Industrial Microbial Testing manufactures assays that use quantitative real-time PCR to rapidly detect and quantify microbes like bacteria and yeasts/molds. Their Total Bacteria Count and Total Yeasts/Molds Count assays can replace standard plate counting methods and provide results in 1 hour versus 48 hours. A collaborative study found their total bacteria count assay accurately quantified bacterial loads within one hour for various applications like enhanced HACCP programs.

1. IMT’s Total Bioburden Assays
IMT’ T t l Bi b d A
Andy Moreno
d
Vice President, Microbiology Sales Engineering
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2. Industrial Microbial Testing
– Based in Newport Beach, Calif., manufactures and
markets assays to support rapid real‐time
quantitative PCR microbial testing.
– IMT's One‐Step Total Bacteria Count (TBC) and
Total Yeasts and Molds Count (YMC)
T t lY t d M ld C t (YMC) quantitative
tit ti
real‐time polymerase chain reaction (qRT‐PCR)
assays replace standard plate count methods.
assays replace standard plate count methods
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3. TRADITIONAL PLATE COUNT (48 HOURS)
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4. TOTAL BACTERIA COUNT BY PCR (1 HOUR)
DNA dilutions after boil prep
for PCR
Real‐Time Standard
Data Curve
C
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5. Slide 5 of 13

6. “Organism Prospecting Package”
• Water/matrix/Ingredients/In Process/Final
Product sample
• Total Bacterial Count (TBC) screen
•Is there bacteria?
•Presence/Absence
Quantitation
•Quantitation
•Gram Negative/Gram Positive
•Total Yeasts and Molds Count (YMC) screen
•Are there yeasts and molds?
•Presence/Absence
•Quantitation
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7. IMT’s TBA: “Internal Investigation Tool”
• Any matrix, post sample preparation
• 30‐minute run time in SmartCycler
30 i t ti i S tC l
• Sensitivity to one (1) cell for TBC
• Presence/Absence
P /Ab
• Quantification
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8. IMT’s OneStep Assays
~30 minute run time
30 minute run time
85 µl molecular‐ 5 µl purified DNA 25 µl Total reaction
grade water added
grade water added Sample volume in
volume in
to primers & probes SmartCylcer tube
included in dried‐
down reagents
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9. Typical QA/QC Protocol
• IMT TBA Assays and Cepheid SmartCycler
IMT TBA Assays and Cepheid SmartCycler
• Minimally‐trained QA Team
• 30 minute easy to use cheap results
30 minute, easy‐to‐use, cheap results
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10. Collaborative Study to evaluate a Total Bacteria Count
Assay using Q
A i Quantitative Real‐Time PCR
tit ti R l Ti PCR
A collaborative study was undertaken to demonstrate the precision and
accuracy of a total bacteria count (TBC) assay developed to run with
accuracy of a total bacteria count (TBC) assay developed to run with
commercially available PCR reagents and a rapid thermocycling
platform. The TBC assay can be completed within one hour. The TBC
assay has many applications for enhanced HACCP applications
The overall conclusions are that all the labs performed well in
constructing the standard curve and had some variance with the
unknown samples. However, the results show that all collaborators
successfully used the TBC assay to quantify bacterial load accurately
f ll d h C if b i ll d l
within one hour. The TBC assay can be used for a rapid real‐time
measure of bacteria in many diverse applications and support HACCP
for real‐time statistical process control.
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11. IMT Testing Assays
‐Screen for presence/absence, quantitation
‐Total Bacteria Count (TBC)
‐Total Yeasts and Molds Count (YMC)
‐Salmonella
‐E. coli species
E li i
‐O15:H7
‐Listeria
‐C. botulisum “A”, “B”, “C” and “E”
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12. Quantitative Real Time PCR Systems
Quantitative Real‐Time PCR Systems
Eppendorf
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13. Questions & Answers
Questions & Answers
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