Testing “technologies”
J. David Legan, PhD.October 03, 2019
United Fresh, 03 Oct. 2019
What?
Testing technologies:• How they work, • practical considerations in use.
2United Fresh, 03 Oct. 2019
But first - Why?Why do we need a test result?• To make a decision• To verify a belief
What do we need from the method?• A reliable result at the right time and the right price.
Never forget:• The method is only one component in the testing enterprise.• The result can never be better than the test portion allows.• Specifications ideally include the test method (but almost never do!)• As much thought should be given to the management of test results as is
given to the mechanics of testing. 3United Fresh, 03 Oct. 2019
The good news
• Today’s program covers the testing enterprise from beginning to end.
• Many good technologies available.
4United Fresh, 03 Oct. 2019
Culture methods
Generally considered “the gold standard”Might involve multiple steps because we’re looking for very low concentrations (usually) and not uniformly distributed
5United Fresh, 03 Oct. 2019
Romaine lettuce By Rainer Zenz CC 3.0
?The oldest class of methods:
conceptually very simple,
Use ability of organisms to grow on appropriate media to make
them visible.
Schematic –Salmonella by FDA BAM
6United Fresh, 03 Oct. 2019
Salmonella indicating (record observations)HE: Blue-green to blue colonies with or without black centers.XLD: Pink colonies with our w/o black centers.CM: Pale pink to mauve colonies
Atypical Salmonella indicating (record observations)HE/XLD: Yellow colonies with or w/o black centersCM: Blue colonies
Report as “Not Salmonella”
Weigh test portion (25 g, 375 g, etc.) Add to primary enrichment broth 1/10 ratio
Salmonella indicating (record observations)TSI: Alkaline (red) slant, acid (yellow) butt, with or without H2S production (blackening of the agar).LIA: Alkaline (purple) butt, with or without H2S production (blackening of the agar).
Report as“Not Salmonella”
24 h results recorded(slant and butt color)
Report as “Not Salmonellaby Vitek 2 GN”
Report as “Salmonella
by Vitek 2 GN”
Incubate 24h @ 35°C
Incubate24h@43°C
HE XLD CM
Transfer 1 mLTo 10 mL TT broth
Incubate24h@42°C
HE XLD CM
Transfer 0.1 mLTo 10 mL RV broth
IncubateHE & XLD, 24h @ 35° C; CM 24 h @ 37°C
Typical or Atypical
Salmonella?
Pick 2 suspect colonies each from HE, XLD and CM
Incubate24h @ 35° C
LIA: acid (yellow) butt?
TSI: acid (yellow) slant & butt?
Vitek®2 GN
NO
YES
NO
YES
NoYes
TSI slantsFrom HE, XLD, CM
LIA slantsFrom HE, XLD, CM
24 h
24 h
24 h
24 h
24 h
3 d
4 d
5 d
“Rapid” methods
7United Fresh, 03 Oct. 2019
Rapid methods are not new!• Methylene Blue reduction test for raw milk – quality not safety (Hastings,
1919, UW Madison: cites Fred, 1912 )
“Rapid” pathogen detection technologies
Are variants of:• Immunological detection• Nucleic acid detection
– DNA– RNA
• Surrogate detection (phage)May be combined with:• A concentration technology• A visualization technologyAOAC RI lists 242 Microbiological tests, from ≈ 50 manufacturers
8United Fresh, 03 Oct. 2019
“Modern Rapid Methods”
• First ELISA method published 1972, principle (immunological detection) published 1971.
• First commercial ELISA method by OrganonTeknika in 1976 for Hepatitis B diagnosis.
• Immunological methods – direct precursor to ELFA
9United Fresh, 03 Oct. 2019
Principle of ELISA / ELFA
10United Fresh, 03 Oct. 2019
substrate
enzyme
antibodies
analyte
reporter
Use of the capture antibodies linked to a surface makes this a “sandwich” ELISA
Schematic –Salmonella by ELFA
11United Fresh, 03 Oct. 2019
Incubate for 16-22 h at 35 °C
Weigh required test portion,add to enrichment broth (matrix
dependent)
Transfer 0.1 mL to SX2 broth
Incubate for 22-26 h at 42 °C
Lysis
Detection
Review and report results (Salmonella spp. presumptive, or
negative)
24 h
26 h
3 h
≈ 54 h
DNA detection technologies• Temperature cycling
– PCR (Polymerase Chain Reaction) with end point detection– Real Time PCR(AKA qPCR)
• Isothermal – several approaches– Loop-mediated isothermal amplification (LAMP)– Strand displacement amplification (SDA)– Helicase-dependent amplification (HDA)– Nicking enzyme amplification reaction (NEAR)
12United Fresh, 03 Oct. 2019
PCR (principle)
13United Fresh, 03 Oct. 2019
Unique DNA fragment found only in the target organism
DNA polymerase
Raising the temperature melts the double-stranded DNA
In the cooling part of the cycle, the primers anneal to the single-stranded DNA
and the polymerase extends the primers
After one cycle there are (theoretically) two copies of the DNA
Primer
Cycle starts again
Image from Hygiena
PCR (end point detection: automated)
14United Fresh, 03 Oct. 2019
Intercalating fluorescent dye
Once amplification is complete, illuminate to
trigger fluorescence.
Re-heat to release the dye and measure the “melting
curve” of the DNA.
Image from Hygiena
dF/dT
Temperature (T)
PCR (real time detection) AKA qPCR
15United Fresh, 03 Oct. 2019
Primer with attached probe (scorpion). F = fluorescent marker, Q = quencher
PrimerDNA polymerase
Raising the temperature melts the double-stranded DNA
In the cooling part of the cycle, the primers anneal to the single-stranded DNA
and the polymerase extends the primers
After one cycle there are two copies of the DNA and one of them has the
scorpion attached.
PCR real time detection, contd..
16United Fresh, 03 Oct. 2019
In the next round of melting the scorpion opens
On cooling, the scorpion anneals to one strand of the DNA, separating the fluorescent reporter from the quencher. At the same time, more scorpions attach to the DNA amplified in the last cycle.
DNA extension continues until there are 4 double-stranded fragments.
As amplification proceeds, the amount of fluorescence
increases.
Detectable fluorescence obtained in about an hour.
A threshold for detection of DNA-based fluorescence is set
3-5 times of the standard deviation of the signal noise
above background.
The number of cycles at which the fluorescence exceeds the
threshold is called the threshold cycle (Ct)
Isothermal amplification
• No thermo-cycler, so equipment can be smaller and less expensive.
• Strands not melted but “unzipped” so less chance of interference.
17United Fresh, 03 Oct. 2019
Loop-Mediated Isothermal AMPlification (LAMP)
18United Fresh, 03 Oct. 2019
3 3c 454c
2
32c3c 5c4
3c3 4c 5c 4
2c 4 5 4c
6c
4 5 6
5c 4
1 2 3
1 2 3
6c
4 5 6
1c 2c 3c 4c 5c4
1 2 3
6c
4 5 6
1c 2c 3c 4c 5c
4c
5c41c 2c 3c
1 32
6c1c 2c 3c 4c 5c
2 3 4 5 61
3
3c 4
54c2
3c
3 4c
5c42c
Six primers recognize 8 distinct regions of target DNA
Bst DNA polymerase, 5´ → 3´ polymerase and strand-displacement activity
Amplifies by “unzipping” DNA.
Complementary sequences on primers form a loop.
New double stranded DNA + strand with loop structure
Amplification continues in the other direction forming a barbell.
Multiple initiation points around loops, allow amplification to continue from each new loop
Rapidly escalating “cascade” of amplification
https://www.youtube.com/watch?v=ZXq756u1msE
3M MDS links amplification to bioluminescence detection
19United Fresh, 03 Oct. 2019 © 3M All Rights Reserved. Used with Permission
True real-time detection
20United Fresh, 03 Oct. 2019
Bioluminescence drops
Exponential amplification of target DNA
© 3M All Rights Reserved. Used with Permission
Schematic –Salmonella by LAMP
21United Fresh, 03 Oct. 2019
22 – 36 h
Weigh 25 ± 0.5 g or mL, prepare enrichment (matrix dependent)
Review and report results (Salmonella spp. presumptive, or
negative)
Lysis
Detection
Incubate 18 – 30 h @ 37 or 42 °C (matrix dependent)
Is LAMP more robust than PCR?
22United Fresh, 03 Oct. 2019
LAMP detected both Listeria and Salmonella in the presence of chocolate or mustardIsolated spices were incubated in enrichment broth for 24 h, then spiked with different concentrations of pathogens. Detection assays were run immediately after spiking to identify amplification inhibition.
LAMP/MDS had fewer invalid results than PCR/Bax, and, in general, detected lower concentrations of cells in the broth. Little evidence of amplification inhibition, and indications that dilution can overcome it. This does not address ability to enrich the organism from these matrices.
Nicking Enzyme Amplification Reaction
• Another isothermal DNA amplification and detection technology.
• Used in Neogen ANSR®– Note – Neogen expert is here!
23United Fresh, 03 Oct. 2019
NEAR principle (1)
24United Fresh, 03 Oct. 2019
5’3’
3’5’
3’5’
5’3’
5’3’
= Nicking Enzyme
= DNA Polymerase
Nicking enzyme nicks at naturally occurring sites
DNA polymerase extends from nick sites…
displacing the existing strand
© Neogen. Used with permission
NEAR – Phase II
Template Binding
• Template’s Region of Complementarity base-pairs with target DNA
Displaced StrandNew DNA
Target DNA
Template ≡ PCR primer
© Neogen. Used with permission
NEAR – Phase IITemplate Extension
• Template’s Region of Complementarity base-pairs with target DNA
• Bst polymerase extends the 3’ end of the template
• Copy of target DNA created
Target DNA
Template
Bst DNA Polymerase
New DNA
© Neogen. Used with permission
2nd layer of Specificity: Beacon Binding
• Target binding ‘unzips’ beacon• Quencher no longer prevents Fluorophore signal
Target Amplicon
FQ
F Q
Complimentaryto Target
Base-pairing forms stable “stem”
© Neogen. Used with permission
RNA detection technologies
• There are several RNA amplification technologies, e.g.– Nucleic acid sequence based amplification (NASBA)– Transcription mediated amplification (TMA)– Reverse transcription (into DNA) linked with a DNA
amplification technology
28United Fresh, 03 Oct. 2019
RNA detection – why bother?
• Some viruses (e.g. Norovirus) only have RNA –and are not culturable.
• Bacterial cells have many (1000-10,000) times more copies of RNA than DNA.
• RNA degrades more easily in the environment so claimed less chance of detecting “dead” cells.
29United Fresh, 03 Oct. 2019
Bacteriophage
30United Fresh, 03 Oct. 2019
Schematic of bacteriophage Lambda by Peterutz. Wikimedia commons, CC 3.0
Originally published by Seesandra, et al (2011)
Phage-based assays• Phage-based capture
– VIDAS-UP– More sensitive substitute for antibody capture
• Phage-based detection– Sample 6. Use phage to inject “reporter genes” that are expressed in
living cells to generate the detection signal (e.g. bioluminescence)• Phage-based amplification
– Use bacteriophage to infect living bacteria. The infection generates massive amplification of phage particles and the assay detects the phage. Mostly seem experimental.
31United Fresh, 03 Oct. 2019
Limit of Detection (LOD) per mL of enrichment broth
TechnologyCFU/mL (after enrichment) Source
ELISA/ELFA 104 - 105 K.M. Lee et al. Food Control 47 (2015) 264 – 276
PCR 104 K.M. Lee et al. Food Control 47 (2015) 264 – 276
LAMP 103 Eurofins internal
32United Fresh, 03 Oct. 2019
Validated LOD / test portion: Salmonella
Principle Method AOAC RI PTMLOD50 MPN or CFU/test portion1 Test portion (g)2
DNA LAMP 3M MDA2 91501 0.35-0.99 25 - 325
DNA PCR BAX 81201 0.85 25-375
DNA PCR BACGene 121501 0.75-1.06 25 - 375
ELFA phage tails VIDAS UP 71101 0.41 - 0.90 25 - 375
ELFA antibody VIDAS SLM 20901 0.37-1.06 25
RNA TMA ATLAS 41303 0.17-1.50 25 - 375
DNA NEAR ANSR 61203 0.45-1.10 25 - 375
Phage detection Sample6 NA NA NA
33United Fresh, 03 Oct. 20191 concentration where probability of detection is 50 %2 portion size in data set submitted for certification
Validated LOD / test portion: Listeria spp.
Principle Method AOAC RI PTMLOD50 MPN or CFU/test portion1 Test portion (g)2
DNA LAMP 3M MDA2 111501 0.27-1.12 25-125
DNA PCR BAX 30502 0.58-2.3 25
DNA PCR BACGene 61702 0.45-1.04 25
ELFA phage tails VIDAS UP NA NA NA
ELFA antibody VIDAS LIS 981202 Not listed Not listed
RNA TMA ATLAS 11201 0.05-0.95 25
DNA NEAR ANSR @ 24 h 101202 0.26-5.8 25 - 125
Phage detection Sample6 41401 Environmental surfaces only
34United Fresh, 03 Oct. 20191 concentration where probability of detection is 50 %2 portion size in data set submitted for certification
Validated LOD / test portion: STEC/O157 E. coli
Principle Method AOAC RI PTMLOD50 MPN or CFU/test portion1 Test portion (g)2
DNA LAMP 3M MDA2 71902 0.49-1.20 200 - 375
DNA PCR BAX 50501 0.32-1.8 25 - 375
DNA PCR BACGene NA NA NA
ELFA phage tails VIDAS UP 60903 1.08-1.15 25 - 375
ELFA antibody VIDAS NA NA NA
RNA TMA ATLAS 11402 0.2-0.8 375
DNA NEAR ANSR 111502 .26-1.2 125-375
Phage detection Sample6 NA NA NA
35United Fresh, 03 Oct. 20191 concentration where probability of detection is 50 %2 portion size in data set submitted for certification
Time to result
Culture > ELISA/ELFA > PCR > rtPCR ≈ LAMP/NEAR ≈ RNA by TMA > ?
Note, for rapid screening methods this time is driven by enrichment.
36United Fresh, 03 Oct. 2019
Interferences / failure to detect (False –ve)
• Antimicrobials – esp. environmentals, spices• High competitive background• PCR inhibitors (especially from spices and
botanicals, red lettuce vs. green lettuce)• Deep colors (some detection technologies)• True false negative rate is “impossible” to know.
37United Fresh, 03 Oct. 2019
Interferences (False +ve)
• True cross-reaction with non-target strain (rare)
• Cross contamination(should be rare – and identifiable)
38United Fresh, 03 Oct. 2019
Non-confirming presumptive• In a well-run food operation, “presumptive” rate
is typically low.• Not all presumptives confirm by culture – doesn’t
mean they are truly “false positive”– Presumptive Listeria spp. by Lamp– Culture –ve– Detected L. monocytogenes DNA in enrichment by
NGS
39United Fresh, 03 Oct. 2019
Method selection: things to consider• Sensitivity• Accuracy• Precision• Robustness• Reliability• Speed• Cost• Regulatory acceptance• Consequences arising from error• Matrix
• Use an accredited lab• Select a method validated for your
matrix
• If you have any concerns ask for additional verification
40United Fresh, 03 Oct. 2019
Thank you
41United Fresh, 03 Oct. 2019 https://commons.wikimedia.org/wiki/File:5aday_salad.jpg