HybriScan® Rapid Test System

HybriScan® Rapid Test System
Deteccion rapida, cualitativa y cuantitativa de microorganismos
por metodo basado en la medida de acido nucleico (rRNA) en
alimentos, bebidas y agua.
•
•
•
•
•
Ahorro de 5 a 7 dias
Alta flexibilidad
Facil manipulacion
Analisis en 2,5 horas
Solo detecta celulas vivas
(rRNA se descompone rapidamente en celulas muertas)
Sensible (hasta 1 CFU/ml con etapa de enriquecimiento)
Alta especificidad de las sondas (minimas reacciones cruzadas)
Sistema robusto, no sensible a la matriz de la muestra
Eficiente coste por analisis (formato de placa de 96-pocillos)
Principios del metodo HybriScan
El metodo HybriScan esta basado en la deteccion de rRNA
via hibridacion, captura especifica y deteccion de sondas.
Hibridacion Sandwich es muy sensible, detectando
atomoles de la molecula de rRNA target. El target ideal
de hibridacion para bacterias y levaduras es rRNA. Estas celulas
contienen una gran cantidad de ribosomas conteniendo rRNA;
una unica celula, por lo tanto, contiene miles de copias de
rRNA, pero solo una de DNA. Hibridacion Sandwich tambien
provee sensibilidad en muestras biologicas crudas porque este no
es susceptible de interferencias matriz.
La especificidad es conseguida por unica diana de
secuencias de rRNA. Una sonda de captura marcada con biotina
es usada para inmobilizar la secuencia diana en un soporte solido
(placa microtiter recubierta con streptavidina ). Una sonda
marcada con digoxigenina provee una senal optica medible.
Los resultados detectados desde la aplicacion son de fragmentos
anti-DIG-horseradish peroxidasa Fab. El complejunido es
visualizado por el sustrato de la peroxidasa
TMB (3,3’,5,5’-tetramethylbenzidine). Los datos fotometricos
son medidos a 450 nm y comparados con un estandar .
El software HybriScan permite facil medida y analisis de datos.
2.
1.
capture step
hybridisation
target rRNA
microtiter plate
3.
- labeling with enzyme
- washing
- detection
binding molecule
CCCCCC
CCCCCC labeled cature probe
DDDDDD
DDDDDD labeled detection probe
enzym with binding molecule
substrate
product
Como trabaja
analysis time: approx. 2 – 2.5 hours
Kits de deteccion para:
Kits de Identificacion para:
Beer spoiling organisms
Campylobacter
Cronobacter spp.
Beverage spoiling organisms
E. coli
Lactobacilli
Legionella
Legionella pneumophila
Listeria
Listeria monocytogenes
Salmonella
Total Bacterial Count
Waste Water Microthrix parvicella
Waste Water Total Bacteria Count
Yeasts
Brettanomyces
Candida albicans
E. coli
Lactobacillus brevis
Lactobacillus buchneri
Lactobacillus lindneri
Legionella pneumophila
Leuconostoc
Listeria monocytogenes
Megasphaera
Pectinatus cerevisiiphilus
Pectinatus frisingensis
Pediococcus damnosus
More Information about HybriScan®
E-mail: [email protected]
Internet: www.sigma-aldrich.com/hybriscan
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Date: 07/2010
HybriScan® –
Rapid Test Systems
for the Detection and Identification of Microorganisms
Rapid, sensitive
and reliable,
without PCR
The technology
The HybriScan® technology is based on the detection of target molecules,
specific for the microorganism of interest, with capture and detection probes by
means of a so-called sandwich hybridisation. The signal read-out is triggered
optically by an enzymatically generated colour change.
Save up to 10 days
in comparison to
cultivation-based
assays
Easy sample preparation:
Live/deaddiscrimination
Easy handling
with standard lab
equipment, like
96-wellplate reader
Quantifiable by
standards
Our Innovation, Your Research – Shaping the Future of Life Science
17
HybriScan Rapid Test Systems
®
Rapid detection, identification and quantification of microorganisms
The new HybriScan® Test System, which uses sandwich hybridisation, provides
fast, sensitive and reliable detection, identification and quantification of spoilage and pathogenic microorganisms. It is ideal for the comprehensive and reliable analysis of microorganism and pathogens. HybriScan® is a simple, timesaving assay that can be performed with standard laboratory equipment.
Benefits over conventional detection methods and PCR
HybriScan® has significant time- and labour-saving benefits over traditional
methods. It also has benefits over PCR and real-time PCR, which, although
highly sensitive, are susceptible to experimental interferences, like template
inhibition from insufficient purification [1], and lack quantification accuracy due
to biases associated with PCR and reverse transcription reactions (a general
accepted error connected to these methods). In contrast, the HybriScan®
method is nearly independent of the influences of sample matrix and is able to
distinguish between live and dead cells. It also permits the detection of nonculturable microbes. Table 1 compares the benefits and disadvantages of the
various methods.
Principles of the HybriScan® method
The HybriScan® method is based on the detection of rRNA via hybridisation
events and specific capture and detection probes (Figure 1). Sandwich hybridisation assays from crude cell samples or in connection to PCR have been extensively used in clinical diagnostics for detection of nucleic acids from bacteria [3,
9, 10] and viruses [11]. Specificity is achieved by targeting conserved or unique
rRNA sequences. A labelled capture probe is used to immobilise the target
sequence on a solid support plate (coated microtiter plate). A labelled detection
probe provides an enzyme-linked optical signal read-out. Detection results
from application of antibody labelled enzyme. The bound complex is visualised
by chromogenic substrate. Photometric data are measured at 450 nm and
compared with standard solutions. The HybriScan® software enables easy measurement and data analysis.
By using specific probes, HybriScan® allows flexible
group- and species-specific detection. It is applicable to
many analytical fields, including monitoring the microbial content of beer, wine, non-alcoholic beverages,
drinking water, a wide variety of foods and wastewater.
HybriScan® rapidly and accurately identifies, detects
and quantifies many important pathogenic species,
including Salmonella, Campylobacter, Listeria and
Legionella including the most relevant species L. pneumophila. [3, 4, 5]
HybriScan® Listeria monocytogenes: Rapid
and innovative test system
One of the most important foodborne pathogens is
Listeria monocytogenes (Figure 2), which poses a
health threat in foods that have long, refrigerated shelf
lives. [6] Listeriosis, caused by ingestion of foods contaminated with Listeria monocytogenes, has increased
dramatically in recent years, causing a great deal of distress and even death. Milk, cheese, ice cream and meat
contaminated with this pathogen have led to recent
outbreaks of listeriosis. [7] L. monocytogenes proliferates at refrigeration temperatures and is able to grow
over a wide pH range from 4.39 to 9.40. These are
important characteristics particularly with regard to
food safety.
Figure 2: Listeria mono Confirmatory Agar (Fluka 92302; in
front Listeria moncytogenes)
2
1
References
Figure 1: Principle of the HybriScan sandwich hybridisation assay.
®
Sensitivity, specificity, flexibility and applicability of
HybriScan® technology
Sandwich hybridisation is very sensitive, detecting attomoles of the respective
target rRNA molecules. [2] The ideal hybridisation target for bacteria and yeast
is rRNA. These cells contain a large number of rRNA-containing ribosomes; a
single cell therefore contains several thousand copies of rRNA but only one
DNA. Sandwich hybridisation also provides sensitivity in crude biological samples because it is not susceptible to matrix interference.
1]
Bustin, S.A. Absolute quantification of mRNA using realtime reverse transcription polymerase chain reaction
assays. J. Mol. Endocrinol. (2000), 25, 169–93.
2]
Tenhunen, J., Eloranta, J., Kallio, A., Soderlund, H. A
solution hybridisation method for quantification of
mRNAs: determining the amount and stability of oncogene
mRNA. Genet. Anal. Tech. Appl. (1990), 7, 228–233.
3]
Huhtamella, S., Leinonen, M., Nieminen, T., Fahnert, B.,
Myllykoski, L., Breitenstein, A., Neubauer, P. RNA-based
sandwich hybridisation method for detection of lactic acid
bacteria in brewery samples. J. Microbiol. Methods (2007),
68(3), 543–53.
4]
Leskela, T., Tilsala-Timisjarvi, A., Kusnetsov, J., Neubauer,
P., Breitenstein, A. Sensitive genus-specific detection of
Legionella by a 16S rRNA based sandwich hybridisation
assay. J. Microbiol. Methods (2005), 62(2), 167–79.
Our Innovation, Your Research – Shaping the Future of Life Science
18
Detection
technology
Advantage
Disadvantage
HybriScan®
• differentiates live/dead cells,
minimal interference by
sample matrix, high specificity
• low cross-reactivity
• easy handling
• cost-efficient read-out devices
• quantitative and qualitative
• high sample throughput
(96-microwell plates)
• detects of non-culturable
microbes
• no differentiation of serotypes or
subspecies
• limited probe design (rRNA target)
• high sample throughput
• sensitive
• quantitative
• no live/dead cell differentiation
• sensitive to matrix interference
(high extraction effort)
• susceptible to polymerase
inhibition
Table 1: Advantages of HybriScan® over other detection techniques.
Conventional culture-based methods to detect L. monocytogenes generally
involve selective enrichment followed by culturing on selective medium, isolation and biochemical identification. [8] This laborious and time-consuming
approach often takes several days to show results. Also, compared to molecular
biological and immunological methods, culture-based methods often give false
negatives.
100
80
50 49
60
38 38
40
27 27
15 15
20
1 1
l
ta
c.
to
is
m
f
g e r ui
ta t /
bl
e
category food
ve
da
ir
y
0
sh
• time consuming (up to 10 days)
• no detection of non-culturable
microbes
• low sample throughput
• laborious
HybriScan
fi
relatively inexpensive
simple
specific
widely accepted method
131130
culture based method
120
t
•
•
•
•
• low sensitivity
• low specificity, higher crossreactivity
• slow and expensive assay
development
140
ea
Conventional
cultivationbased
methods
• differentiation of serotypes or
subspecies
• high sample throughput
(96-microwell plates)
• quantitative and qualitative
3
m
ELISA
HybriScan® kits are the result of a joint project between
Sigma-Aldrich and Scanbec GmbH. For more details
please visit us at www.sigma-aldrich.com/hybriscan
No. L.monocytogenes positive samples
PCR
Two versions are available. HybriScan® I Listeria monoytogenes is used for the extremely rapid, sensitive and
economical identification of suspect colonies of L.
monocytogenes. HybriScan® D Listeria monocytogenes
is used for the detection, identification and quantification of L. monocytogenes in different food matrixes.
Figure 3: Validation of HybriScan® Listeria monocytogenes.
355 food samples were analysed and compared to culturebased method according to § 64 LFGB. The blue values are
the number of L. monocytogenes positive-tested analysed
food samples in each category. Validation was according to
ISO 16140:2003 (ASU L00.00-22).
Prod. no.
Description
Reactions
96343
HybriScan® D E. coli
96
59744
HybriScan® D Lactobac
96
16593
HybriScan® D Legionella
96
07190
HybriScan® D Legionella
pneumophila
96
55661
HybriScan® D Listeria
96
49699
HybriScan® D Listeria
monocytogenes
96
55662
HybriScan® D Salmonella
96
02349
HybriScan® D Total Bacterial
Count
96
61397
HybriScan® D Yeast
96
19503
HybriScan® I Candida albicans
48
76545
HybriScan® I E. coli
48
49417
HybriScan® I Legionella
pneumophila
48
49712
48
Rautio, J., Barken, KB., Lahdenpera, J., Breitenstein, A.; Molin, S., Neubauer, P. Sandwich
hybridisation assay for quantitative detection of yeast RNAs in crude cell lysates. Microb.
Cell Fact. (2003), 2(1), 4–12.
HybriScan® I Listeria
monocytogenes
89384
48
6]
Mellefont, L.A., McMeekin, T.A., Ross, T. Effect of relative inoculum concentration
on Listeria monocytogenes growth in co-culture. Int. J. Food Microbiol. (2008), 121,
157–68.
HybriScan® I Pectinatus
cerevisiiphilus
73582
HybriScan® I Pectinatus
frisingensis
48
7]
McLauchlin, J. The relationship between Listeria and listeriosis. Food Control (1996),
7, 187–93.
67289
HybriScan® I Pediococcus
damnosus
48
8]
Tasara, T. et al. Incorporation of Reporter Molecule-labeled Nucleotides by
DNA Polymerases. I. Chemical Synthesis of Various Reporter Group-labeled
2’-deoxyribonucleoside-5’-triphosphates. Nucleic Acid Research, Vol. 31, No. 10
(2003), 2630–5.
44492
HybriScan® Software
HybriScan® Listeria monocytogenes is an excellent alternative to lengthy culture-based methods. It is as reliable and comprehensive as classical methods,
but permits rapid detection and quantification with results available within 48
hours. The species-specific probe permits direct detection of L. monocytogenes,
thereby eliminating false positives caused by other Listeria species. Even more
compelling, suspected single colonies can be identified within one hour, using
the HybriScan® I identification kit without need for further cultivation.
Figure 3 shows the validation results of HybriScan Listeria monocytogenes.
Food samples were analysed with the HybriScan® method and compared to the
culture-based method according to § 64 LFGB. Five different food categories
were tested. Results of validation showed a relative accuracy of 99.2%, relative
specificity of 98.5% and relative sensitivity of 99.6%.
®
5]
9]
Giller, G. et al. Incorporation of Reporter Molecule-labeled Nucleotides by DNA
polymerases. II. High-density labelling of natural DNA. Nucleic Acids Research, Vol.
31, No. 31 (2003), 2636–46.
Table 2: HybriScan® products (HybriScan® D = detection kit;
HybriScan® I = identification kit)
10] Buschmann, V. et al. Spectroscopic Study and Evaluation of Red-Absorbing
Fluorescent Dyes. Bioconjugate Chem. 14 (2003), 195–204.
11] Hinze, K., et al. Triple-Colour Coincidence Analysis: One Step Further in Following Higher
Order Molecular Complex Formation. Biophysical Journal, Vol. 86 (2004), 506–16.
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