validation of the thermo scientific suretect real-time pcr...
TRANSCRIPT
Conclusion
The SureTect Salmonella species Assay was shown to be an
accurate and user-friendly method, due to the use of pre-
dispensed lysis reagent, tableted PCR reagents and automatic
interpretation of results. Results from a wide range of foods,
including challenging matrices, demonstrated the assay was
able to reliably detect the presence of Salmonella.
References
1. AOAC International, Method Committee Guidelines for
Validation of Microbiological Methods for Food and
Environmental Surfaces 2012.
2. ISO, Microbiology of Food and Animal Feeding stuffs-
Horizontal Method for the Detection of Salmonella spp. ISO
6579:2002.
Overview
Purpose: To validate the Thermo Scientific™ SureTect™
Salmonella species Assay according to AOAC Research
Institute (RI) Performance Tested MethodsSM validation
criteria.
Methods: The SureTect method was compared to the
reference method detailed in ISO 6579:2002.
Results: The SureTect Salmonella species assay reliably
detected the presence of Salmonella in a wide variety of
matrices.
Introduction
The Thermo Scientific SureTect Salmonella species
Assay (PT0100A) is a new Real-Time PCR test for the
detection of Salmonella from food, animal feeds and
environmental samples, which combines pre-dispensed
lysis reagent and lyophilised, tableted PCR reagents to
simplify and improve assay handling, along with dedicated
software to run the assays as well as interpret and display
PCR results. This study was conducted using the AOAC
RI Performance Tested MethodsSM program1 to validate
the SureTect Salmonella species assay in comparison to
the reference method detailed in ISO 6579:20022 with a
variety of food matrices.
Methods
Sample Preparation
Bulk samples of foods were screened for natural
contamination with Salmonella before splitting into three
samples; unspiked (control), low spiked (0.2-2 CFU/25g
and high spiked (2-5 CFU/25g) samples. Once spiked, all
samples were allowed to equilibrate as per AOAC
instructions.
Surface samples of stainless steel were spiked with a
suspension of Salmonella. Where samples were not
paired as in the case of surface samples and raw ground
beef analysis with the 8h enrichment protocol, additional
separate samples were prepared.
SureTect Assay Method
25g samples of foods and surface sponges were added to
225ml of room temperature Buffered Peptone Water
(BPW) (ISO), with the exception of raw ground beef with
the short 8h protocol, where pre-warmed BPW (ISO) was
used. Samples of raw ground beef analysed with the
short protocol were incubated at 41.5°C for 8h, non-fat
dried milk, environmental surfaces, raw beef and liquid
egg were incubated at 37°C for 18h and cooked shrimps,
Frankfurters, lettuce and raw chicken for 20h at 37°C.
Following enrichment 10µl of each sample was added to
the prefilled SureTect Lysis Tubes (prepared by
additionally adding Proteinase K Reagent) and the sample
lysed according to the SureTect lysis protocol (37°C for 10
minutes followed by 95°C for 5 minutes).
© 2013 Thermo Fisher Scientific Inc. All rights reserved. API is a trademark of bioMérieux SA. Performance Tested
Methods is a service mark of AOAC-RI. All other trademarks are the property of Thermo Fisher Scientific Inc and its
subsidiaries. This information is not intended to encourage use of these products in any manner that might infringe the
intellectual property rights of others.
Folio number LT2085A, 05/13
.
FIGURE 3. Inclusivity of the SureTect Salmonella
species Assay.
Validation of the Thermo Scientific SureTect Real-Time PCR Method for Detection of Salmonella in
Food and Environmental Samples
Jonathan Cloke1, Dorn Clark2, Roy Radcliff2, Carlos Leon-Velarde3, Nathan Larson3, Keron Dave3
1Thermo Fisher Scientific, Basingstoke, Hampshire, RG24 8PW, UK, 2Marshfield Food Safety, 1000 North Oak Avenue,
Marshfield, Wisconsin, 54449, USA, and 3Agriculture and Food Laboratory, University of Guelph, Guelph, Ontario, N1H
8J7, Canada
Results
Inclusivity and exclusivity
All 117 Salmonella isolates were detected as positive by the
SureTect Software. None of the 36 exclusivity isolates were
detected by the SureTect Software.
Serotype Number % Positive
Salmonella bongori 4 100%
Salmonella enterica subsp. salamae 5 100%
Salmonella enterica subsp. arizoniae 3 100%
Salmonella enterica subsp.
diarizoniae4 100%
Salmonella enterica subsp. enterica 101 100%
Matrix/Inoculating
OrganismLevel
MPN/
25g
No. Test
portionsISO
SureTect
Presum* Con*
Raw chicken breast
Salmonella Indiana
Low 1.2 20 13 12 12
High 2.3 5 5 5 5
Raw ground pork
Salmonella Livingstone
Low 0.68 20 11 11 11
High 2.5 5 4 4 4
Non-fat dried milk/
Salmonella Infantis
Low 1.2 20 15 15 15
High 4.5 5 5 5 5
Pork Frankfurters
Salmonella Poona
Low 0.38 20 8 8 8
High 0.55 5 3 3 3
Cooked shrimp
Salmonella SaintPaul
Low 1.3 20 13 13 13
High 4.5 5 5 5 5
Stainless steel surface
Salmonella Newport & E.
coli
Low N/A 20 16 16 16
High N/A 5 5 5 5
Ready to eat meal
Salmonella Enteritidis
Low 1.3 20 15 15 15
High 4.5 5 5 5 5
Lettuce
Salmonella Anatum
Low 1.0 20 14 13 14
High 1.5 5 4 4 4
Raw ground beef (short
8h protocol)
Salmonella Typhimurium
Low 0.63 20 10 8 11
High 2.3 5 5 4 5
Raw ground beef
(standard protocol)
Salmonella Typhimurium
Low 0.63 20 10 9 11
High 2.3 5 5 5 5
Pasteurised liquid egg
Salmonella Virchow
Low 0.58 20 8 8 8
High 1.35 5 3 3 3
FIGURE 3. Method Developer Results for the ISO and
SureTect Salmonella species Methods.
Matrix/Inoculating
OrganismLevel
MPN/
25g
No. Test
portionsISO
SureTect
Presum* Con*
Lettuce
Salmonella Thompson
Low 0.73 20 10 10 9
High 2.97 5 5 5 5
Pork Frankfurters
Salmonella Vellore
Low 0.48 20 7 7 7
High 2.97 5 5 5 5
Stainless steel surface
Salmonella Berta & E.
coli
Low N/A 20 14 14 14
High N/A 5 5 5 5
* Presumptive (Presum) and Confirmed (Con) SureTect results
* Presumptive (Presum) and Confirmed (Con) SureTect results
FIGURE 4. Independent Laboratory Results for the ISO
and SureTect Salmonella species Methods.
Once lysed, 20µl of the lysate was added to the SureTect
PCR Tubes, which contain lyophilised PCR reagents before
running on the Thermo Scientific™ PikoReal™ Real-Time
PCR instrument.
Assay results were automatically interpreted as “positive” or
“negative” by the SureTect Software.
All SureTect results were confirmed culturally using the
SureTect confirmation method of direct plating onto Oxoid™
Brilliance™ Salmonella Agar and confirming presumptive
positive purple colonies with the Oxoid™ Salmonella Latex
Kit (DR1108A) and additionally using the reference method
confirmation protocol.
ISO Reference Method
The reference method detailed in ISO 6579:2002 was
followed, using Brilliance Salmonella Agar as the second
plating medium. Confirmations were performed using the
Remel™ microID™ kit or bioMérieux API™ 20E kit, Triple
Sugar Iron (TSI) slants and poly-O and poly-H antisera.
Inclusivity
One-hundred and seventeen Salmonella isolates covering a
wide variety of O- serogroups and subspecies were cultured
in BPW (ISO) and analysed at a level of approximately 104
CFU/ml using the SureTect assay protocol according to
AOAC-RI PTM requirements.
Exclusivity
Thirty-six exclusivity isolates were cultured in TSB for 18-24
hours and analysed at a level of approximately 108CFU/ml
using the SureTect assay protocol according to AOAC-RI
PTM requirements.
Food Matrix Analysis
No statistically significant difference, by probability of detection
analysis (POD), was seen for any of the ten food matrices and
the environmental surface evaluated in this PTM study during
either the method developer or independent laboratory studies
between the ISO reference method or the SureTect Salmonella
species assay.
Add 10µl of Proteinase K to the SureTect
Lysis Tube (pre-filled with Lysis Reagent 1)
Add 10µl of BPW (ISO) enriched sample
Lyse sample (37°C 10 min followed by 95°C
for 5 min)
Add 20µl of lysate to SureTect PCR Tube
(pre-filled with lyophilised reagent)
Run samples in PikoReal Instrument and
read off results
FIGURE 2. SureTect Assay Workflow.
FIGURE 1. The Thermo Scientific SureTect System.
Significance The Thermo ScientificTM SureTectTM Salmonella spp Real-Time PCR Assay is a reliable method for Salmonella spp detection in food
and pet food samples, and offers important economic savings by reducing time to result and handling time.
Introduction The Thermo ScientificTM SureTectTM Salmonella spp species Real-Time PCR Assay is a new detection method based on real-time
PCR. The oligonucleotides target unique DNA sequences found only in the analyte and use PCR technology to amplify and detect them. If present, the
target DNA is amplified and the increasing fluorescent signal is detected by the Thermo Scientific PikoReal Real-Time PCR instrument and interpreted
by the Thermo Scientific SureTect. Only 8 h incubation time is required for raw beef meat analyses.
Method comparison studyRelative accuracy, relative specificity and
relative sensitivity study
442 samples were analyzed. The paired data
below show 10 negative deviations and 8 positive
deviations. The performances of the compared
methods are equivalent:
10 laboratories from various European countries
were involved. Ground beef was inoculated with
S. Typhimurium. 8 h incubation time was run in
each involved laboratory.
The paired data, the relative accuracy,
sensitivity, specificity, as well as the
accordance, concordance and odds ratio clearly
show that the alternative method precision is
equivalent to the ISO method:
Performance Assessment of the Thermo ScientificTM SureTectTM Salmonella spp
Real-Time PCR Assay according to the ISO 16140 Standard
for Salmonella spp Detection in Food and Pet Food Samples
J. Baguet, M. Bernard, C. Bernez, C. Le Doeuff, S. Peron, M. Rannou, D. SohierADRIA, expert laboratory at AFNOR certification, AOAC-RI and MicroVal
Purpose An independent study was conducted at ADRIA, to validate this new method in comparison to the ISO 6579 standard, as part of the
NF Validation approval process and according to the ISO 16140 standard.
Inter-laboratory study
Alternative
method
ISO 6579
Reference method TOTAL
+ -
+ PA = 159 PD = 1 160
- ND = 0 NA = 80 80
TOTAL 159 81 240
PA :Positive Agreement NA: Negative Agreement
PD: Positive Deviation ND: Negative Deviation
Alternative
method
ISO 6579
Reference method TOTAL
+ -
+ PA = 188 PD = 8 196
- ND = 10 NA = 236 246
TOTAL 198 244 442
PA :Positive Agreement NA: Negative Agreement
PD: Positive Deviation ND: Negative Deviation
Relative detection levels
Various (matrix/strain) pairs were tested, with
4 inoculation levels. The relative detection limits
of the alternative method vary from 0.2 to
1.2 CFU/25 g, those of the ISO standard vary
from 0.2 to 1.1 CFU/25 g:
Matrices
Relative detection levels
Reference
method
Alternative
method
Poultry meat 0.6 [0.3 ; 1.1] 0.8 [0.4 ; 1.5]
Whole egg 0.5 [0.2 ; 0.8] 0.5 [0.3 ; 0.8]
Produces 0.4 [0.2 ; 0.7] 0.4 [0.2 ; 0.7]
Raw milk 0.7 [0.4 ; 1.1] 0.7 [0.4 ; 1.2]
Pet Food 0.4 [0.3; 0.7] 0.4 [0.2 ; 0.7]
Ground beef 0.6 [0.3; 1.0] 0.43[0.2 ; 0.5]
Inclusivity / exclusivity
30 non target strains and 51 target strains were tested. No cross reaction was observed with the non
target strains, all the Salmonella spp strains were detected.
N° attestation UNI 03/07-11/13
ALTERNATIVE ANALYTICAL METHODS
FOR THE FOOD INDUSTRY
www.afnor-validation.com
Belén Moreno1, Ruth Rodríguez1, Patricia Uribarren1, Carmen Oria2, Nagore Errazti3 y Juncal Artieda4
1 Laboratorio de Salud Pública de Gipuzkoa, Gobierno Vasco, [email protected] 2Unidad de Sanidad Alimentaria, Subdirección de Salud de
Gipuzkoa, Gobierno Vasco. 3 Comarca de Salud Pública Tolosa-Goierri, Gobierno Vasco. 4Unidad de Epidemiología, Subdirección de Salud de Gipuzkoa,
Gobierno Vasco
MUESTREO sistemático de todo el producto clasificado y envasado por el centro de embalaje. El número de muestras
correspondientes a cada una de las manadas y procedencias se estimó en base al número de medias docenas clasificadas y envasadas
el día del muestreo. Cada muestra seleccionada estaba compuesta por seis huevos.
MÉTODO: detección de Salmonella spp. tanto en la cáscara como en el interior de los huevos, mediante el kit de detección de
Salmonella spp. SureTectTM y el equipo PikoReal Real Time PCR.
Comparación del método inmunoenzimático empleado habitualmente en el laboratorio y acreditado por ENAC, VIDAS Salmonella
(VIDAS SLM), con el método Thermo Scientific SureTectTM Salmonella spp. PCR Assay. Ambos métodos están validados por AFNOR.
Tras varios brotes de gastroenteritis aguda por Salmonella Enteritidis se inició el estudio
epidemiológico y ambiental. Los huevos frescos de una explotación fueron el alimento implicado.
Se empleó en el estudio del brote un nuevo método de detección de Salmonella spp., Thermo
ScientificTM SureTectTM, basado en tecnología de PCR, para amplificar y detectar secuencias
únicas de DNA.
El objeto del estudio es evaluar el sistema SureTectTM para la detección de Salmonella spp. por
PCR en tiempo real para su implantación en un laboratorio acreditado.
Los resultados fueron concordantes y se detectó, por ambos métodos, Salmonella Enteritidis,
cepa no vacunal, en 3 de las 60 muestras de cáscara de huevo (5 %).
Al evaluar los dos métodos de análisis, el laboratorio se decantó por utilizar el método de
detección mediante PCR al presentar las siguientes ventajas:
- Método más rápido, resultados negativos a las 24 h
- Flujo de trabajo más fácil, menor tiempo de manipulación y probabilidad de errores
- Incremento de la productividad del laboratorio
Añadir 10µl de Proteinasa K al tubo de lisis SureTectTM,
que contiene el Reactivo de lisis 1
Pesar la muestra y añadir APT en proporción 1:10
Incubar a 37ºC durante 22 ± 2 h
Añadir 10µl del preenriquecimiento de la muestra en
APT
Lisado de la muestra (37°C 10 min y 95°C 5 min)
Añadir 20µl del lisado al tubo de PCR SureTectTM, que
contiene los reactivos liofilizados
Inicio de Confirmación de resultados positivos en
SureTect PCR según ISO 6579
Llevar a cabo la reacción de PCR en el instrumento
PikoReal y leer los resultados (1h, 20 min)
Día 1
Día 2
Día 1
Día 2
Día 3
Pesar la muestra y añadir APT en proporción 1:10
Incubar a 37ºC durante 16-20 h
Añadir 1 ml de cada caldo M en un tubo y calentar a 100ºC
durante 15± 1min
Inicio de Confirmación de resultados positivos en VIDAS
SLM según ISO 6579
Añadir 1 ml a un tubo con
10 ml de MKTTn
Incubar 6-8h a 37 ± 1ºC
Añadir 0,1 ml a un tubo
con 10 ml de RVS
Incubar 6-8h a 41,5 ± 1ºC
Añadir 0,5 ml en la tira de VIDAS SLM y llevar a cabo la
reacción (45 min). Leer los resultados
Añadir 1 ml a un tubo con
10 ml de Caldo M
Incubar 16-20h a 41,5 ± 1ºC
Añadir 0,1 ml a un tubo
con 10 ml de Caldo M
Incubar 16-20h a 37 ± 1ºC
Detección de Salmonella spp. en huevos frescos mediante el método de PCR a tiempo real SureTectTM
INTRODUCCIÓN
MATERIAL Y MÉTODOS
OBJETIVO
RESULTADOS Y CONCLUSIONES
XIX CONGRESO NACIONAL DE MICROBIOLOGÍA DE LOS ALIMENTOS, Zaragoza Septiembre de 2014
PROTOCOLO VIDAS SLM PROTOCOLO SureTectTM
Conclusión
El ensayo SureTect Listeria monocytogenes es un método preciso y
fácil
de
usar,
al
presentar
los
reactivos
de
lisis
predispensados,
los
reactivos
de
PCR
liofilizados
y
permitir
informar
los
resultados
de
forma
automática. Los
resultados
en
una
gran
variedad
de
alimentos,
incluyendo
matrices
dificiles
cómo
el
salami
y
el
salmón
ahumado,
demostraron
que
este
ensayo
detecta
de
forma
fiable
la
presencia de L. monocytogenes en alimentos y superficies.
Referencias
1.
AOAC
International,
Method
Committee
Guidelines
for
Validation
of
Microbiological
Methods
for
Food
and
Environmental Surfaces 2012.
2.
ISO,
Microbiology
of
Food
and
Animal
Feeding
stuffs‐Horizontal
Method for the Detection of Listeria monocytogenes. ISO 11290‐
1:1996.
Resumen
Objetivo:
Validar
el
ensayo
Thermo
Scientific™
SureTect™
Listeria
monocytogenes
según
los
criterios
de
validación
Performance
Tested
MethodsSM
del
AOAC
Research
Institute
(RI).
Métodos:
El
método
SureTect
se
comparó
con
el
método
de
referencia detallado
en
la
ISO
11290‐1:1996
y
su Modificación
1:2004.
Resultados:
El
ensayo SureTect Listeria monocytogenes detecta
de manera fiable la presencia de L. monocytogenes en una gran
variedad de matrices.
Introducción
El
ensayo
Thermo
Scientific
SureTect
Listeria
monocytogenes
(PT0300A)
es
un
nuevo
ensayo
de
PCR
en
Tiempo
Real
para
la
detección
de
Listeria
monocytogenes
en
alimentos
y
muestras
ambientales, que combina los reactivos de lisis predispensados
y
los
reactivos
de
PCR
liofilizados
para
simplificar
y
mejorar
la
manipulación
en
el
ensayo,
además
de
un
software
dedicado
para analizar e interpretar los resultados por PCR. El estudio de
validación del ensayo SureTect se realizó
según especificaciones
del
programa1
AOAC
RI
Performance
Tested
MethodsSM
en
comparación con el método de referencia ISO 11290‐1.
Métodos
Preparación de la Muestra
Con
el
objetivo
de
detectar
contaminación
natural
por
Listeria
spp,
se
analizaron
muestras
de
alimentos
dividiendo
seguidamente cada una en tres porciones: muestra sin inocular
(control), con
bajo
nivel
de
contaminación
(0.2‐2
CFU/25g)
y
con
alto
nivel
de
contaminación (2‐5
CFU/25g). Una
vez
dopadas,
las
muestras
se
dejaron
equilibrar
según
las
especificaciones técnicas indicadas por AOAC . Las muestras de
superficie
de
acero
inoxidable
y plástico
se
inocularon
con
una
suspensión de L. monocytogenes.
Método del Ensayo SureTect
Tras diluir 25g de las muestras y cada una de las esponjas de
superficie
en
225
ml
del
caldo
Oxoid™
24
LEB con
Tampón
24
LEB y
suplementos
selectivos,
y
atemperado
previamente, se
incubaron
todas
las
muestras
a 37°C
durante
22
horas.
Transcurrido
este
tiempo,
se
añadió
10
µl
de
cada
uno
de
los
enriquecimientos a los tubos con solución de lisis, Proteinasa K
y
Reactivo
de
Lisis
2.
El
protocolo
de
lisado
se
llevó
a
cabo
mediante
calentamiento
en
termobloque
a
37°C
durante 10
minutos seguido de 95°C durante 5 minutos. A continuación se
añadieron 20
µl
del
lisado
en
tubos de
PCR
SureTect
que
contienen
predispensados
y liofilizados
los
reactivos
de
PCR
y
se
procesaron en
el
equipo
de
PCR
Real‐Time
PikoReal™
de
Thermo
Scientific™.
El
Software
SureTect
interpretó
los
resultados automáticamente
como
“positivo”
o
“negativo”.
Todos
estos
resultados
se
confirmaron mediante
siembra
directa
en placa Oxoid™
Brilliance™
Listeria
Agar
y
galería
de
identificación bioquímica de Oxoid™
Microbact™
12L así
como
por las descritas por el método
ISO de referencia.
FIGURA 1. El Sistema Thermo Scientific SureTect
© 2013 Thermo Fisher Scientific Inc. All rights reserved. Performance Tested Methods is a service mark of AOAC‐RI. All other
trademarks are the property of Thermo Fisher Scientific Inc and its subsidiaries. This information is not intended to encourage
use of these products in any manner that might infringe the intellectual property rights of others.
Folio number LT2086A, 05/13
.
Validación del Método Thermo Scientific SureTect Real-Time PCR para la detección de Listeria monocytogenes en Alimentos y Muestras medioambientales
Jonathan Cloke1, Carlos Leon-Velarde2, Nathan Larson2, Keron Dave2, Helen Simpson1, Craig Hopper1, Katharine Evans1, David Crabtree1, Annette Hughes1, Milena Oleksiuk1, Sophie Withey1
1Thermo Fisher Scientific, Basingstoke, Hampshire, RG24 8PW, UK and 2Agriculture and Food Laboratory, University of Guelph, Guelph, Ontario, N1H 8J7, Canada
Matriz/microorganismo
inoculadot y serotypoNivel
MPN/
25g
No.
Porciones
del testISO
SureTect
Presun* Con*
Melon CantaloupeL. monocytogenes 3c
Bajo 0.70 20 11 13 13
Alto 3.00 5 5 5 5
Salami L. monocytogenes 1/2a
Bajo 0.29 20 4 10 11
Alto 0.40 5 3 4 4
Salmón ahumadoL. monocytogenes
Bajo 0.60 20 8 7 8
Alto 1.25 5 2 4 4
Espinacas frescasL. monocytogenes 3b
Bajo 0.38 20 6 8 8
Alto 0.39 5 5 5 5
Pavo cocidoL. monocytogenes 4b
Bajo 1.06 20 12 19 19
Alto 4.37 5 5 4 4
Salchichas de cerdoL. monocytogenes 4e
Bajo 0.72 20 9 14 14
Alto 1.00 5 5 5 5
HeladoL. monocytogenes 1/2b
Bajo 0.24 20 1 12 12
Alto 0.64 5 0 4 4
Gambas cocidasL. monocytogenes 4b
Bajo 1.00 20 14 15 15
Alto 1.88 5 4 4 4
Queso procesadoL. monocytogenes 1/2a
Bajo 0.53 20 9 6 6
Alto 1.48 5 4 4 4
Carne cruda picada de
terneraL. monocytogenes 1/2c
Bajo 1.13 20 14 13 13
Alto 1.88 5 4 4 4
Superficie acero inoxidableL. monocytogenes 3a
Bajo N/A 20 12 11 11
Alto N/A 5 4 5 5
Superficie de plásticoL. monocytogenes 3a
Bajo N/A 20 15 16 16
Alto N/A 5 5 4 4
FIGURA 3. Resultados por ISO y SureTect durante el desarrollo
del método
Matrix/Inoculating Organism
Level MPN/ 25g
No. Test portions
ISO SureTect
Presum* Con*
Salchichas de cerdo L. monocytogenes 4b
Bajo 0.59 20 8 12 12
Alto 2.97 5 5 5 5
Lechuga frescaL. monocytogenes 4b
Bajo 0.37 20 5 10 10
Alto 2.19 5 5 5 5
Superficie acero inoxidableL. monocytogenes 1/2a
Bajo N/A 20 8 8 8
Alto N/A 5 4 5 5
Resultados
Inclusividad
Los 53 cultivos de L. monocytogenes de distintos serotipos
fueron detectados con el ensayo y software
SureTect.
Exclusividad
Las 38
cepas,
fueron detectados como
no‐
Listeria
monocytogenes
con el ensayo y software SureTect.
Análisis des las Matrices Alimentarias
Durante
el
desarrollo
del
método,
se
constató
una
diferencia
estadísticamente
significativa,
mediante análisis
de
la
probabilidad
de
detección (POD), para las
matrices
de
salami,
salmón
ahumado,
espinacas,
pavo
cocido
loncheado,
salchichas
de
cerdo,
helado,
gambas
cocidas
y
acero
inoxidable
a
favor
del
método SureTect
y
en
salchichas
de
cerdo
y
lechuga
durante
el
estudio
del
laboratorio
independiente,
demostrando
que
el
ensayo SureTect
Listeria
monocytogenes es
más
fiable
que
le
método de referencia ISO para la detección de contaminación por
L.
monocytogenes
en
muestras
de
alimentos
y
superficies. Los
resultados
para
el
resto
de
las
matrices
no
mostraron
una
diferencia
estadísticamente
significativa
por
análisis
POD
entre
los métodos ISO y SureTect.
* Presumptive (Presum) and Confirmed (Con) SureTect results
*Resultados presuntivos (Presun) y confirmados (Con) por SureTect
FIGURA 4. Resultados del Laboratorio Independente por
métodos ISO y SureTect para Listeria monocytogenes.
Método de referencia ISO
Todos
los
experimentos
fueron
llevados
a
cabo
según
las
especificaciones
técnicas indicadas
en
el
método
de
referencia
ISO
11290‐1:1996,
incluída
la
Modificación
1:2004
y
empleando
el Agar Oxford como medio secundario
de siembra
y
las
pruebas
confirmatorias
de
tinción
de
Gram,
catalasa,
hemólisis,
prueba
CAMP , ramnosa y xilosa.
Inclusividad
Se
emplearon
53
cultivos
puros
de
L.
monocytogenes
de todos
los serotipos conocidos excepto el 4ab, incubados en el caldo 24
LEB,
suplementado de
forma
selectiva,
y a una concentración
de
104 CFU/ml con el método SureTect.
Exclusividad
Se
inocularon
38 cultivos
puros en
Caldo
Triptona
Soja
y
se
analizaron
a
una
concentración
aproximada de 108CFU/ml
con
el ensayo SureTect y siguiendo las especificaciones del Programa
Performance Tested de AOAC‐RI.
FIGURA 2. Flujo de trabajo del ensayo SureTect .
Añadir 10µl de Proteinasa K y 10µl del Reactivo
de Lisis 2 al tubo de lisis de SureTect que contiene
predispensado el Reactivo Lisis 1
Añadir 10µl de la muestra enriquecida en 24 LEB
Lisar la muestra 37°C 10 min seguido de 95°C 5 min
Añadir 20µl de lisado al tubo de PCR SureTect
(incluye el reactivo liofillizado)
Analizar las muestras en el equipo PikoReal
Significance The Thermo ScientificTM SureTectTM Listeria monocytogenes species Real-Time PCR Assay is a reliable method for Listeria
monocytogenes detection in food and environmental samples, and offers important economic savings by reducing time to result and handling time.
Introduction The Thermo ScientificTM SureTectTM Listeria monocytogenes species Real-Time PCR Assay is a new detection method based on
real-time PCR. The oligonucleotides target unique DNA sequences found only in the analyte and use PCR technology to amplify and detect them. If
present, the target DNA is amplified and the increasing fluorescent signal is detected by the Thermo Scientific PikoReal Real-Time PCR instrument and
interpreted by the Thermo Scientific SureTect.
Method comparison studyRelative accuracy, relative specificity and
relative sensitivity study
339 samples were analyzed. The paired data
below show 26 negative deviations and
34 positive deviations. The performances of the
compared methods are equivalent:
10 laboratories from various European countries
were involved. Cheese was inoculated with
L. monocytogenes.
The paired data, the relative accuracy,
sensitivity, specificity, as well as the
accordance, concordance and odds ratio clearly
show that the alternative method precision is
equivalent to the ISO method:
Performance Assessment of the Thermo ScientificTM SureTectTM
Listeriamonocytogenes Real-Time PCR Assay according to
the ISO 16140 Standard for Listeria monocytogenes Detection
in Food and Environmental SamplesJ. Baguet, M. Bernard, C. Bernez, C. Le Doeuff, S. Peron, M. Rannou, D. Sohier
ADRIA, expert laboratory at AFNOR certification, AOAC-RI and MicroVal
Purpose An independent study was conducted at ADRIA, to validate this new method in comparison to the ISO 11290-1 standard, as part of the NF
Validation approval process and according to the ISO 16140 standard.
Inter-laboratory study
Alternative
method
ISO 11290-1
Reference method TOTAL
+ -
+ PA = 152 PD = 2 154
- ND = 6 NA = 80 86
TOTAL 158 82 240
PA :Positive Agreement NA: Negative Agreement
PD: Positive Deviation ND: Negative Deviation
Alternative
method
ISO 11290-1
Reference method TOTAL
+ -
+ PA = 97 PD = 34 131
- ND = 26 NA = 182 208
TOTAL 123 216 339
PA :Positive Agreement NA: Negative Agreement
PD: Positive Deviation ND: Negative Deviation
Relative detection levels
Various (matrix/strain) pairs were tested, with
4 inoculation levels. The relative detection limits
of the alternative method vary from 0.2 to
1.2 CFU/25 g, those of the ISO standard vary
from 0.2 to 1.1 CFU/25 g:
Matrices
Relative detection levels
Reference
method
Alternative
method
Rillettes 0.3 [0.2 ; 0.5] 0.5 [0.3 ; 0.9]
Smoked salmon 0.4 [0.2 ; 0.7] 0.3 [0.2 ; 0.5]
Raw milk 0.4 [0.2 ; 0.8] 0.6 [0.3 ; 1.2]
Produces 0.6 [0.3 ; 1.1] 0.6 [0.4 ; 1.0]
Process water 0.5 [0.4; 0.8] 0.6 [0.5 ; 0.8]
Inclusivity / exclusivity
30 non target strains and 50 target strains were tested. No cross reaction was observed with the non
target strains, all the Listeria monocytogenes strains were detected.
N° attestation UNI 03/08-11/13
ALTERNATIVE ANALYTICAL METHODS
FOR THE FOOD INDUSTRY
www.afnor-validation.com