 1.5 times the weight of the tomatoes. For instance, tomatoes weighing 454 g will probably need a volume of approximately 675 ml lactose broth to float. Add more lactose broth, if necessary. Place the plastic bag, with tomatoes and lactose broth, into a non-sterile 5 liter beaker, or other appropriate container, for support during incubation. Allow the open-end flap of the plastic bag to "fold over" so as to form a secure, but not air-tight, closure during incubation.
  3. Sample preparation/method: Incubate each, individual tomato sub-sample at 35oC for 24 ± 2 h.

    After pre-enrichment, tomato sub-samples are to be selectively enriched as described below. The selective enrichment strategy is dependent on whether the culture method or a rapid method is to be used. Only rapid methods, for Salmonella, listed in the memo entitled, "Guidance for the Use of Rapid Methods for Food Microbiology" dated April 24, 1998, may be used. If the laboratory does not have a copy of the memo, they should request a copy from the Division of Field Science, HFC-140.

    For the Salmonella culture method (method 2000.06), use Rappaport-Vassiliadis (RV) medium and tetrathionate (TET) broth. For the rapid methods 989.14, 990.13, 992.11, 993.08, or 996.08 use selenite cystine (SC) and TET broths. Please note that method 996.08 (Vidas SLM method) may be changed in the near future: SC broth may be replaced with RV medium. In the event that RV medium is approved for use, with method 996.08, use TET and RV medium for this method.

    After incubation, manually mix the contents of the bags containing the tomatoes and the pre-enrichments. The sub-sample pre-enrichments are then to be "wet composited". From each of 5 incubated sub-samples, remove 1.0 ml lactose broth pre-enrichment and place into a tube or flask containing 50 ml TET broth. For the other 5 incubated sub-samples, remove 1.0 ml lactose broth from each sub-sample and place into a tube or flask containing 50 ml TET broth. Incubate the 2 TET broth composites at 35oC for 24 h.

    In addition to sub-culturing the sub-sample pre-enrichments to TET broth, for the rapid methods listed above, these sub-sample pre-enrichments are to be sub-cultured to SC broth. From each of 5 incubated sub-samples, remove 1.0 ml lactose broth pre-enrichment and place into a tube containing 50 ml SC broth. For the other 5 incubated sub-samples, remove 1.0 ml lactose broth from each sub-sample and place into a tube containing 50 ml SC broth. Incubate the 2 SC broth composites at 35oC for 24 h.

    In addition to sub-culturing the sub-sample pre-enrichments to TET broths, for the culture method and possibly for method 996.08, these sub-sample pre-enrichments are to be sub-cultured to RV medium. From each of 5 incubated sub-samples, remove 0.1 ml lactose broth pre-enrichment and place into a tube containing 50 ml RV medium. For the other 5 incubated sub-samples, remove 0.1 ml lactose broth from each sub-sample and place into a tube containing 50 ml RV medium. Incubate the 2 RV medium composites at 42 ± 0.2oC in a circulating, thermostatically-controlled water bath for 24 h.

    After incubation of the RV and TET composites, continue as directed in the BAM Online.

    Please note that none of the rapid method test kits listed in the memo entitled, "Guidance for the Use of Rapid Methods for Food Microbiology", dated April 24, 1998, have been validated for the analysis of tomatoes. Thus, should the analyst use one of these rapid methods, then it will be necessary to do a comparative seeding study as described in the guidance memo of April 24, 1998 and in ORA-LAB1.

    If a sample is a presumptive positive based on the test kit, then perform confirmation analysis as described in the BAM Online. It should be noted that the use of this soak method does not allow a most probable number determination of Salmonella for those samples that are qualitatively positive for this pathogen.

    This procedure involves wet compositing of incubated sample pre-enrichments as opposed to dry compositing as described in the BAM.

    For positive samples refer to Salmonella Isolates on page 8 of the Assignment.

 

 Attachment E

Domestic Produce Assignment FY 2005 DFP # 05-20
Protocol for the Detection of Viruses in Produce

The following protocol was developed for the detection of foodborne viruses in produce such as scallion, and is probably applicable to other produce material such as cilantro or loose-leaf lettuce.

Materials required:

10X Tris-Glycine buffer Sigma T-4904
Polyethylene Glycol (PEG, Average Mol. Wt. 8000) Sigma 81268
Ribonucleic acid (Transfer) (10mg/ml) Sigma R8508
Tri Reagent MRC TR118
RNase/DNase free water Any  
AMV reverse transcriptase Promega M9004
10X reverse transcriptase buffer Promega A3561
Magnesium Chloride 25mM Promega A3511
PCR nucleotide mix (10mM) Promega C1141
Taq DNA polymerase in storage buffer B
Oligonucleotide primers (see note 1).
Promega M1661

Procedure:

  1. Chop 50g or fewer scallions (leaf, stalk and bulb) into 2cm long pieces with a sterile razor blade and transfer the contents to a sealable plastic bag. Prepare as many samples as you want or can handle at a time. Add 50ml of 1X Tris-glycine buffer (diluted from 10X stock buffer with RNase/Dnase free water) to each bag. Close the bag securely. If a positive control is needed, add 20-200 pfu of virus to a sample prior to the addition of 1X buffer.
  2. Place the bags on a tilting platform and rock at maximum speed for 20 min. Decant the liquid to a sterile 50 ml centrifuge tube (we recommend Corning 25330). Discard the solid plant material.
  3. Centrifuge the tubes at 9000Xg (approximately 7500 rpm in a Sorvall GSA rotor with Delrin adapters) for 30 min at 4°C.
  4. Decant the liquid into a fresh tube, avoiding any pelleted material.
  5. To 10 to 20ml of the clarified extract, add BSA (fatty acid free, Promega W384A) from a 10mg/ml stock solution (50µl for every ml of extract), and sodium chloride from a 5M stock solution (87µl for every ml of extract, Ambion 9760G). Mix well by inversion. Calculate the total volume of the extract. To each extract, add equal volume of a 17% stock solution of PEG (prepared in nuclease free water and stored at 4°C). Mix thoroughly by inversion and incubate overnight at 4°C.
  6. Centrifuge at 9000Xg for 30 min at 4°C. Completely drain the liquid from each tube. Dissolve the pellet thoroughly in 0.75ml Tri Reagent containing 100mM beta-mercaptoethanol. Make sure to dissolve the material adhering to the side of the tube using a 1 ml Eppendorf type pipetor. Transfer the material to a 1.5 ml nuclease free eppendorf tube. Leave all the samples at room temperature for 5min.
  7. Add 0.2 ml Chloroform to each tube. If you have multiple samples, add chloroform to all tubes close the caps tightly, place them together in a small rack, and shake vigorously by hand for 1min. Let stand at room temperature for 5min, then centrifuge for 10 min at 4°C in a microfuge at top speed.(14,000 rpm).
  8. Remove the upper clear colorless phase to a fresh tube. Add 40µl 3M sodium acetate (Sigma S7899), mix by vortexing, Add 1µl (10µg) tRNA to each sample, mix, and add an equal volume of isopropanol. Mix well, and incubate in a -20°C freezer for 2 hours.
  9. Centrifuge at 14000 rpm at 4°C for 20min. Drain thoroughly, add 800µl 70% ethanol, and centrifuge again as above. Discard the supernatant, and drain inverted on paper towel until completely dry (do not use SpeedVac or similar equipment to dry the tubes).
  10. Dissolve the RNA in 90µl water by vortexing to cover the entire inner bottom surface of the tube. Add 10µl of 3M sodium acetate, and 250 µl 100% ethanol. Mix and incubate at -20°C for 2h to overnight.
  11. Centrifuge 14000 rpm at 4°C for 20min. Discard the supernatant. Add 0.8ml of 70% ethanol to each tube and centrifuge as above. Discard supernatant, and dry the tubes inverted on paper towel. Do not use SpeedVac. Dissolve the RNA in 20µl nuclease free water, by flicking and vortexing to cover the inner surface of the bottom part of the tube. Centrifuge briefly to collect the liquid at the bottom.
  12. For reverse transcription mix in a 0.5ml eppendorf tube up to 9.6µl of the RNA sample and 1µl (50 to 100 ng) reverse transcription (RT ) primer such as random hexamers available from any supplier. Alternatively, 0.5 to 1 µg Oligo dT15 can be used. Heat the mixture at 70° for 5min in a thermal cycler and chill on ice. Centrifuge briefly.

    Add 9.4µl of the following reaction mixture:

    !0X reverse transcription buffer (Promega) 2µl
    100mM DTT 2µl
    10mM deoxynucleotide mix 1µl
    25mM MgCl2 2.4 µl
    RNasin RNase inhibitor 20 -40 units perµl 1µl
    AMV reverse transcriptase 20u/µl 1µl
    All the above reagents are from Promega  

    Heat the reactions in a thermal cycler at 22° for 10min followed by 42° for 90 min, and 95° for 5min. Chill immediately on ice and centrifuge at 14,000 rpm for 5 min. Proceed immediately to PCR or freeze at -20°.

  13. Perform PCR with up to 10µl of the sample according to the protocol below. Rest of the samples may be stored frozen at -20°C.
  14. PCR protocol

    Buffer 50 mM Tris-HCl, pH ; 75 mM KCl
    dNTPs 200 µM each
    MgCl2 3 mM
    Primers 50 pmol of forward and reverse primers
    Template 5-10 µl of reverse transcription reaction
    Taq polymerase 0.5 Units (Promega)
    Total volume 50 µl  

    Note 1:
    Primers

    BG 7 (5'-CCGAAACTGGTTTCAGCTGAGG-3')
    BG 8 (5'-CCTCTGGGTCTCCTTGTACAGC-3')

    The expected size of an amplicon generated with BG7/BG8 is 276 bp. This is considered a presumptive positive result.

    PCR products should be sent under refrigeration temperature to the attention of Yuan Hu, NRL, to perform DNA sequencing. An e-mail should also be sent to Yuan to alert him of the sample:

    Yuan Hu, Microbiologist
    FDA - Northeast Regional Laboratory
    158 - 15 Liberty Avenue
    Jamaica, New York 11433-1034

    Developed by Biswendu B. Goswami, PhD, Division of Molecular Biology, OARSA