using solus listeria elisa. · 2018. 3. 8. · detection of listeria in pasteurised and raw cow’s...

Detection of Listeria in pasteurised and raw cow’s

milk cheese using Solus Listeria ELISA.

Deliverables Report Ref. LCH-01

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Detection of Listeria in pasteurised and raw cow’s milk cheese

using Solus Listeria ELISA.




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TABLE OF CONTENTS

1. EXECUTIVE SUMMARY / OVERALL CONCLUSIONS ..................................................................................... 3

2. INTRODUCTION .................................................................................................................................................. 4

3. OBJECTIVE ......................................................................................................................................................... 4

4. MATERIALS ......................................................................................................................................................... 4

5. EQUIPMENT ........................................................................................................................................................ 5

6. METHODS ............................................................................................................................................................ 5

6.1. Solus Listeria ELISA vs ISO Protocol ....................................................................................................... 5

6.2. ELISA Assay protocol ................................................................................................................................ 7

7. RESULTS ............................................................................................................................................................. 7

7.1. Seeding Levels ............................................................................................................................................ 7

7.2. Primary Enrichment .................................................................................................................................... 8

7.3. Secondary Enrichment ............................................................................................................................. 10

8. RECOMMENDATIONS ...................................................................................................................................... 13




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1. EXECUTIVE SUMMARY / OVERALL CONCLUSIONS

Positive results were achieved from both pasteurised and raw milk cheese

samples at the shortest incubation times advised.

To offer more assurance to customers it is recommended to use the longest

incubation parameters for samples perceived to be more challenging.




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2. INTRODUCTION

In 2013, a comparison project was conducted by three third party laboratories. The project involved testing

for Listeria sp. in sub-samples of unpasteurised blue cheese. One laboratory used the RayAl Listeria

method and this was compared to the ISO Listeria method used by the other two laboratories.

It was found that, at the time, the RayAl testing method was not detecting Listeria spp. in all instances of

the cheese samples when used at that laboratory.

Recently, the efficacy of our Solus Listeria ELISA system for the detection of Listeria in cheeses has been

questioned again with reference back to the incidents of 2013.

We wished to carry out further testing to investigate how the Solus system currently performs and to

provide data to help reassure our customers.

3. OBJECTIVE

The objective of this investigation is to test the capability of the Solus Listeria ELISA system to detect

Listeria species in pasteurised and unpasteurised milk cheeses.

4. MATERIALS

Materials used for LCH-01 experimentation were prepared in accordance with standard operating procedures and

documented via batch history records.

Listeria ELISA Assay Protocol

Supplier Supplier code Reagent Batch number

E&O Laboratories Ltd. PP0280 TSA Plates 90mm PK/10 (P090) 04140013

E&O Laboratories Ltd. PP0005 Palcam Agar (10pk) (P090) 04140161

E&O Laboratories Ltd. RPM007 RELM - Ready to use (50 x 10ml) (M002) 04075258

E&O Laboratories Ltd. BM0640 Fraser Broth 50 x 10ml Tubes (E&O) (M002) 04074699

E&O Laboratories Ltd. BM0760 MRD 9ml pk/50 (M002) 04075075

Lab M LAB049 Brain Heart Infusion 1132

Solus Scientific Solutions Ltd. AGAR-OA020S Solus Listeria Agar-OA (20 plates) 200467

Solus Scientific Solutions Ltd. FBH002 Half Fraser Broth (5kg) 200014/347

Solus Scientific Solutions Ltd. LIS-0480S Solus Listeria ELISA (480 wells) Kit EL094




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5. EQUIPMENT

The following equipment were used to generate test results at the East Kilbride R&D facilities.

Model Serial number Description

BioMAT EXH MC5765/6 Class 2 safety hood

MIR-554 11080222 Sanyo Incubator

SBB AQUA 12 MJ1026005 Grant Instruments Water bath

Dynex-DS2 1DSA0729 Automated ELISA system

Multiskan FC - Type 357 357-901089 Plate reader

6. METHODS

The following methods were used in LCH-01.

6.1. Solus Listeria ELISA vs ISO Protocol

‘Live’ bacteria cultures (with serotype details) were prepared as follows:

Listeria species description Identification

Listeria innocua LMG 11387

Listeria monocytogenes 1/2b LMG 16780

Listeria monocytogenes 4a ATCC 19114

The following cheese samples were prepared in triplicate for each culture plus one sample that remained

uninoculated and served as a blank control, resulting in 10 test samples of each cheese.

Short name Full name Milk treatment

Stilton Colston Bassett Stilton Pasteurised

Castello Castello Danish Blue Pasteurised

Raw Reblochon Fermier Unpasteurised




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Overnight sub-culturing of a microbank bead of bacteria sp. onto TSA (Tryptone Soya Agar, E&O Labs

Product code: PP0280, Lot: 04140013) at 37°C.

Colony transferral from the TSA plate into 10ml BHI (Brain Heart Infusion, LabM Products code: LAB049,

Lot: #1132). Cultured for 18 hours at 37°C for Listeria spp.

The cultures were diluted in MRD – Maximum Recovery Diluent (E&O Labs Product code: BM0760, Lot:

04075075) to achieve an approximate 1-3cfu/100µl suspension for Listeria spp. Actual counts were

determined by spread plate methods, onto TSA plates which were subsequently incubated for 48 hours at

37°C.

Within a Class 2 safety hood, 100µl Listeria cell suspension was aliquoted directly onto 25g of each cheese

sample in an associated stomacher bag.

Samples were incubated for 72 hours at 4°C prior to the addition of 225ml Half-Fraser Broth (Solus Scientific

Solutions Ltd. Product code: FBH002 Batch 1167) to each stomacher bag and stomached for 30 seconds

each and subsequently incubated for 22 hours at 30°C.

100µl primary enrichment cultures were spread on OA-Agar (Solus Scientific Solutions Ltd. Product code:

AGAR-OA020S, Lot: 200467) and Palcam agar (E&O Labs Product code: PP005, Lot: 04140161), and

subsequently incubated for 24-48 hours at 37°C for confirmation of Listeria spp. isolation from the primary

enrichment cultures.

Primary enrichment aliquots were also heated to 85-100°C for 15-20 minutes in a water bath. Following

heating, the samples were allowed to cool to room temperature prior to running the ELISA immunoassay.

In addition, 100µl primary enrichment cultures were aliquoted into 10ml Full Fraser (E&O Labs Product code:

BM0640, Lot: 04074699 and 200µl primary enrichment cultures were aliquoted into 10ml RELM (E&O Labs

Product code: RPM007, Lot: 04075258). Secondary enrichment samples were then sub-cultured for 22 hours

at 37°C (Full Fraser) or 22 hours at 30°C (RELM).

10µl secondary enrichment samples were streaked on OA-Agar and Palcam agar and subsequently

incubated for 24 hours at 37°C for confirmation of Listeria spp. isolation from the secondary enrichment

cultures.

Secondary enrichment aliquots were also heated to 85-100°C for 15-20 minutes in a water bath. Following

heating, the samples were allowed to cool to room temperature prior to running the ELISA immunoassay.




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6.2. ELISA Assay protocol

100l Sample loaded

30 minute incubation at 37°C

5 Wash cycle in the Dynex-DS2

100l Conjugate diluent loaded

30 minute incubation at 37°C

5 Wash cycle in the Dynex-DS2

100l Substrate (TMB) loaded

30 minute incubation at Room temperature

100l Stop solution loaded

Read after 1 minute on the Multiskan plate reader

7. RESULTS

7.1. Seeding Levels

The seeding level for each of the Listeria strains after growth in BHI and subsequent dilution in MRD was assessed

by growth on 5x TSA agar plates. The aim was to achieve average seeding levels of 1-4 cfu/100ul. The results are

shown in Table 1.

Table 1: Listeria seeding populations

Bead Location D8 D9 G10

Listeria ID ATCC19114 LMG 16780 LMG 11387

Plate number cfu/100ul cfu/100ul cfu/100ul

1 6 3 5

2 1 5 3

3 2 4 4

4 2 2 4

5 2 3 3

Average 2.6 3.4 3.8

The results suggest that each cheese sample was inoculated with approximately 1-6 cfu.

With three replicates of each 25g cheese sample, the average 100ul inoculation can be assumed to have

been between 2-4 cfu.




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7.2. Primary Enrichment

The primary enrichment broth was assessed to determine if each cheese sample had been inoculated and if growth

of the target organism had occurred.

The results are shown in Table 2.

Table 2: Primary enrichment broth growth

Primary enrichment ELISA results Primary enrichment agar plate growth

@22 hours

Primary enrichment agar plate growth

@48 hours

Sample Id Raw Data

Threshold Final

Results OA Palcam OA Palcam

Negative 0.047 NC1 NC1

Positive 2.548 PC1 PC1

BLANK BLANK BLANK BLANK


@22 hours


@48 hours

Sample Id Raw Data

Threshold Final


Stilton D8 1 0.059 - - +H + +H +

Stilton D8 2 0.060 - - +H + +H +

Stilton D8 3 0.062 - - +H + +H +

Stilton D9 1 0.060 - - - - - -

Stilton D9 2 0.071 - - - - - -

Stilton D9 3 0.066 - - +H + +H +

Stilton G10 1 0.065 - - + + + +

Stilton G10 2 0.065 - - + + + +

Stilton G10 3 0.063 - - + + + +

Stilton BLANK 0.063 - - - - - -




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@22 hours


@48 hours

Sample Id Raw Data

Threshold Final


Castello D8 1 0.060 - - - - - -

Castello D8 2 0.062 - - - NG - NG

Castello D8 3 0.071 - - +H + +H +

Castello D9 1 0.060 - - +H + +H +

Castello D9 2 0.063 - - NG NG NG NG

Castello D9 3 0.064 - - - NG - NG

Castello G10 1 0.063 - - + + + +

Castello G10 2 0.067 - - + + + +

Castello G10 3 0.065 - - + + + +

Castello BLANK 0.061 - - - - - -


@22 hours


@48 hours

Sample Id Raw Data

Threshold Final


Raw D8 1 0.057 - - +H + +H +

Raw D8 2 0.055 - - +H + +H +

Raw D8 3 0.058 - - - - - -

Raw D9 1 0.061 - - +H + +H +

Raw D9 2 0.061 - - - - - -

Raw D9 3 0.058 - - +H + +H +

Raw G10 1 0.052 - - + + + +

Raw G10 2 0.054 - - + + + +

Raw G10 3 0.054 - - + + + +

Raw BLANK 0.056 - - - - - -

Each of the ‘blank’ plates showed non-target growth. However, this was expected as the primary enrichment is not selective.

The majority of sample plates showed positive Listeria growth indicating that inoculation had occurred successfully.

The ELISA results are uniformly negative. This is to be expected at this point as the protocol is designed to be a 2-enrichment-step process to allow time for the target organisms to grow to detectable levels.




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7.3. Secondary Enrichment

During secondary enrichment, selective media is used to support the growth of the target organism whilst inhibiting the development of background microflora. The final ELISA and growth plates should be able to detect the Listeria in the cheese samples if the cheese itself has not prevented the growth of the Listeria or interfered with the efficacy of the test in another way.

The results are shown in Table 3.

Table 3: Secondary enrichment broth growth

Secondary enrichment ELISA results

Secondary

enrichment growth

@22 hours

Sample Id Secondary enrichment Raw Data Final Results OA Palcam

Negative 0.043 NC1

Positive 2.518 PC1

BLANK BLANK BLANK


Secondary enrichment

growth @22 hours


Stilton D8 1 Fraser 1.685 POS ++++H ++++



Stilton D9 1 Fraser 1.135 POS ++H ++

Stilton D9 2 Fraser 0.083 - NG NG

Stilton D9 3 Fraser 0.110 - NG NG

Stilton G10 1 Fraser 2.254 POS ++++ ++++



Stilton BLANK Fraser 0.085 - NG ---




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growth @22 hours


Castello D8 1 Fraser 0.082 - NG NG


Castello D8 3 Fraser 0.411 POS +++H +++

Castello D9 1 Fraser 0.607 POS +++H +++



Castello G10 1 Fraser 0.911 POS +++ +++

Castello G10 2 Fraser 2.583 POS ++++ ++++

Castello G10 3 Fraser 2.756 POS ++++ ++++

Castello BLANK Fraser 0.079 - NG NG



growth @22 hours


Raw D8 1 Fraser 2.508 POS ++++H ++++

Raw D8 2 Fraser 2.199 POS ++++H ++++

Raw D8 3 Fraser 0.080 - NG NG

Raw D9 1 Fraser 1.137 POS ++H +++

Raw D9 2 Fraser 0.078 - NG NG

Raw D9 3 Fraser 0.719 POS ++H ++

Raw G10 1 Fraser 2.547 POS ++++ ++++

Raw G10 2 Fraser 2.248 POS ++++ ++++

Raw G10 3 Fraser 1.758 POS +++ +++

Raw BLANK Fraser 0.081 - NG NG




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growth @22 hours


Stilton D8 1 RELM 0.374 POS ++H ++

Stilton D8 2 RELM 1.854 POS +++H +++

Stilton D8 3 RELM 2.097 POS ++++H +++

Stilton D9 1 RELM 0.621 POS ++H ++

Stilton D9 2 RELM 0.055 - NG NG

Stilton D9 3 RELM 0.063 - NG NG

Stilton G10 1 RELM 2.426 POS ++++ ++++

Stilton G10 2 RELM 3.000 POS ++++ ++++

Stilton G10 3 RELM 1.993 POS +++ +++

Stilton BLANK RELM 0.064 - - ---



growth @22 hours


Castello D8 1 RELM 0.045 - NG -

Castello D8 2 RELM 0.057 - - NG

Castello D8 3 RELM 3.000 POS +++H +++

Castello D9 1 RELM 2.952 POS +++H +++

Castello D9 2 RELM 0.063 - NG NG

Castello D9 3 RELM 0.062 - NG NG

Castello G10 1 RELM 3.000 POS ++++ ++++



Castello BLANK RELM 0.051 - - NG




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growth @22 hours


Raw D8 1 RELM 2.289 POS +++H +++

Raw D8 2 RELM 0.793 POS +++H ++

Raw D8 3 RELM 0.051 - NG NG

Raw D9 1 RELM 1.349 POS +++H +++

Raw D9 2 RELM 0.070 - NG NG

Raw D9 3 RELM 0.890 POS ++H ++

Raw G10 1 RELM 1.251 POS ++++ +++

Raw G10 2 RELM 1.995 POS ++++ +++

Raw G10 3 RELM 1.562 POS ++++ ++++

Raw BLANK RELM 0.074 - NG NG

When Listeria is present in the sample according to the ISO method, the ELISA method also returns a positive result.

ELISA did not give a positive when there were high levels of non-target growth indicating specificity.

ELISA did not give any ‘false’ results in this assay.

8. RECOMMENDATIONS

Positive results were achieved from both pasteurised and raw milk cheese samples at the shortest incubation

times advised.

To offer more assurance to customers it is recommended to use the longest incubation parameters for

samples perceived to be more challenging.

using solus listeria elisa. · 2018. 3. 8. · detection of listeria in pasteurised and raw cow’s...

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