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Nucleic Acid Based Detection Methods in Food Microbiology

Monday, June 23 (Lecture Series Day 1):   

Morning:    Arrival in Fort Collins
       
2:00 p.m.  Welcome and introduction

2:15 p.m.  Lecture 1: “Fundamentals: Nucleic Acids, DNA Replication, Transcription and Translation and their Application to Assay Development”

3:00 p.m.  “Concepts in Microbial Taxonomy and Diversity and Implications for Molecular Detection”

3:30 p.m.  Break

3:45 p.m.  “PCR: Fundamentals and Variations (PCR; Nested PCR; Multiplex PCR; Real-Time PCR: Reverse Transcriptase PCR; Sequencing-Based Applications for Identification of Bacteria as well as Yeasts and Molds; Controls and GLPs)”
Including: Hands-on Primer Design Activity
       
6:30 p.m.  Opening Reception and Dinner Followed by Keynote Speaker (Proposed Topic: “Molecular Detection in Regulatory Oversight and Public Health Investigations”)


Tuesday, June 24, (Lecture Series Day 2):

8:00 a.m.  “Assay Validation and Approval, Including Use and Misuse of the Sensitivity and Specificity concepts”

8:30 a.m.   The Steps Before PCR: Sampling and Enrichment, Concentration Procedures, Compositing, True Real Time PCR without Enrichment
   
9:00 a.m.   “The Ins and Outs of Selected Commercially Available DNA-Based Detection Assays”

10:00 a.m.  Break/Vendor Exhibits/Poster Session

10:30 a.m.  15-20 Minute Presentations from 4 to 5 Selected Companies on Their Assays

12:00 p.m.  Working Lunch/Question and Answer Session with Company Representatives and Workshop Instructors on Selection of the Molecular Detection Assay that Fits Your Needs

1:30 p.m.  Case Studies on Interpretation of Results from Nucleic Acid Based Detection Methods (Including Questions such as ”What Does a Positive PCR Assay Mean?” and “How to Deal with a PCR Positive Culture Negative Result?”) / Break-out into Groups and Troubleshoot

2:30 p.m.  Considerations for Application of Molecular Detection Methods (Integration of Molecular and Cultural Methods, PCR Analysis on Enrichments, PCR on IMS beads, Quality Control)

3:30 p.m.   Round Table Discussion and Wrap-up Question and Answer Session

4:30 p.m.   Vendor Exhibits/Poster Session and Cocktail Hour

6:30 p.m.   Dinner on Your Own


Wednesday, June 24 (Hands-on Workshop Day 1):

8:00 a.m.   Introduction to DNA Sequence Databases, Uploading/Downloading Sequence Data, Alignment Programs (Additional Time for Database Searches during Downtime)

9:00 a.m.   Design PCR Assay for Target Organism of Interest (e.g., Salmonella, E. coli O157:H7, L. monocytogenes, Listeria spp. Pseudomonas fluorescence, Paenibacillus spp., Alicyclobacillus) and Order Primers.

10:30 a.m.  Prepare Lysates and Design PCR Protocol for Amplification of hly Sequence Unique to L. monocytogenes (primers and sequences supplied) Using Detection by SYBR Green and Conventional PCR and Gel Electrophoresis

12:00 p.m.  Lunch on your own

1:30 p.m.   Set-up PCR

3:00 p.m.   Design Multiplex PCR, Taqman Primers and Probes

4:00 p.m.   PCR Controls, Internal Positive Controls, Live Positive Controls, etc.

5:30 p.m.   Dinner on your own


Thursday, June 25 (Hands-on Workshop Day 2): 

8:00 a.m.    Run Gel and SYBR Green Detection
       
11:00 a.m.  Set-up Custom PCR

12:00 p.m.  Lunch on Your Own

1:00 p.m.    Run Gel from Custom PCR, Troubleshooting

2:00 p.m.    Lecture and Discussion  “DNA Sequencing”

3:00 p.m.    Purification of Custom PCR Products and Send for Sequencing

6:30 p.m.    Workshop Closing Dinner, Location TBA


Friday, June 27 (Hands-on Workshop Day 3):
   
8:00 a.m.    Taqman PCR (ABI, Master Mix Kit)
       
10:00 a.m.  16S-Based Detection Strategies

11:00 a.m.  Lecture and Discussion “mRNA Detection”

12:00 p.m.  Lunch on Your Own

1:00 p.m.    Assemble and Proofread Sequence Data and perform BLAST Searches

3:00 p.m.    Optional Wrap-up Discussion Session and Individual Discussion with Instructors

5:00 p.m.    Informal Happy Hour, Location TBA