respiFISH Masterpanel - miacom diagnostics GmbH

Transcription

respiFISH
Masterpanel
Kit for the differentiation of bacterial strains directly from respiratory
samples
M-60-007
72 tests
miacom diagnostics GmbH, Düsseldorf, Germany
respiFISH Masterpanel - Package Insert
I - Intended Use
Miacom´s respiFISH Masterpanel is a qualitative nucleic acid hybridization assay indicated
for use as an aid in the diagnosis of pneumonia. It is intended for the identification of the
following species / genera / families from respiratory secretions:
Gram positive
!
!
!
!
Gram negative
!
!
!
!
!
!
!
!
!
!
Staphylococcus spp.
Staphylococcus aureus
Streptococcus spp.
Streptococcus pneumoniae
Enterobacteriaceae
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilis
Serratia marcescens
Pseudomonas aeruginosa
Acinetobacter spp.
Stenotrophomonas maltophilia
Haemophilus influenzae
Moraxella catarrhalis
For susceptibility testing or further identification, subculturing may be necessary.
In addition, the kit may be used for the identification of bacteria from cultures
II - Summary and Explanation
The respiFISH Masterpanel identifies particular bacteria within approximately 30 minutes
directly from respiratory secretions. To prepare the respiratory specimen a special buffer is
applied that liquefies the samples and reduces background fluorescence.
The methodology of this test is based on fluorescence in-situ hybridization (FISH) (Amman et
al. 1990). Miacom diagnostics GmbH has combined the classical FISH technology with the
usage of fluorescence labeled molecular beacons (Tyagi and Kramer 1996). Miacom´s
molecular beacons consist of a DNA sequence folded into a hairpin-like structure that is
linked to a fluorophore on the 5´end and to a quencher on the 3´end. A part of the DNA
sequence on both ends forms a stem region through complementary base-pairing. This
structure keeps the fluorophore and quencher in close proximity. In this state, the generation
of a fluorescent signal is inhibited. The DNA sequence between the stem regions is
complementary to a rRNA counterpart that is unique at the family, genus or species level.
Because each bacterial cell includes more than 10.000 copies of rRNA, no amplification step
is necessary. The DNA sequences of the beacons that are specific to the target region form a
loop-like structure and are able to bind to their rRNA targets after passing through the
bacterial cell wall and membrane. In a bound state, the fluorophore of the beacon is spatially
separated from its quencher and is able to emit light if it is excited with an adequate light
source.
Every rRNA copy with a bound beacon contributes to a fluorescent signal and the cell can be
detected as a shining object under a fluorescence microscope. In addition, to the fluorescent
signal the cells intact morphology can be examined to confirm the result.
Unbound beacons do not fluoresce and do not generate a signal so that a washing step
commonly used in FISH technologies can be omitted.
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III - Procedure Principle
A solution of fluorophore labeled molecular DNA beacons is dispensed on fixed and partially
perforated cells prepared from respiratory secretions. The hybridization is carried out at 52+/1°C for 10 minutes. Submerging the slide into a bath containing a stop solution ceases the
reaction. Adding a drop of Mounting Medium to each field prevents fading of fluorescence.
After applying a coverslip, the slide is ready for examination using fluorescence microscopy.
IV - General Kit Precautions
!
For professional use, only by personnel trained in laboratory techniques and
experienced in performing fluorescence microscopy.
!
All patient samples, biological reagents and other materials used for conducting the
test have to be regarded as potentially infectious and should be treated and disposed
of accordingly.
!
Establish precautions against microbiological hazards.
!
Comply with federal, state and local regulations concerning safety.
!
Dispose of reagents in accordance with federal, state and local regulations.
!
The kit and all instruments / devices used in combination have to be validated by the
laboratory complying to federal, state and local regulations.
!
Important: A diagnosis must not be based solely on this test. In addition to the result
obtained by this test the clinical picture and possibly culture or other diagnostic results
have to be taken into consideration.
V - Reagents
Reagents are provided at fixed concentrations. Assay performance may be affected if the
reagents are modified in any way or are not stored under the recommended conditions as
detailed in „Storage of kit components“.
Before reconstitution, store all kit components at 15-25°C. They must be kept in a dark and
dry place.
Reconstituted and prepared solutions stored refrigerated have to be brought to room
temperature (15-25°C) prior to use.
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Table 1: Storage conditions of components
Ingredients /
Name
Explanation
Stop Solution
50ml
(10x conc.)
Tris buffered saline
Stop Solution
Tris buffered saline
(1x prepared)
Ethanol
8ml photo bleaching
Mounting
inhibitor in glycerol
Medium
solution
Clearance Buffer
50ml
(10x conc.)
Trade secret
Clearance Buffer
500ml
(1x prepared)
Lysis
Reconstitution
8ml
Buffer
8ml immersion oil suited
Immersion oil
for fluorescence
microscopy
fluorescently labeled
DNA beacons in Tris
Beacon Cartridge
bufferd saline +
formamide
Lysis Cartridge
Storage
Conditions
Safety information
15-25°C
n.a.
tightly sealed
15-25°C
highly flammable
15-25°C
contains DABCO –
see safety sheet and
MSDS
15-25°C
n.a.
15-25°C
n.a.
15-25°C
n.a.
15-25°C
15-25°C
after opening at
2-8°C
15-25°C
after reconstitution at
2-8°C
Lysis Buffer
contains benzyl
benzoate – see safety
sheet and MSDS
contains 20%
formamide – see
safety sheet and
MSDS
n.a.
Technical Precautions regarding the reagents:
!
Avoid microbial contamination of reagents.
!
Avoid any cross-contamination of samples and reagents, as this may give rise to
erroneous results.
!
Do not allow dropper bottle tip to touch the sample as this may cause cross
contamination of material between slides/fields, or cause contamination of the
reagent.
!
Do not use microscope slides other than the microscope slides provided by miacom
diagnostics GmbH.
!
Reagents and slides must not be used after the expiration date printed on each label.
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Directions for reconstitution and dilution:
!
Important: Use clean glassware and clean pipette tips.
!
Stop Solution: Dilute 50ml 10x concentrated Stop Solution with 200ml double
distilled water and 250ml ethanol (≥ 95 %, methylated or pure); mix thoroughly; store
in a tightly sealed container at room temperature 15-25°C. Shelf life after
reconstitution: 4 months.
!
Lysis Cartridge: Reconstitute lyophilized lysis buffer by adding 800µl Lysis
Reconstitution Buffer to each well; close cartridge tightly; leave at room temperature
(15-25°C) for 10 min; mix thoroughly by inverting the cartridge 10-15 times; store at 28°C in the dark. Shelf life after reconstitution: 4 months.
!
Beacon Cartridge: Beacons are ready to use. Store at 2-8ºC in the dark. Shelf life
after opening: 4 months.
!
Clearance Buffer: Dilute 50ml 10x concentrated Clearance Buffer with 450ml double
distilled water; mix thoroughly; store in a tightly sealed container at room temperature
(15-25°C). Shelf life after reconstitution: 4 months.
VI - Required Instruments
Microscope:
! A fluorescence microscope equipped with a 100x oil immersion objective and a 10x
eyepiece lense (1000x magnification is needed).
! Filterset A for ATTO550-label: absorption max 554nm / emission max 576nm.
! Filterset B for FAM-label: absorption max 494nm / emission max 520nm.
! Adequate illumination device comparable to 100W HBO lamp, correctly adjusted.
Bulb not aged beyond its specified lifetime.
Hotplate:
! The hotplate must be maintained at an operating temperature of 52+/-1°C.
! A hotplate combined with a hybridization chamber is available from miacom
diagnostics and its use is strongly recommended.
! Temperature consistency has to be validated.
! Maintain the hotplates at regular intervals according to manufacturer’s specifications.
VII - Standard Specimen Collection and Preparation
!
Important: Test should be run as shortly as possible after specimen is taken. Not
later than 24 hours. Specimen should be stored refrigerated (2-8°C).
! This kit is to be used after the Gram stain shows Gram-negative and / or Grampositive bacteria.
! Do not use the test if there is not sufficient sample material available.
! Take a small aliquot (approx. 200µl) of the clinical sample into an empty tube and mix
sample with one volume of Clearance Buffer. Vortex and/or shake vigorously for
approx. 30 sec or until sample is sufficiently liquefied. Transfer 100µl of the liquefied
sample into an empty tube and add 400µl of double distilled water and shake again.
Start testing within one hour
Pipette 10µl of the dilution on each well of the microscope slide.
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VIII - Assay Procedure
Provided Material:
Each kit contains sufficient material for 72 tests
!
Microscope Slides (72 pieces)
!
Lysis Reconstitution Buffer (8ml)
!
Coverslips (~100 pieces)
!
Cartridge with beacons, ready to use
!
Hybridization Covers (72 pieces)
!
Cartridge with lyophilized lysis buffer
!
Immersion Oil suited for fluorescence
microscopy (8ml)
!
Clearance Buffer (10x concentrated
50ml)
!
Mounting Medium (8ml)
!
!
Stop Solution (10x concentrated; 50ml)
Package insert, including
sentences (see section XVIII)
H&P
Important: Reagents must not be interchanged with components of any other type of
miacom kit or self-manufactured solutions.
Important: Microscope Slides, Hybridization Covers and Coverslips are disposable and for
single use only.
Required material not included in the kit:
!
Precision micropipettes suited for pipetting the following volumes / volume ranges:
10µl, 20-100µl, 200-1000µl.
!
Pipette tips.
!
2ml screw cap reaction tubes.
!
3 clean horizontal staining dishes (suited for a volume of 100ml).
!
Double distilled or deionized water (750ml per Kit).
!
Ethanol (≥95% methylated or pure, ~1-2 L per Kit).
!
2 clean 500ml glass bottles.
!
Use of a multichannel pipette is recommended, also available from miacom
diagnostics.
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Assay procedure step by step:
Total turn-around time is approximately 30 minutes.
Prior to first use:
!
Important: Prepare 1x Stop Solution by adding 200ml ddH2O and 250ml Ethanol
(see section ”Directions for reconstitution and dilution”).
!
Important: Prepare 1x Clearance Buffer by adding 450ml ddH2O (see section
”Directions for reconstitution and dilution”).
!
Important: Reconstitute lyophilized lysis buffer by adding 800µl of the Lysis
Reconstitution Buffer to each well of the respective cartridge (see section ”Directions
for reconstitution and dilution”).
!
Prepare two ethanol baths (> 95% methylated or pure) by adding ~100ml Ethanol into
a clean horizontal staining dish for each bath and label them properly.
!
Prepare one stop bath by adding ~100ml 1x Stop Solution into a clean horizontal
staining dish and label it properly.
Prior to use (daily):
!
Allow kit components stored in the refrigerator to adapt to room temperature (1525°C).
!
Replace ethanol in ethanol bath 1 and 2 by fresh ethanol; keep horizontal staining
dishes closed when they are not in use.
!
Replace stop solution in stop bath if it is older than 5 days or a total of more than 15
slides have been immerged into the solution; keep horizontal staining dishes closed
when they are not in use.
1. Fixation:
!
Note ID of specimen.
!
Important: Label slide only with pencil.
!
Take a small aliquot (approx. 200µl) of the clinical sample into an empty tube and mix
sample with one volume of Clearance Buffer. Vortex and/or shake vigorously for
approx. 30 sec or until sample is sufficiently liquefied.
!
Transfer 100µl of the liquefied sample into an empty tube and add 400µl of double
distilled water and shake again. Start testing within one hour.
!
Transfer 10µl of the pretreated specimen to each well of an adequately labeled
microscope slide.
!
Add 10µl of reconstituted Lysis Solution to each field of the microscope slide
(Important: Solution contained in well 1 of the cartridge has to be added to well 1 of
the microscope slide and so on). Keep in mind to change the pipette tips after each
slide.
!
Place slide on a heating plate pre-warmed to 52+/-1°C.
!
Allow samples to dry completely.
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2. Dehydration of cells:
!
Transfer slide to ethanol bath 1.
!
Incubate slide for 5 minutes in ethanol bath 1.
!
Take slide out of ethanol bath 1 and thoroughly dry bottom side with a piece of clean
absorbent paper.
!
Place slide on a second piece of paper and allow ethanol to evaporate. Take care
that you do not place slide on heating plate for drying.
3. Hybridization:
!
Careful: Beacon Solution contains formamide.
!
Add 10µl of Beacon Solution to each field of the microscope slide (Important:
solution contained in well 1 of the cartridge has to be added to well 1 of the
microscope slide and so on).
!
Place Hybridization Cover on the microscope slide.
!
Transfer covered microscope slide to the hotplate hybridization chamber (52+/-1°C).
!
Incubate for 10 minutes.
4. Stop:
!
Take slide from hotplate hybridization chamber, remove Hybridization Cover and
transfer slide into the stop bath.
!
Incubate for 1 minute +/- 10 seconds (Important: This step is time-critical! Do not
exceed specified incubation time!).
!
Take slide out of the stop bath and dip in ethanol bath 2 to rinse off Stop Solution.
!
Take slide out of ethanol bath 2 and thoroughly dry bottom side with a piece of clean
absorbent paper.
!
Place slide on a second piece of paper and allow ethanol to evaporate. Take care
that you do not place slide on heating plate for drying.
5. Mounting:
!
Add one small drop of Mounting Medium (approximately 5µl) to the center of each
field of the microscope slide - see Figure 1.
!
Add coverslip with caution; avoid air bubbles.
Protect slide from direct sunlight or other strong light sources to prevent fluorescence
bleaching. Examine slide as described under readout section within 2 hours.
Figure 1: Example for Mounting Medium application
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6. Readout:
!
Place a small drop of immersion oil onto the coverslip and examine slide under a
fluorescence microscope fulfilling the specifications described in ” Section VI,
Required Instruments”
!
Use the Teflon rim on each field as an aid to find the focus level in the bright field and
check for fixed bacterial cells.
!
Switch off bright field illumination and scan the complete field for fluorescing bacterial
cells using both filters.
!
A true positive result is defined as a positive fluorescent signal in only one of the
channels. If one individual particle/cell fluoresces in both channels it is not counted as
a true positive signal.
!
Keep the expected cell morphology in mind when interpreting fluorescent signals.
7. Interpretation of Results:
!
Field 1 serves as a positive control. If positive, it shows fluorescent cells in red and
no fluorescent cells in green.
!
Results should be interpreted in conjunction with Gram-stain results.
!
Results are read as cells of the expected morphology fluorescing in either the red or
the green channel and the bacterial load is semi-quantified as high, medium or low.
Cells/particles fluorescing in both channels are not counted.
o
Enterobacteriaceae other than E. coli, K. pneumoniae, P. mirabilis and S.
marcescens, are identified by red fluorescence only in Field 2.
o
E. coli is identified by red fluorescent cells in Field 2 and 3.
o
K. pneumoniae is identified by red fluorescent cells in Field 2 and 4.
o
P. mirabilis is identified by red fluorescent cells in Field 2 and 5.
o
S. marcescens is identified by red fluorescent cells in Field 2 and 6.
o
P. aeruginosa is identified by green fluorescent cells in Field 2.
o
Acinetobacter spp. is identified by green fluorescent cells in Field 3.
o
S. maltophilia is identified by green fluorescent cells in Field 4.
o
H. influenzae is identified by green fluorescent cells in Field 5.
o M. catarrhalis is identified by green fluorescent cells in Field 6.
o
Staphylococci other than S. aureus are identified by red fluorescent cells only
in Field 7.
o
S. aureus is identified by red fluorescent cells in Field 7 and 8.
o
Streptococci other than S. pneumoniae are identified by green fluorescent
cells only in Field 7.
o
S. pneumoniae is identified by green fluorescent cells in Field 7 and 8.
Mixed cultures are reported when two or more types of cells fluorescing in different
channels and/or fields are observed. Mixed specimen containing ‘other’ organisms (not
further identified) are reported when significantly more and/or morphological different
cells are observed with the Positive Control (Field 1).
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Field
Red Channel
Green Channel
1
Positive control
Negative control
2
Enterobacteriaceae
3
Escherichia coli
Acinetobacter spp.
4
Klebsiella pneumoniae
5
Proteus mirabilis
6
Serratia marcescens
Moraxella catarrhalis
7
Staphyolococcus spp.
Streptococcus spp.
8
Table 2: Panel composition respiFISH Masterpanel
Figure 2: Example for red and green fluorescent bacteria.
IX - Quality Control
!
Control material should be tested in accordance with guidelines or requirements of
local, state and /or federal regulations or accrediting organizations.
!
Quality control with one of the three QC strains has to be tested on a daily basis and
the strain should be rotated:
o
Escherichia coli
o
o
The QC strains may be cultured on solid medium.
X - Troubleshooting
!
!
!
If no fluorescent signal appears in the positive control field, check if you see cells in
the bright field. If no cells are visible in the bright field, either there are no bacteria in
the sample, or the sample is below the detection limit.
If there is a fluorescent signal in the negative control field the test is called invalid.
In rare cases cells exhibit an autofluorescence, which is visible in all fields. In this
case the test is called invalid.
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XI - Limitations
!
!
!
!
!
!
!
!
The probe against Escherichia coli does not distinguish between Escherichia coli and
Shigella spp. and will also detect E. albertii and E. fergusonii due to sequence
identity. Enterobacteriaceae beacon detects some Aeromonas species. Klebsiella
pneumoniae beacon detects also K. variicola. Serratia marcescens beacon detects
Pseudomonas fluorescence. Pseudomonas aeruginosa beacon detects P. fulva and
P. mendocina. Haemophilus influenzae beacon detects H. haemolyticus.
The Enterobacteriaceae beacon detects among others: Citrobacter spp.,
Enterobacter spp., Hafnia spp., Morganella spp., Pantoea spp., Escherichia spp.,
Klebsiella spp., Proteus spp., Salmonella spp., Serratia spp., Shigella spp. and
Yersinia spp.
Streptococcus spp. beacon also detects Lactococci.
According to databases, the Streptococcus and Staphylococcus spp. beacons detect
all species for which sequences are available.
Fluorescence may vary due to microscope equipment and rRNA content of the
examined cells (fitness of the cells).
The detection limit was found to be 105 CFU/mL (determined by serial dilution
experiments).
False positive autofluorescence may occur if other than the specified filtersets are
used or due to autofluorescent particles being present in the sample such as dust
particles, hair or fungi.
Important: The product has not been validated with specimens other than respiratory
secretions.
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XII - Expected Values
The SENTRY antimicrobial surveillance program listed the frequencies of occurrence in
North America, Europe and Latin America of bacterial pathogens associated with HAP
and VAP infections in participating medical centers. Over 30,000 isolates were evaluated,
giving the following results:
Table 4: Data* extracted from: Jones R.N.
Organism
Klebsiella spp.
Escherichia coli
Acinetobacter spp
Enterobacter spp.
Percent
Organism
Serratia spp.
Other
Total
28.0
21.8
9.8
6.9
6.8
6.3
Percent
3.5
3.1
2.9
2.7
8.2
100
*The organisms and percentages will vary depending on institution and patient population.
XIII - Specific Performance Characteristics
Results of performance studies:
Performance data has been collected in one independent study. The results of this study
yielded a sensitivity of 96% and a specificity of 98%.
A total quantity of 125 specimen were screened. Results have been compared to those of
conventional routine methods.
Analytical sensitivity:
For the tested reference strains a detection limit of approximately 105 colony-forming units
per ml was determined by serial dilution experiments.
Analytical specificity:
respiFISH Masterpanel has been tested on at least 2 ATCC strains of the respective
beacons. Studies for cross-reactions have been conducted that included closely related
species. See section XI – Limitations regarding known cross reactions.
XIV - Bibliography
Amann, R. I., Krumholz, L. and Stahl, D. A. (1990) Fluorescent oligonucleotide probing of
whole cells for determinative, phylogenetic, and environmental studies in microbiology. J.
Bacteriol.172: 762-770.
Jones, R.N. (2010) Microbial etiologies of hospital-acquired bacterial pneumonia and
ventilator-associated bacterial pneumonia. Clin Infect Dis. 51: Suppl 1:S81-7.
Tyagi, S. and Kramer, F. R. (1996) Molecular Beacons: Probes hat fluoresce upon
hybridization. Nature Biotechnology 14: 303-308.
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XV - Definitions
Explanation of symbols and abbreviations:
Product code / Catalog number
Batch code
Use by date
Temperature limitation for storage
Manufactured by / Manufactured for
Consult instructions for use
Do not reuse
Contains sufficient for n tests
Protect from direct sun light
In vitro diagnostic medical device
Breakable
Hazard symbols
See material safety data sheet (MSDS).
Also available on www.miacom-diagnostics.com.
XVI - Technical Advice and Customer Service
miacom diagnostics GmbH
Merowingerplatz 1 a
40225 Düsseldorf
Germany
phone: +49 (0)211 3015 5795
email: [email protected]
web:
www.miacom-diagnostics.com
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XVII - Supplements
Supplement: Hazardous Substances / Mixtures
Beacon Reconstitution Buffer / Probes (dissolved in Beacon
Reconstitution Buffer)
Classification
Symbol
Signal Word
H-Phrase(s)
H360D: May damage the unborn child.
„Danger“
Reproductive toxicity, Cat. 1B
P-Phrase(s)
P201 Obtain special instructions before use.
P308 + P313 IF exposed or concerned: Get medical advice/attention.
Hazardous substance(s)
Name
Formamide
CAS-Number
75-12-7
Concentration
≥ 15 < 25%
Mounting Medium:
Classification
Symbol
Signal Word
„Warning“
H-Phrase(s)
H302 Harmful if swallowed
H315 Causes skin irritation.
H319 Causes serious eye irritation.
H335 May cause respiratory irritation.
H412: Harmful to aquatic life with long lasting effects
P-Phrase(s)
P280 Wear protective gloves/protective clothing/eye protection/face protection
P273 Avoid release to the environment
Name
CAS-Number
Concentration
DABCO (Triethylenediamine, 1,4280-57-9
2.5%
Diazabicyclo [2.2.2]octane)
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Immersion oil:
Classification
Symbol
Signal Word
H-Phrase(s)
H315 Causes skin irritation.
H317 May cause an allergic skin reaction.
H302 Harmful if swallowed. (Acute toxicity,
Category 4, oral)
H411 Toxic to aquatic life with long lasting
effects. (Hazardous to the aquatic
environment, Chronic Category 2)
„Warning“
P-Phrase(s)
P273 Avoid release to the environment.
P280 Wear protective gloves/protective clothing/eye protection/face protection
P302 + P352 IF ON SKIN: Wash with soap and water
P301+312: IF SWALLOWED: Call a POISON CENTER or doctor/physician if you feel unwell
P305+351+338: IF IN EYES: Rinse continuously with water for several minutes. Remove contact
lenses if present and easy to do – continue rinsing
Name
Benzyl benzoate
CAS-Number
120-51-4
Concentration
≥ 25 < 50%
General guidelines:
All chemicals are potentially dangerous. They should only be handled by specially trained personnel.
Do not eat, drink, smoke, apply cosmetics, store or prepare foods within the designated work area.
Dispose of reagents in accordance with federal, state and local regulations.
Reagents must not be used after the expiration dates printed on the labels.
Avoid microbial contamination of reagents.
Version 150409-1
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respiFISH Masterpanel - miacom diagnostics GmbH

Transcription

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