Detection of Listeria monocytogenes and Salmonella spp. in plant-based foods
Jennifer Pelowitz1, Joshua Whitworth1, Mike Clark1, Astrid Cariou2.
1
Bio-Rad Laboratories, Hercules, CA, 2Bio-Rad Laboratories, Marnes-la-Coquette, France
FDA BAM Protocol

As commercial markets grow for plant-based substitutes for foods such as meatand dairy products, producers need reliable methods for the detection of Listeria
monocytogenes and Salmonella spp. with rapid and accurate results. In thisstudy, iQ-Check Listeria monocytogenes II and iQ-Check Salmonella II methodswere tested and compared to the United States Food and Drug Administration’s
Bacteriological Analytical Manual (FDA BAM) reference method.

Alternative Method
Detection of Salmonella spp.

Matrix

Enrichment broth dependent
on matrix being tested
Pre-enrichment

Selectiveenrichment

Ground Meat AlternativeSample

A plant-based alternative to ground meat was inoculated with L. monocytogenes ATCC 13932
or S. Typhimurium ATCC 14028 from lyophilized pellets, at high and fractional inoculum levels orleft uninoculated. After a food stabilization period, 25 g test portions (20 fractional, 5 high, and5 uninoculated) were enriched at 1:10 in Listeria Special Broth (LSB, Bio-Rad Laboratories, catalog#3564703) for L. monocytogenes at 30°C for 24 hr or Buffered Peptone Water Standard (BPW, Bio‑RadLaboratories, catalog #12013260) for Salmonella at 37°C for 20 hr while FDA BAM reference samples(20 fractional, 5 high, and 5 uninoculated at 25 g, as well as 5 g, 10 g, and 50 g portions for MPNs)
were enriched in Buffered Listeria Enrichment Broth (BLEB, Hardy Diagnostics for L. monocytogenes) orLactose Broth (LB, Hardy Diagnostics) for Salmonella using FDA BAM protocols. Test method samples
were analyzed using iQ-Check PCR kits and all samples were culture confirmed using FDA BAM
protocols (Fig.1 and 2).

35°C
24 ± 2 hrandBPWTetrathionate

42 ± 0.2°C
24 ± 2 hr

37 or 41.5ºC
8–21 hr

Bacterial lysis and DNA extraction

43 ± 0.2°C
or 35 ± 2.0°C*
24 ± 2 hr

Plating

Real-time PCR amplification and detection withiQ-Check Salmonella IIBismuthSulfite
XLD

35°C
24 ± 2 hr

Confirmation

iQ-Check Listeria monocytogenes II real-time PCR Kit

Buffered Listeria Enrichment Broth* Temperature based on low or high microbial load.

Oxford oror Palcam andmodified Oxford(MOX)orAL (according toOttaviani and Agosti)

37°C
24 hr (+ 24 hr)

Bacterial lysis and DNA extraction standard(Half Fraser and LSB) or easy (LSB)

RAPID'L.mono

37°C
24 hr (+ 24 hr)

Real-time PCR amplification and detection with
iQ-Check Listeria monocytogenes II

Reading, isolation, and confirmation+Listeria spp.
Trypticase soy agar with yeast extract, catalase, Gram test, mobility

95% CI

500.000.00, 0.43

00.00

20

120.600.39, 0.78

6551.00

0.57, 1.00

50.0

A

0.025
(0.016, 0.040)
0.14(0.067, 0.28)
Spike levela

Nb

0.0
0.025
(0.016, 0.040)
0.14(0.067, 0.28)

xPODcp5020
5Confirmation of positive results withRAPID'Salmonella

0.025(0.016, 0.040)
0.14(0.067, 0.28)

or*	
NF Validation on iQ-Check Salmonella II Real-Time PCR Kit only.
**	Can be used as part of an overall confirmation method.

BResults
PCR results for L. monocytogenes (Table 1) and Salmonella (Table 2) test method samples were identicalto culture confirmation. When compared to the reference methods, the POD was found to be identical for
the Salmonella test method and statistically equivalent for the L. monocytogenes test method.

iQ-Check Method Presumptivec0.0

LSB or Half Fraser
30°C
18–26 hr (LSB) or 25 ± 1 hr
(Half Fraser broth and LSB)

PODre

Automatic results analysis and interpretation

Fig. 2. FDA BAM protocol for detection of Salmonella spp. in foods compared with iQ-Check Salmonella II real-time PCR Kit
protocol..
30°C
24 ± 2 hr (48 ± 2 hr*)xc

NbMatrix–iQ-Check S. Typhimurium+ supplements

95% CIdPODcg

95% CIi

0.00, 0.43

0.00

-0.43, 0.43

0.30

0.15, 0.52

0.30

-0.01, 0.54

1.00

0.57, 1.00

0.00

-0.43, 0.43

dPODcph95% CIi

iQ-Check Method Confirmed

95% CIcx

PODcd

95% CI

0.00

0.00, 0.43

00.00

0.00, 0.43

0.00

-0.43, 0.43

120.600.39, 0.78

120.600.39, 0.78

0.00

-0.28, 0,28

5

1.00

0.57, 1.00

51.00

0.57, 1.00

0.00-0.43, 0.43

fTable 2. iQ-Check method confirmed results compared to reference method (top) and iQ-Check method presumptive
results compared to iQ-Check method confirmed results (bottom) for Salmonella.

Confirmation of species** with
iQ-Check S. Enteritidis

TSI and LIA
Biochemical and serological confirmation

Detection of Listeria monocytogenes

SampleReference Method Confirmed

PODcd

Nb

Spike levela+Hektoen

Selective isolation

Alternative Method

30°C
4 hriQ-Check Method Confirmed
xcSpike levela*Methods

SampleiQ-Check Salmonella real-time PCR KitSample

RV made fromingredients

FDA BAM Protocol

Table 1. iQ-Check method confirmed results compared to reference method (top) and iQ-Check method presumptive
results compared to iQ-Check confirmed results (bottom) for Listeria monocytogenes.

Ground Meat AlternativeIntroduction

Reference Method Confirmed

cxPODc50205

Spike levela

Nb

0.0
0.025
(0.016, 0.040)
0.14(0.067, 0.28)

iQ-Check Method Confirmed

dPODcg

95% CIi

0.00, 0.43

0.00

-0.43, 0.43

0.30

0.15, 0.52

0.30

-0.01, 0.54

1.00

0.57, 1.00

0.00

-0.43, 0.43

dPODcph95% CIi

95% CIcx

PODre

95% CI

0.00

0.00, 0.43

00.00

120.600.39, 0.78

65

1.00

0.57, 1.00

5d

iQ-Check Method Presumptive

iQ-Check Method Confirmed

xc

PODcpf

95% CIxc

PODcd

95% CI

500.000.00, 0.43

00.00

0.00, 0.43

0.00

-0.43, 0.43

20120.600.39, 0.78

120.600.39, 0.78

0.00

-0.28, 0,28

55

1.00

0.57, 1.00

51.00

0.57, 1.00

0.00

-0.43, 0.43

Spike Level in cfu/g, calculated using MPN including 95% CI
N = Number of test portions
c x = Number of positive test portions
d POD = Candidate method confirmed positives divided by Nce POD = Reference method confirmed positives divided by Nrf POD
cp = Candidate method presumptive positives divided by N
g dPOD = Difference between the candidate method confirmed positives and reference method confirmed positives POD
ch dPOD
cp = Difference between the candidate method presumpitve positives and candidate method confirmed positives POD
i 95% CI = If the confidence interval of the dPOD includes zero, then the difference between the methods is not significant.
abSignificanceThe results of this study demonstrated that iQ-Check real-time PCR methods are less time-consuming
and perform equivalent to the FDA BAM reference method for the detection of L. monocytogenes andSalmonella in plant-based ground meat substitutes. These data have been submitted to AOAC for amethod extension of validation.

–Automatic results analysis and interpretation

Listeria monocytogenes
Trypticase soy broth with yeast extract, hemolysis, carbohydrates, CAMP testorReal-time PCR

Confirmation of positive results withRAPID'Listeria spp.
orRAPID'L.mono* If low or absent colony growth is observed after 24 hr.

A

BFig. 1. FDA BAM protocol for detection of Listeria monocytogenes in foods compared with iQ-Check Listeria monocytogenes II
real-time PCR Kit protocol.

BIO-RAD is a trademark of Bio-Rad Laboratories, Inc. in certain jurisdictions. IQ-CHECK is a trademark of Bio-Rad Europe GmbH incertain jurisdictions. All trademarks used herein are the property of their respective owner.
22-2125

Visit us at bio-rad.com/FoodScience

Ver A

Bio-Rad Laboratories, Inc. | Food Science Division | 2000 Alfred Nobel Drive | Hercules, CA 94547 USA