Novel Real-Time PCR Assay for Detection of Listeria species in … · Listeria monocytogenes is a...
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CONFIDENTIAL. For Internal Use Only. © 2010 Life Technologies Patrick M. Zoder* 1 , Paolo Vatta 1 , Craig Cummings 1 , Lily Wong 1 , Arlene Nuñez 1 , Priya Balachandran 1 , Manohar Furtado 1 , Olga Petrauskene 1 , Pius Brzoska 1 , Erin Crowley 2 , Patrick Bird 2 , Marianne Torontalli 2 , David Goins 2 and Robert Tebbs 1 , Life Technologies TM , 850 Lincoln Centre Drive, Foster City, California 1 , Q Laboratories, Inc., Cincinnati, OH, USA 2 ABSTRACT Our novel real-time PCR assay for detection of Listeria species was specifically designed and evaluated to be used in both food and environmental safety testing. This lyophilized assay targets the conserved rnpB gene and includes an internal positive control. The assay detected all strains of all Listeria species tested and did not detect other ubiquitous food pathogens such as Salmonella, Staphylococcus and Escherichia coli. The PCR efficiency was close to 100% with a limit of detection at 10 genomic copies. The assay was validated on 25g of food matrices (Hot Dog, Roast beef, Smoked Salmon, Milk and Infant Formula) and 5 different environmental surfaces (stainless steel, rubber, plastic, ceramic, sealed concrete). All samples were enriched in Buffered Listeria Enrichment Broth (BLEB) for either 24 hours (food matrices) or 28 hours (environmental samples). Robust detection of Listeria species was observed, with perfect correlation with a control culture confirmation method. Detection using the real-time PCR assay reduced the time-to-result to less than 31 hours. In conclusion, our results demonstrate that rapid pathogen detection systems are invaluable tools for ongoing monitoring of environmental safety at food processing and production sites. INTRODUCTION Listeria monocytogenes is a gram positive, intracellular bacterial pathogen which has been implicated as the causative organism in several major outbreaks of food borne disease, which result in approximately 2,500 illnesses and 500 deaths in the United States annually (Farber et al. 1991). L. monocytogenes bacteria are found frequently in the food-processing environment and can form biofilms on solid surfaces commonly found in food processing plants, including stainless steel and rubber (Berrang et al. 2010). Traditional culture confirmation methods used in environmental safety testing are lengthy and time-to-result can be greatly reduced by using rapid molecular methods. The genus Listeria is composed of eight species; L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi and two recently discovered new species, given the provisional names L. marthii and L. rocourtiae (Leclercq et al. 2009). The presence of Listeria species in a food processing plant indicates potential co- contamination with Listeria monocytogenes. MATERIALS AND METHODS Seven Listeria strains were sequenced by the SOLiD TM system. The new sequences, together with publicly available sequences were used for multiplex assay development. One assay targets the conserved rnpB gene in L.monocytogenes, L.innocua, L. ivanovii, L. welshimerii, L. marthii and L. seeligeri, while the other targets the more divergent L. grayi, in a single reaction, which also includes an internal positive control. The assay was evaluated for the detection of Listeria species on a variety of environmental surfaces and foods. Food samples (25 g) were spiked with Listeria spp. to generate fractional positive recovery and enriched in Buffered Listeria Enrichment Broth for 24 hr. Enriched samples were prepared using PrepSEQ™ sample preparation and Real- time PCR was run on the 7500Fast instrument and results analyzed using Rapid Finder Express software. In addition, the ISO 11290-1 Listeria detection method was performed and the two data sets were evaluated by Chi-square analysis. The surfaces (ceramic, stainless steel, plastic, rubber and sealed concrete) were spiked with 100 cfu of bacteria and allowed to equilibrate overnight at ambient temperature, a procedure that mimics the normal conditions for which the assay will be used. RESULTS Figure 5. Assay Specificity – Inclusivity & Exclusivity Panel • The Listeria spp. assay showed 100% specificity to all inclusivity targets analyzed. • An exclusion panel consisting of gram positive and gram negative bacteria were not detected even at 10 9 cfu/ml. • The MicroSEQ® Listeria spp. Kit successfully detected down to 0.2- 2 cfu of bacteria for all food matrices tested. • No significant difference was observed between the MicroSEQ® method and the reference method for dry infant formula, lox, milk and roast beef (Chi-square < 3.84) • For Hot Dog samples, the MicroSEQ® method was superior to the reference culture method. Table 1. Food Matrices Results • The MicroSEQ® Listeria spp. Kit successfully detected fractional positive samples from all surfaces. • With Chi-square values of < 3.84 no significant difference was observed between the MicroSEQ® method and the reference method. Figure 3. Assay Level of Detection Detection limit for new RT-PCR Listeria spp. assay, tested on L. monocytogenes and Listeria grayi gDNA, is 10 genomic copies Figure 4. Assay Efficiency Assay efficiencies of 100% ± 10% were determined using serially diluted L. monocytogenes and L. grayi genomic DNA. Assay efficiency is determined by the slope and 3.3 represents a ten-fold difference in target concentration. The exhibited 100 ± 10% efficiency was over a 5 log range of target concentrations. CONCLUSIONS Shortened time-to-result: The streamlined workflow offers a great advantage over existing culture confirmation methods by reducing the time-to-result to under 3h following pre- enrichment. Traditional culture confirmation can take as long as a week. Highly sensitive assay: Lyophilized assay is very robust and allows for addition of more sample volume. In addition, lyophilized assays provide a convenient ease-of-use for laboratory operators. High throughput or Low throughput options: High quality DNA can be prepared by manual or automated methods, depending on number of samples being tested. Our novel Listeria spp. RT-PCR assay matched the high sensitivity of the ISO 11290-1 Listeria testing method. High-background food matrices, such as hot dog, roast beef and smoked salmon, allowed detection comparable to low-background, pasteurized matrices such as milk and infant formula. The use of BLEB and selective supplements during the 24 hour (food) and 28 hour (environmental) enrichment step assured maximum recovery of stressed and damaged Listeria bacteria and optimized time-to-result performance. Efforts are currently underway to improve the growth and recovery of Listeria and to incorporate additional assay targets for Listeria rocourtiae, a newly discovered Listeria species (Leclercq et al. 2009), in order to further enhance the sensitivity of this assay. REFERENCES 1. Farber, J. M., Peterkin, P.I. (1991). Listeria monocytogenes, a food-borne pathogen. Microbiol Mol Biol Rev., 55(3), 476-511. 2. Berrang, M.E., Frank, J., Meinersmann, R.J. 2010. Listeria monocytogenes Biofilm Formation on Silver Ion Impregnated Cutting Boards. Food Protection Trends., 30(3): 168 - 171. 3. Leclercq, A., Clermont, D., Bizet, C., Grimont, P. A. D., Le Fleche-Mateos, A., Roche, S.M., Buchrieser, C., Cadet Daniel, V., Le Monnier, A., Lecuit, M., Allerberger, F. Listeria rocourtiae sp. Nov. Int J Syst Evol Microbiol., 2009 0: ijs.0.017376-0 ACKNOWLEDGEMENTS We would like to thank Brooke Schwartz, Yan Cao and Karl Meyer (LIFE Technologies) for their significant contributions. Table 2. Environmental Surfaces Results Novel Real-Time PCR Assay for Detection of Listeria species in Food & Environmental Samples 30 th Food Microbiology Symposium, University of Wisconsin-River Falls L. monocytogenes 10 6 10 5 10 4 10 3 10 2 10 IPC L. grayi 10 6 10 5 10 4 10 3 10 2 10 IPC y = -3.4801x + 42.046 R 2 = 0.9937 0 10 20 30 40 1 2 3 4 5 6 log Avg. CT 94% Assay Efficiency: L. grayi y = -3.2658x + 41.42 R 2 = 0.9993 0 10 20 30 40 1 2 3 4 5 6 Log Avg. CT 102% Inclusivity Test 15 20 25 30 35 40 L. m onocytog e nes L.m on oc yo tge ne s L.m on oc y toge ne s L. i n noc u a L .in noc ua L. s eel i geri L . we l shimerii L. gra y i L. gra y i Avg. Ct Target IPC Exclusivity Test 15 20 25 30 35 40 B. c e re u s B. chole r ae B. th u ringi e nsis E. coli S. au r eus S . en t eri t id i s S. a g alactia e V. p a rah e m o l y ti c u s V. v ul n i fi c u s Avg. Ct Target IPC • 25g or 25ml, spiked with 0.2-2 cfu or 2-10 cfu • Samples homogenized with 225ml BLEB • Incubation at 37 o C • Listeria Selective Supplements added at 4h • Samples further incubated for a total of 24 h. MicroSEQ ® Listeria spp. PrepSEQ TM NA Extraction PrepSEQ TM RapidSpin Culture confirmation by ISO • Pre-defined area spiked with low or high levels of Listeria (in the presence of high background) • Surface allowed to dry. • Samples collected with sponges or swabs soaked in D/E Neutralizing Broth. • Samples enriched at 37 o C with 100ml(sponge) or 10ml (swab) of BLEB (supplements added at 4h) •Samples further incubated for a total of 28 h. MicroSEQ ® Listeria spp. PrepSEQ TM NA Extraction PrepSEQ TM RapidSpin Culture confirmation by ISO Environmental Samples 13 Listeria genomes and the L. innocua pLI100 plasmid were sequenced using SOLiD TM . A Listeria pan-genome was determined. Figure 1. Listeria pan-genome This figure shows the phlyogenetic tree for gene rnpB, the target for the Listeria spp. real-time PCR assay. Figure 2. Listeria assay target phylogenetic tree
Transcript of Novel Real-Time PCR Assay for Detection of Listeria species in … · Listeria monocytogenes is a...
CONFIDENTIAL. For Internal Use Only. © 2010 Life Technologies
Patrick M. Zoder* 1, Paolo Vatta 1, Craig Cummings 1, Lily Wong 1, Arlene Nuñez 1, Priya Balachandran 1, Manohar Furtado 1, Olga Petrauskene 1, Pius Brzoska 1, Erin Crowley 2, Patrick Bird 2, Marianne Torontalli 2, David Goins 2 and Robert Tebbs 1, Life Technologies TM, 850 Lincoln Centre Drive, Foster City, California 1, Q Laboratories, Inc., Cincinnati, OH, USA 2
ABSTRACTOur novel real-time PCR assay for detection of Listeria species was specifically designed and evaluated to be used in both food and environmental safety testing. This lyophilized assay targetsthe conserved rnpB gene and includes an internal positive control. The assay detected all strains of all Listeria species tested and did not detect other ubiquitous food pathogens such as Salmonella, Staphylococcus and Escherichia coli. The PCR efficiency was close to 100% with a limit of detection at 10 genomic copies. The assay was validated on 25g of food matrices (Hot Dog, Roast beef, Smoked Salmon, Milk and Infant Formula) and 5 different environmental surfaces (stainless steel, rubber, plastic, ceramic, sealed concrete). All samples were enriched in Buffered ListeriaEnrichment Broth (BLEB) for either 24 hours (food matrices) or 28 hours (environmental samples). Robust detection of Listeria species was observed, with perfect correlation with a control culture confirmation method. Detection using the real-time PCR assay reduced the time-to-result to less than 31 hours. In conclusion, our results demonstrate that rapid pathogen detection systems are invaluable tools for ongoing monitoring of environmental safety at food processing and production sites.
INTRODUCTIONListeria monocytogenes is a gram positive, intracellular bacterial pathogen which has been implicated as the causative organism in several major outbreaks of food borne disease, which result in approximately 2,500 illnesses and 500 deaths in the United States annually (Farber et al. 1991). L. monocytogenes bacteria are found frequently in the food-processing environment and can form biofilms on solid surfaces commonly found in food processing plants, including stainless steel and rubber (Berrang et al. 2010). Traditional culture confirmation methods used in environmental safety testing are lengthy and time-to-result can be greatly reduced by using rapid molecular methods. The genus Listeria is composed of eight species; L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, L. ivanovii, L. grayi and two recently discovered new species, given the provisional names L. marthiiand L. rocourtiae (Leclercq et al. 2009). The presence of Listeriaspecies in a food processing plant indicates potential co-contamination with Listeria monocytogenes.
MATERIALS AND METHODSSeven Listeria strains were sequenced by the SOLiDTM system. The new sequences, together with publicly available sequences were used for multiplex assay development. One assay targets the conserved rnpB gene in L.monocytogenes, L.innocua, L. ivanovii, L. welshimerii, L. marthii and L. seeligeri, while the other targets the more divergent L. grayi, in a single reaction, which also includes an internal positive control. The assay was evaluated for the detection of Listeria species on a variety of environmental surfaces and foods. Food samples (25 g) were spiked with Listeriaspp. to generate fractional positive recovery and enriched in Buffered Listeria Enrichment Broth for 24 hr. Enriched samples were prepared using PrepSEQ™ sample preparation and Real-time PCR was run on the 7500Fast instrument and results analyzed using Rapid Finder Express software. In addition, the ISO 11290-1 Listeria detection method was performed and the two data sets were evaluated by Chi-square analysis. The surfaces (ceramic, stainless steel, plastic, rubber and sealed concrete) were spiked with 100 cfu of bacteria and allowed to equilibrate overnight at ambient temperature, a procedure that mimics the normal conditions for which the assay will be used.
RESULTS
Figure 5. Assay Specificity –Inclusivity & Exclusivity Panel
• The Listeria spp. assay showed 100% specificity to all inclusivity targets analyzed.• An exclusion panel consisting of gram positive and gram negative bacteria were
not detected even at 109cfu/ml.
• The MicroSEQ® Listeria spp. Kit successfully detected down to 0.2- 2 cfu of bacteria for all food matrices tested.
• No significant difference was observed between the MicroSEQ® method and thereference method for dry infant formula, lox, milk and roast beef (Chi-square < 3.84)
• For Hot Dog samples, the MicroSEQ® method was superior to the reference culturemethod.
Table 1. Food Matrices Results
• The MicroSEQ® Listeria spp. Kit successfully detected fractional positive samples from all surfaces.
• With Chi-square values of < 3.84 no significant difference was observed between the MicroSEQ® method and the reference method.
Figure 3. Assay Level of Detection
Detection limit for new RT-PCR Listeriaspp. assay, tested on L. monocytogenesand Listeria grayi gDNA, is 10 genomic copies
Figure 4. Assay Efficiency
Assay efficiencies of 100% ± 10% were determined using serially diluted L.monocytogenes and L. grayi genomic DNA. Assay efficiency is determined by the slope and 3.3 represents a ten-fold difference in target concentration. The exhibited 100 ± 10% efficiency was over a 5 log range of target concentrations.
CONCLUSIONSShortened time-to-result: The streamlined workflow offers a great
advantage over existing culture confirmation methods by reducing the time-to-result to under 3h following pre-enrichment. Traditional culture confirmation can take as long asa week.
Highly sensitive assay: Lyophilized assay is very robust and allows for addition of more sample volume. In addition, lyophilized assays provide a convenient ease-of-use for laboratory operators.
High throughput or Low throughput options: High quality DNA can be prepared by manual or automated methods, depending on number of samples being tested.
Our novel Listeria spp. RT-PCR assay matched the high sensitivity ofthe ISO 11290-1 Listeria testing method. High-background foodmatrices, such as hot dog, roast beef and smoked salmon, alloweddetection comparable to low-background, pasteurized matrices suchas milk and infant formula. The use of BLEB and selectivesupplements during the 24 hour (food) and 28 hour (environmental)enrichment step assured maximum recovery of stressed anddamaged Listeria bacteria and optimized time-to-result performance.
Efforts are currently underway to improve the growth and recovery ofListeria and to incorporate additional assay targets for Listeriarocourtiae, a newly discovered Listeria species (Leclercq et al. 2009),in order to further enhance the sensitivity of this assay.
REFERENCES1. Farber, J. M., Peterkin, P.I. (1991). Listeria monocytogenes, a food-bornepathogen. Microbiol Mol Biol Rev., 55(3), 476-511.
2. Berrang, M.E., Frank, J., Meinersmann, R.J. 2010. Listeria monocytogenesBiofilm Formation on Silver Ion Impregnated Cutting Boards. Food ProtectionTrends., 30(3): 168 - 171.
3. Leclercq, A., Clermont, D., Bizet, C., Grimont, P. A. D., Le Fleche-Mateos, A.,Roche, S.M., Buchrieser, C., Cadet Daniel, V., Le Monnier, A., Lecuit, M.,Allerberger, F. Listeria rocourtiae sp. Nov. Int J Syst Evol Microbiol., 2009 0:ijs.0.017376-0
ACKNOWLEDGEMENTSWe would like to thank Brooke Schwartz, Yan Cao and Karl Meyer (LIFETechnologies) for their significant contributions.
Table 2. Environmental Surfaces Results
Novel Real-Time PCR Assay for Detection of Listeriaspecies in Food & Environmental Samples
30th Food Microbiology Symposium, University of Wisconsin-River Falls
L. monocytogenes
106 105 104 103 102
10
IPC
L. grayi
106 105 104 103 102
10IPC
y = -3.4801x + 42.046R2 = 0.9937
0
10
20
30
40
1 2 3 4 5 6
log
Avg
. CT
94%
Assay Efficiency: L. grayi
y = -3.2658x + 41.42R2 = 0.9993
0
10
20
30
40
1 2 3 4 5 6Log
Avg
. C
T
102%
Inclusivity Test
152025303540
L. mon
ocyto
gene
s
L.monoc
yotgen
es
L.monoc
ytogen
esL.
innocu
aL.in
nocu
aL.
seelig
eri
L. welsh
imeri
iL.
gray
iL.
gray
i
Avg
. C
t
Target
IPC
Exclusivity Test
152025303540
B. cer
eus
B. cho
lerae
B. thu
ringie
nsis
E. coli
S. aur
eus
S. ente
ritidi
sS. a
galac
tiae
V. par
ahem
olytic
usV. v
ulnific
us
Avg
. C
t
Target
IPC
• 25g or 25ml, spiked with 0.2-2 cfu or 2-10 cfu
• Samples homogenized with 225ml BLEB• Incubation at 37 oC• Listeria Selective Supplements added at 4h• Samples further incubated for a total of 24 h.
MicroSEQ ® Listeria spp.
PrepSEQTM
NA ExtractionPrepSEQTM
RapidSpinCulture
confirmationby ISO
• Pre-defined area spiked with low or high levels of Listeria (in the presence of high background)• Surface allowed to dry.
• Samples collected with sponges or swabs soaked in D/E Neutralizing Broth.• Samples enriched at 37 oC with 100ml(sponge) or 10ml (swab) of BLEB (supplements added at 4h) •Samples further incubated for a total of 28 h.
MicroSEQ ® Listeria spp.
PrepSEQTM
NA ExtractionPrepSEQTM
RapidSpinCulture
confirmationby ISO
Environmental Samples
13 Listeria genomes and the L. innocuapLI100 plasmid were sequenced using SOLiDTM. A Listeria pan-genome was determined.
Figure 1. Listeria pan-genome
This figure shows the phlyogenetic tree for gene rnpB, the target for the Listeria spp. real-time PCR assay.
Figure 2. Listeria assay target phylogenetic tree
