USER MANUAL

USER MANUAL
USER MANUAL
PCP
ref. 03-20A
Kit for the detection of DNA of
Pneumocystis carinii
Including all the reagents for PCR amplification
and visualisation by agarose gel electrophoresis
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1.
KIT CONTENT
3
2.
STORAGE AND STABILITY OF REAGENTS
4
3.
PRECAUTIONS FOR USE
4
4.
SAFETY RULES
5
4.1.
General safety rules
5
4.2.
Safety rules about the kit
6
5.
MATERIALS REQUIRED, BUT NOT PROVIDED
7
5.1.
Reagents
7
5.2.
Instruments
7
5.3.
Materials
7
6.
PREPARATION OF REAGENTS
8
7.
INTRODUCTION
9
8.
TEST PRINCIPLE
10
9.
PRODUCT DESCRIPTION
10
10.
COLLECTION, MANIPULATION AND PRE-TREATMENT OF SAMPLE
12
10.1.
Respiratory specimens
12
10.2.
Histological samples
13
10.3.
Blood
13
11.
11.1.
AMPLIFICATION PROTOCOL
14
DNA EXTRACTION
14
11.2.
DNA AMPLIFICATION
11.2.1. TST DNA Amplification
11.2.2. Pneumocistis carinii DNA amplification
15
15
15
11.3.
VISUALIZATION OF AMPLIFICATION PRODUCTS
11.3.1. Agarose gel electrophoresis
11.3.2. Sample loading
11.3.3. Interpretation of the results: TST and Pneumocistis carinii DNA amplification
16
16
16
17
1
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12.
TROUBLESHOOTING
19
13.
DEVICE LIMITS
21
14.
DEVICE PERFORMANCES
21
14.1.
Specificity
21
14.2.
Diagnostic and analytic sensitivity
21
15.
BIBLIOGRAFIA
22
16.
INFORMATION FOR ORDERS
23
16.1.
Related products
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23
2
1.
KIT CONTENT
BOX P
DESCRICTION
STORE AT – 20°C
LABEL
COLOUR OF
TUBE (T)
OR LID
25 test
50 test
8 test
Green
(T)
25
50
8
Blue (T)
25
50
8
Red
1 x 40 μL
1 x 80 μL
1 x 15 μL
Monodose premix tube PCP
Monodose premix TST
(thiosulfate sulfurtransferase rhodanese) tubes
Taq DNA polymerase
thermostable
AB TAQ
5 U/μL
SMALL BAG
Plasmid DNA containing
a portion of P.carinii genome
STORE AT – 20°C
Positive control
PCP
Blue
BOX F
1 x 30 μL
1 x 50 μL
1 x 10 μL
STORE AT +2°-+8°C
Electrophoresis Buffer for
sample loading
Blue 6X
Blue
1 x 200 μL
1 x 350 μL
1 x 100 μL
Ethidium Bromide solution
(2,5 mg/mL)
Ethidium
Bromide
Red
1 x 150 μL
1 x 250 μL
1 x 80 μL
Yellow
1 x 150 μL
1 x 250 μL
1 x 50 μL
DNA molecular weight marker
TOXIC
R 23 68
S 36/37 45
MW Marker
BOX A
STORE AT +15°-+25°C
Agarose for molecular biology
AGAROSE
1 x 15 g
1 x 27 g
1x5g
Electrophoresis Buffer
TRIS-Acetate-EDTA pH: 8,00
TAE 50X
1 x 60 mL
1 x 100 mL
1 x 20 mL
3
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2.
STORAGE AND STABILITY OF REAGENTS
Each component of the kit should be stored according to the directions
indicated on the label of the single boxes.
In particular:
Box P
Small bag
Box F
Box A
Store at – 20°C
Store at – 20°C
Store at + 2°-+ 8°C
Store at + 15°-+ 25°C
When stored at the recommended temperature, all test reagents are stable
until their expiration date.
3.
PRECAUTIONS FOR USE
• The kit should be handled by investigator qualified through education and
training in molecular biology techniques applied to diagnostics;
• Before starting the kit procedure, read carefully and completely the
instruction manual;
• Keep the product out of heating sources;
• Do not use any part of the kit if over the expiration date;
• In case of any doubt about the storage conditions, box integrity or method
application,
contact
AB
ANALITICA
technical
support
at:
[email protected] .
In the amplification of nucleic acids, the investigator has to take the
following special precautions:
• Use filter-tips;
• Store the biological samples, the DNA extracted, positive control included
in the kit and all the amplification products in different places from where
amplification reagents are stored.
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4
• Organise the space in different pre- and post-PCR units; do not share
consumables (pipets, tips, tubes, etc) between them.
• Change the gloves frequently;
• Wash the bench surfaces with 5% sodium hypochloride;
• Thaw the PCR premixes at room temperature before use.
Add the Taq DNA polymerase and purified DNA very quickly at room
temperature or in an ice-bath.
•
4.
SAFETY RULES
4.1.
General safety rules
• Wear disposable gloves to handle the reagents and the clinical samples
and wash the hands at the end of work.
• Do not pipet with mouth.
• Since no known diagnostic method can assure the absence of infective
agents, it is a good rule to consider every clinical sample as potentially
infectious and handle it as such.
• All the devices that get directly in touch with clinical samples should be
considered as contaminated and disposed as such. In case of accidental
spilling of the samples, clean up with 10% Sodium Hypochloride. The
materials used to clean up should be disposed in special containers for
contaminated products.
• Clinical samples, materials and contaminated products should be disposed
after decontamination by:
immersion in a solution of 5% Sodium Hypochloride (1 volume of 5% Sodium
Hypochloride solution every 10 volumes of contaminated fluid) for 30 minutes
OR
autoclaving at 121°C at least for 2 hours (NOTE: do not autoclave solutions
containing Sodium Hypochloride!!)
5
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4.2.
Safety rules about the kit
The risks for the use of this kit are related to the single components:
Dangerous components:
ETHIDIUM BROMIDE
3,8-diamino-1-ethyl-6-phenylphenantridiumbromide (Ethidium Bromide) <2%
Description of risk:
T (Toxic)
RISK SENTENCES AND S SENTENCES
R 23 and R 68
S 36/37 45
Toxic for inhalation.
Risk of irreversible effects.
Wear laboratory coat and disposable gloves.
In case of accident or discomfort, seek for medical
assistance and show the container or label.
R and S sentences refer to the concentrated product, as provided in the kit.
In particular, for Ethidium Bromide, until the diluition in the agarose gel.
In manipulating concentrated Ethidium Bromide, use a chemical dispensing
fume cabinet. Always wear disposable gloves and laboratory coat in
manipulating the diluited Ethidium solution as well.
The product can not be disposed with the common waste. It can not reach the
drainer system. For the disposal, follow the local law.
In case of accidental spilling of Ethidium Bromide, clean with Sodium
hypochloride and water.
Safety data sheet (MSDS) of Ethidium Bromide is available upon request.
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5.
MATERIALS REQUIRED, BUT NOT PROVIDED
5.1.
Reagents
• Sterile DNase and RNase free water;
• Distilled water.
5.2.
•
•
•
•
•
•
•
•
•
•
•
Laminar flow cabinet (use is recommended while adding TAQ
polymerase to the amplification premix to avoid contamination; it would
be recommended to use another laminar flow cabinet to add the
extracted DNA);
Micropipettes (range: 0,2-2 µL; 0,5-10 µL; 2-20 µL);
Thermalcycler;
Balance;
Magnetic heating stirrer or microwaves;
Microcentrifuge (max 12-14.000 rpm);
Chemical cabinet (use is recommended in handling Ethidium Bromide);
Horizontal electrophoresis chamber for agarose gel;
Power supply (50-150 V);
UV Transilluminator;
Photo camera or image analyzer.
5.3.
•
•
•
•
•
Instruments
Materials
Disposable gloves;
Disposable sterile filter-tips (range: 0,2-2 µL; 0,5-10 µL; 2-20 µL);
Graduated cilinders (1L) for TAE diluition;
Pyrex bottle or Becker for agarose gel preparation;
Parafilm.
7
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6.
PREPARATION OF REAGENTS
To make 1 L of TAE Buffer (1X):
Mix 20 mL of 50X TAE and 980 mL of distilled water.
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7.
INTRODUCTION
Pneumocystis carinii is an ubiquitous pathogen that is the most frequent
etiologic agent of pneumonia in immunosuppressed patients (organ
engrafted, oncologic or AIDS patients). P.carinii is responsible also of a
particular and serious interstitial form of pneumonia that mainly affects kids in
the first years of age (never at birth) (H. M. Ginzburg, 1996).
P.carinii has a corpuscular form with spheroidal or small egg-shaped cells
surrounded by a capsule enriched in neutral mucopolisaccharides.
In the first stadium (endocellular phase) the pathogen is phagocytized by the
alveolar cells that are stimulated to proliferation and desquamated in the
pulmonary alveoli. In the second stadium, due to a disgregative phenomenon
of the alveolar cells, a sharp eosinophil reticulum with a spongy aspect is
found at the endoalveolar and endobronchiolar level.
The parasite cultural analysis is usually difficult and histological methods are
not very specific and sensitive (cito-histological specimen staining with
Grocott-Gomori, toluidine blueO, Giemsa or Wright staining). The use of
monoclonal antibodies (in immunofluorescence) increased test specificity
even though the sensitivity is still uncertain.
For many years P. carinii infection was clinically diagnosed by analysis of
samples obtained by invasive procedures; the development of PCR
techniques allowed the use of pathological samples easy to obtain and the
amplification of specific DNA sequences. This method is highly sensitive and
specific for the detection of this parasite (A. E. Wakefield et al., 1990).
In this kit PCR techniques is used to detect P.carinii infection in different
biological tissues: pulmonary biopsies (fresh, frozen or embedded and
included in paraffine), bronchoalveolar lavages (BAL), induced sputum,
bronchoaspirates, expectorates, nasopharyngeal aspirates, blood and serum
(for extrapulmonary infection).
9
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8.
TEST PRINCIPLE
PCR (Polymerase Chain Reaction) has been the first method of DNA
amplification described in literature. (Saiki RK et al., 1985). It can be defined
as an in vitro amplification reaction of a specific part of DNA (target
sequence) by a thermo-stable DNA polymerase.
In the reaction three segments of nucleic acid are involved: the double strand
DNA template to be amplified (target DNA) and two single-strand
oligonucleotides “primers” that, annealing in a specific way with the template
DNA.
The DNA polymerase begins the synthesis process at the region marked by
the primers and synthesizes new double stranded DNA molecules, identical
to the original double stranded target DNA region, by facilitating the binding
and joining of the complementary nucleotides that are free in solution
(dNTPs). After several cycles, we can get millions of DNA molecules which
correspond to the target sequence.
The sensitivity of this test makes it particularly suitable to the application in
laboratory diagnostic.
Besides the amplification reaction can be executed from a wide range of
biological sample and because it is able to amplify very small DNA segments,
the starting DNA can also be partially degraded.
9.
PRODUCT DESCRIPTION
The method of PCP kit consists in the amplification of a highly conserved
rRNA 5S region of P. carinii genome, that is specific for the detection of this
pathogenic micro-organism.
The kit also allows to evaluate the suitability of the extracted DNA for the
amplification, by amplification of the TST- thiosulfate sulfurtransferase rhodanese gene (amplification control). A negative result in TST gene
amplification indicates either the presence of inhibitors of the amplification
reaction or that the DNA is highly degraded. This method helps the operator
to recognize possible false negative results.
The kit also provides with positive controls of amplification. When the
amplification of the positive control is successful, it is guaranteed that the
reaction worked correctly. These controls are not dangerous for the operator
because they are plasmid DNA containing only a part of P. carinii genome.
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The PCP kit is in premix format: all the reagents for the amplification are premixed and aliquoted in monodose test tubes (0,2 or 0,5 mL test tubes) to
which Taq polymerase and the extracted DNA will be added.
This premix format allows the reduction of the manipulation steps in preamplification, with a considerable time saving for the operator, the repeated
freezing/thawing of reagents (that could alter the product performances) is
avoided and, above all, this format reduces at minimum the risk of
contamination, so the risk to get false positive results. For this purpose it is
always recommended to use all the proper amplification controls.
11
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10. COLLECTION,
MANIPULATION
TREATMENT OF SAMPLE
AND
PRE-
The samples used for the determination of P. carinii infection by
microbiological analysis and molecular biology, are respiratory specimens
(bronchoalveolar lavages (BAL), induced sputum, expectorates, excretions,
bronchial secretions obtained by bronchoscopy) and other non respiratory
(pulmonary and lymphonodal biopsies, blood and serum).
10.1.
Respiratory specimens
For the operator’s safety during the biological specimen collection, it is a good
rule to consider every clinical sample as potentially infectious and handle it as
such.
The expectorate collection and bronchoscopy, which could cause diffusion in
the environment of infected micro-organisms, should be performed in suitable
rooms and the staff should use appropriate protection barriers.
Respiratory samples (excreta) could be fluidified by dilution with N-acetil-Lcysteine (1,5%) or dithiothreitol (0,1%), the solvent should be added in equal
volume with the excretum (directly in the collection box) and left at 37°C for
15-30 min according to the consistency of the material.
Generally, respiratory samples are decontaminated by adding 4% NaOH
solution, then, after centrifugation, neutralised by HCl. The pellet obtained
could be resuspended in PBS.
The collection boxes should be sterile, with screw cap and disposable. To
have a significant exam, the sample should not be saliva, but should be a
sample from the deep respiratory tract.
The fresh or decontaminated samples should be stored at + 2 / + 8°C for
short time (not more than 48 hours), or at -20°C for longer period (also for
some months).
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10.2.
Histological samples
The histological samples are pulmonary, and lymphonodal biopsies, fresh,
frozen formalin fixed or paraffin embedded.
The biopsy sample collection should be carried on as routine.
The fresh biopsy could be treated within few minutes from sampling, or
quickly frozen with liquid nitrogen and successively stored at -80°C until
mechanic disgregation by sterile cutter, followed by enzymatic digestion.
In case that the biopsy is fixed and paraffine-embedded, it is suggested the
use of formalin buffer at pH 7 with sodium and potassium salts at 10%, as
Lilie formula. Tissue fixation with not-buffered formalin in Bouin, Holland or
other acidic fixatives (osmic acid, for istance) are not suitable for subsequent
DNA extraction because that substances cause cross-links in the tissue,
making it not-digestible.
In case of fresh or frozen histological sample (up to about 50 mg), it is
suggested to proceed immediately with mechanic disgregation of tissue by
using sterile cutter. Do these operations on a clean glass slide, adding an
aliquote of 1X PBS buffer. Then transfer the minced tissue in a tube by a
Pasteur pipette and proceed with sample digestion.
In case that the histological sample is fixed and paraffine-embedded, proceed
with the paraffin removal and then with sample enzymatic digestion
10.3.
Blood
The P. carinii searching could be done starting from blood, which could be the
best material in case of extrapulmonary or diffused infection.
Sample collection should follow all the usual sterility precautions. Use sterile
boxes for transport, without transport medium.
Blood should be treated with EDTA. Other anticoagulation agents, as heparin,
are strong inhibitors of TAQ polymerase and so they could alter the efficiency
of the amplification reaction.
Fresh blood can be stored at +2/+8°C (processed within 4 hours from the
collection); if DNA is not extracted in short time, it’s necessary to freeze the
sample.
Before DNA extraction, a preliminary step for lymphocytes isolation by FicollHypaque system or erythrocyte lysis. is necessary.
13
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11. AMPLIFICATION PROTOCOL
11.1.
DNA EXTRACTION
For DNA extraction from respiratory specimens and histological samples, AB
ANALITICA suggests to use CLEAN DNA kit (AB ANALITICA, cod. 05-41),
with which the kit has been standardized.
This method involves the use of Proteinase K for the digestion of fresh, frozen
or fixed and embedded tissues. Tissue digestion should proceed overnight.
It’s possible to use another extraction method.
In this case it is necessary to remember that the volumes of extract indicated
in paragraph 11.2.1 and 11.2.2 for TST (10 μL) and P.carinii (10 μL)
amplification are referred to the extracts obtained with AB ANALTICA
methods.
Using an alternative extraction methods, if the DNA amount to amplify is
less than 10 μL, it will be necessary adjust the amplification mix volume
by adding water.
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14
11.2.
11.2.1.
DNA AMPLIFICATION
TST DNA Amplification
Add to each premixed test tube (blue tubes):
0,5 μL
10 μL
AB Taq
Extracted DNA
It is a good practice always to amplify, with the samples to analyze, a
negative control (add water instead of DNA to the mix) and 10 µL of positive
control (any DNA genomic).
11.2.2.
Pneumocistis carinii DNA amplification
Add to each premixed test tube (green tubes):
0,5 μL
10 μL
AB Taq
Extracted DNA
It is a good practice always to amplify, with the samples to analyze, a
negative control (add water instead of DNA to the mix) and 10 µL of positive
control (included in the kit).
Centrifuge shortly and put the test tubes (TST and Pneumocistis carinii) in the
thermalcycler programmed as below:
1 cycle
40 cycles
1 cycle
95°C
5 min
95°C
1 min
60°C
1 min
72°C
2 min
72°C
5 min
Amplification fragments length:
TST:
202 bp
Pneumocistis carinii:: 120 bp
15
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11.3.
VISUALIZATION OF AMPLIFICATION PRODUCTS
11.3.1.
Agarose gel electrophoresis
Preparation of a 3% agarose gel: weight 1,5 g of Agarose and pour it into 50
mL of 1X TAE.
Leave the solution on a magnetic stirring heater or in a microwave until the
solution becomes clear. Allow the gel to cool to “hand warm” (3-5 min), then
add 10 µL of Ethidium Bromide solution.
NOTICE: Ethidium Bromide is a strong mutagenic agent: Always wear
gloves and preferably work under a chemical safety cabinet during the
handling of this reagent or gels containing it.
Place the gel into the appropriate gel casting tray, with the comb placed in
and allow the gel to cool at room temperature or in a fridge until the gel
becomes solified.
When the gel is solified, remove carefully the comb (pay attention to not
damage the gel wells) transfer the tray into an electrophoresis chamber and
pour the appropriate amount of TAE buffer so that it covers completely the gel
(about 1-2 mm over the gel surface).
11.3.2.
Sample loading
Mix into a tube or directly on a parafilm layer:
2 μL
10 μL
6X Blue
PCR product
or DNA molecular weight marker (MW Marker)
Load the mixture in the gel wells; switch on the power supply and set the
voltage between 80-100 V.
Run the gel for about 30-45 min, then place the gel on an UV transilluminator
and analyze the results by comparing the size of the amplification products
with the reference Molecular Weight Marker.
*DNA Molecular Weight Marker (Marker MW):
501-489, 404, 353, 242, 190, 147, 110, 89, 67, 34, 26 bp.
NOTE: In a 3% agarose gel the 501-489 bp bands usually are not clearly
resolved and appear as an unique band; the 26 and 34 bp bands are
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16
sometimes too small to be visible in a 3% agarose gel (because of their low
molecular weight).
NOTICE: UV rays are dangerous for skin and, above all, eyes: always
wear gloves and safety glass or make use of the protection screen of
UV transilluminator.
11.3.3.
Interpretation of the results: TST and Pneumocistis
carinii DNA amplification
The included controls should show the following results:
CONTROL
Positive
Control
Negative
Control
RESULT
INTERPRETATION
POSITIVE
The amplification works correctly
NEGATIVE
Absence of contaminations
The interpretation of the bands on agarose gel follows the table below:
RESULT
INTERPRETATION
TST band absent
Sample not suitable for amplification
(repeat the DNA extraction)
Amplificable sample
P. carinii negative
Amplificable sample
P. carinii positive
TST band present
P. carinii band absent
TST band present
P. carinii band present
It’s possible that the positive samples for the screening give aspecific
fragments; this could depend on the typology of starting clinical sample (for
example paraffin embedded material), which do not interfere the analysis.
For any further information contact AB ANALITICA technical support e-mail:
[email protected]; fax N +39 049-8709510.
17
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120 bp
Figure 1.
Agarose gel electrophoresis
1.
2.
3.
DNA Molecular weight marker
Positive control
Positive sample for P. carinii
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12. TROUBLESHOOTING
1. Neither amplification products, nor positive control DNA band
• TAQ polymerase was not correctly added to the premix
– Use pipets and tips with suitable volumes (pipet range 0,2 - 2 μL);
– Check visually that TAQ polymerase diffuse in the premix: this is easy
because the enzyme is dissolved in glycerol that has a higher density;
– Alternatively, check visually the drop of TAQ polymerase put on the
tube wall, then centrifuge briefly.
• The thermalcycler was not programmed correctly
– Check the conformity of the thermalcycler program and the temperature
profile in the instruction manual.
• The kit doesn’t work properly
– Store the premix, TAQ polymerase and positive control at -20°C;
– Avoid repeated freezing/thawing of the premix and the reagents.
2. No amplification bands nor for TST neither P. carinii in the tested
sample, but a good band for positive controls
• Problems during the extraction step
– Be sure that the extraction kit is adequate and that you follow correctly
all the instructions;
– Consult the troubleshooting section of the extraction kit;
– Repeat the DNA extraction starting from a new sample.
• The amplification was inhibited
– Dilute the starting sample with distilled water and TE;
– Repeat the DNA extraction from a smaller amount of clinical sample;
– Use an adeguate extraction system.
19
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3. Presence of aspecific
electrophoresis
or
extraband
after
the
agarose
gel
• The thermalcycler perform temperature changes too slowly
– Do a thermalcycler revision.
• The preparation of the amplification reaction at room temperature took too
long.
– Accelerate the work time at room temperature;
– Work on an ice bath.
• The extracted DNA has not been purified
– Use an extraction system which allows good sample purification.
• The starting sample contained degraded DNA
– Repeat the extraction step using another specimen;
– Be sure that the sample had been collected and stored in appropriate
way.
For any further problem contact AB ANALITICA technical support (e-mail:
[email protected]; fax N 049-8709510.
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13. DEVICE LIMITS
The kit can have reduced performances if:
The clinical sample is not suitable for this analysis (using of alternative
fixatives instead of neutral formalin buffer, incorrect sample storage).
The DNA is not amplificable because of the presence of inhibitors of the
amplification reaction or due to an inadequate extraction system.
The kit has not been stored at the suggested temperature.
14. DEVICE PERFORMANCES
14.1.
Specificity
Primer sequence alignment in the most important databanks shows the
absence of unspecific alignment, and it has guaranteed the amplification of
Pneumocistis carinii. Cross reaction with genomic DNA or nucleic acid of
other pathogenic microorganism have not been revealed.
14.2.
Diagnostic and analytic sensitivity
For the molecular analysis (PCR) the optimal clinical sample for the research
of Pneumocistis carinii is the bronchoalveolar lavage (BAL) and pulmonary
biopsies. In this case the sensitivity and specificity of the test is nearly 100%.
In case of clinical samples such as induced sputum (IS) and tracheobronchial
aspirates the specificity and sensitivity of the test decrease until 86,2% and
60% respectively.
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15. BIBLIOGRAFIA
H. M. Ginzburg, Emerging Infectious Diseases; Vol. 2, No. 2, April-June, 1996
A. E. Wakefield et al., Detection of Pneumocistis carinii with DNA
amplification; Lancet, Vol. 36, 336: 451-53, 1990
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16. INFORMATION FOR ORDERS
PCP:
Kit for the detection of Pneumocystis carinii DNA by amplification in the
region rRNA 5S.
Cod.
Product
Pkg.
03-20A-25
PCP - Pneumocystis carinii
25 test
03-20A-50
PCP - Pneumocystis carinii
50 test
16.1.
Related products
CLEAN DNA:
CLEAN DNA: Kit for quick DNA extraction and purification with a spin-column
method, without use of phenol or chloroform, from histological samples, fresh,
frozen and formalin-fixed and paraffin-embedded, from different biological
fluids, from microbial cellular culture in solid or liquid media.
Cod.
Product
Pkg.
05-41-50
CLEAN DNA
50 test
05-41-100
CLEAN DNA
100 test
23
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AB ANALITICA srl - Via Svizzera 16 - 35127 PADOVA, (ITALIA)
Tel +39 049 761698 - Fax +39 049 8709510
e-mail: [email protected]
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