Testing “technologies”

J. David Legan, PhD.October 03, 2019

DavidLegan@EurofinsUS.com

United Fresh, 03 Oct. 2019

What?

Testing technologies:• How they work, • practical considerations in use.

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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.

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Culture methods

Generally considered “the gold standard”Might involve multiple steps because we’re looking for very low concentrations (usually) and not uniformly distributed

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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

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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

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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

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“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

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Principle of ELISA / ELFA

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substrate

enzyme

antibodies

analyte

reporter

Use of the capture antibodies linked to a surface makes this a “sandwich” ELISA

Schematic –Salmonella by ELFA

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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)

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PCR (principle)

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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)

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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

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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..

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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.

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Loop-Mediated Isothermal AMPlification (LAMP)

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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

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Bioluminescence drops

Exponential amplification of target DNA

© 3M All Rights Reserved. Used with Permission

Schematic –Salmonella by LAMP

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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?

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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!

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NEAR principle (1)

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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

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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.

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Bacteriophage

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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.

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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

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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.

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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.

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Interferences (False +ve)

• True cross-reaction with non-target strain (rare)

• Cross contamination(should be rare – and identifiable)

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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

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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

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Thank you

41United Fresh, 03 Oct. 2019 https://commons.wikimedia.org/wiki/File:5aday_salad.jpg