natgillin/translations-raw huggingface dataset:
Wells shall be washed with Phosphate buffered saline (PBS) and fixed by incubation with 80 % acetone for 20 minutes at room temperature.
Preparati se dehidrirajo tako, da se potopijo v stopenjske serije etanola in nato posušijo na zraku.
Plates shall be air-dried and stained immediately or stored at 0 to 6 °C for no more than 24 hours prior to staining.
Rezine se inkubirajo s 100 μl hibridizacijskega pufra (4 × SSC [standardna raztopina slanice in citrata], 50-odstotni formamid, 1 × Denhardtova raztopina, 250 μg ml– 1 tRNK iz kvasovk, 10-odstotni dekstran sulfat), ki vsebuje 10 ng (1 μl reaktantov PCR, pripravljenih v skladu s točko I.3.2 z uporabo začetnih oligonukleotidov CCG-GTG-CCA-GGT-ATA-TCT-CG in TTC-GGG-TGG-TCT-TGA-AAG-GC) sonde, označene z digoksigeninom.
Replicate wells shall be stained with a mix of monoclonal antibodies (MAb) 3H6F8 and 1OC9F5 against ISAV, or other MAb of proven efficacy and specificity, diluted in PBS and incubated at 37 ± 4 °C for 30 minutes.
Rezine se prekrijejo s plastičnimi pokrivnimi stekelci in situ in za pet minut položijo na grelno enoto pri 95 °C.
MAb shall be removed and plates washed three times with 0,05 % Tween 20 in PBS.
Preparati se nato eno minuto hladijo na ledu, preden se čez noč hibridizirajo v vlažni komori pri 42 °C.
Anti-mouse IgG Fluorescein isothiocyanate (FITC) conjugate diluted in PBS shall be added to each well and incubated at 37 ± 4 °C for 30 minutes.
Rezine se spirajo dvakrat po pet minut v 2 × SSC pri sobni temperaturi in enkrat po 10 minut v 0,4 × SSC na 42 °C.
The dilutions of different batches of MAb and FITC conjugate shall be optimised in each laboratory.
Postopki dokazovanja se izvajajo v skladu z navodili proizvajalca.
Antibody shall be removed and plates shall be washed three times with 0,05 % Tween 20 in PBS.
Preparati se nato sperejo v sterilni destilirani vodi (dH2O).
Wells shall be examined immediately using an inverted microscope set up for fluorescence microscopy with a suitable filter for excitation of FITC.
Rezine se nasprotno pobarvajo z Bismarck rjavo-rumenim barvilom, sperejo v dH2O, pokrivna stekelca pa se namestijo z uporabo vodenega medija s pokrivnimi stekelci.
A test shall be considered positive if fluorescent cells are observed.
Pozitivne in negativne kontrole so rezine iz znanih okuženih oz. neokuženih živali gostiteljic.
For a test to be valid, the positive controls shall score positive and the negative controls shall score negative.
Pozitiven rezultat je dokazan z vijoličasto-črno označenimi celicami Marteilia refringens v znanih ciljnih tkivih, pri čemer so vse negativne kontrole negativne in vse pozitivne kontrole pozitivne.
Examination of other tissues
Sekvenciranje
The technique referred to in point II.2.6 may be applied to other fish tissues such as liver, spleen and heart providing a reasonable quantity of endothelial cells, leucocytes or lymphocytes can be deposited on the slide.
Sekvenciranje se izvede kot eden od zadnjih korakov za potrditveno diagnostiko.
The staining procedure shall remain the same for each tissue, although for some tissues it may be preferable to omit the propidium iodide staining and rely on the phase illumination to identify the cell types present in the imprint.
Ciljni regiji sta mala podenota rDNK in ITS1.
Histology
Natančne diagnostične metode in postopki za nadzor in potrditev okužbe z Marteilia refringens
Paraffin-embedded sections shall be cut at 5 μm and stained using haematoxylin and eosin.
Za namene programov nadzora in za potrditev prisotnosti okužb z Marteilia refringens ali izključitev suma na navedeno bolezen s seznama v skladu z zahtevami iz oddelka II dela 4 Priloge I se uporabijo diagnostične metode in ustrezni postopki v skladu s smernicami iz tabele 4.1:
Histological changes in clinically diseased Atlantic salmon are variable, but may include the following:
Tabela 4.1
numerous erythrocytes in the central venous sinus and lamellar capillaries of the gills, where erythrocyte thrombi also may be formed;
Smernice za uporabo diagnostičnih metod za programe nadzora in za potrditev ali izključitev okužbe z Marteilia refringens
multifocal to confluent petechiae or hepatocyte necrosis or both at some distance from larger vessels in the liver; multifocal accumulation of erythrocytes in dilated hepatic sinusoids;
Metoda
accumulation of erythrocytes in blood vessels of the intestinal lamina propria and eventually haemorrhage into the lamina propria;
Ciljno usmerjen nadzor
spleen stroma distended by erythrocyte accumulation;
Domnevna diagnoza
slight multifocal to extensive diffuse interstitial haemorrhage with tubular necrosis in the haemorrhagic areas, erythrocyte accumulation in the glomeruli in the kidney;
Potrditvena diagnoza
erythrophagocytosis in the spleen and secondary haemorrhages in liver and kidney.
Odtisi prebavne žleze
Immunohistochemistry (IHC)
X ali
Polyclonal antibody against ISAV nucleoprotein shall be used on paraffin sections from formalin-fixed tissue.
Histopatologija
The organs to be examined shall be mid-kidney and heart (transitional area including all three chambers and valves).
Hibridizacija in situ
Suspected cases due to pathological signs shall be verified with a positive IHC.
X in
Histological sections shall be prepared in accordance with standard methods.
NATANČNE DIAGNOSTIČNE METODE IN POSTOPKI ZA NADZOR IN POTRDITEV OKUŽBE Z BONAMIA OSTREAE
Preparation of tissue sections
I. Postopki za diagnozo okužbe z Bonamia ostreae
The tissues shall be fixed in neutral phosphate-buffered 10 % formalin for at least 1 day, dehydrated in a graded ethanol series, cleared in xylene and embedded in paraffin, according to standard protocols.
Pri vzorčenju in laboratorijski preiskavi za namene pridobitve ali ohranitve določenega zdravstvenega statusa glede Bonamia ostreae, kot je določeno v oddelku I dela 5 Priloge I, ali za potrditev prisotnosti ali izključitev suma na navedeno bolezen s seznama v skladu s členom 57(b) Direktive 2006/88/ES z diagnostičnimi metodami iz oddelka II dela 5 Priloge I se uporabljajo natančne diagnostične metode in postopki iz naslednjih točk I.1, I.2 in I.3.
Approximately 5 μm thick sections (for IHC placed on poly-L-lysine-coated slides) shall be heated at 56 °C to 58 °C (maximum 60 °C) for 20 minutes, dewaxed in xylene, rehydrated through a graded ethanol series, and stained with haematoxylin and eosin for pathomorphology and IHC in accordance with point (2).
Postopek vzorčenja
Staining procedure for IHC
Ko se opravi vzorčenje ostrig, se te živali hranijo pri 4 °C ali na ledu v hladilniku največ 24 ur, če vzorci vključujejo odprte mehkužce, oz. 72 ur, če ne, v plastični vrečki skupaj z etiketo s podatki o naravi in poreklu ostrig.
All incubations shall be carried out at room temperature on a rocking platform, unless otherwise provided for in this decision:
Za diagnostiko Bonamia ostreae s histologijo se uporabijo 3 mm do 5 mm debele rezine tkiv, vključno s tkivom škrg in srca.
antigen retrieval shall be done by boiling sections in 0,1 M citrate buffer pH 6,0 for 2 × 6 minutes followed by blocking with 5 % non-fat dry milk and 2 % goat serum in 50 mM TBS (TBS; Tris/HCl 50 mM, NaCl 150 mM, pH 7,6) for 20 minutes;
Po sušenju tkiv škrg ali srca na vpojnem papirju se na steklenih preparatih naredi več odtisov.
sections shall then be incubated overnight with primary antibody (monospecific rabbit antibody against ISAV nucleoprotein) diluted in TBS with 1 % non-fat dry milk, followed by three washes in TBS with 0,1 % Tween 20;
Preparati se posušijo na zraku, fiksirajo v metanolu ali absolutnem etanolu in obarvajo s komercialno dostopnim kompletom za barvanje krvi, kot je Diff-Quik®/Hemacolor®, v skladu z navodili proizvajalca.
for detection of bound antibodies, sections shall be incubated with Alkaline phosphatase-conjugated antibodies to rabbit IgG for 60 minutes.
Preparati se najprej preučujejo s povečavo × 200, nato pa potopljeni v olje s povečavo × 1000.
Following a final wash, Fast Red (1 mg ml– 1) and Naphthol AS-MX phosphate (0,2 mg ml– 1) with 1 mM Levamisole in 0,1 M TBS (pH 8,2) shall be added to develop for 20 minutes.
Rezultat je pozitiven, če so v hemocitih prisotni majhni okrogli ali ovalni organizmi (široki 2 μm do 5 μm).
Sections shall then be washed in tap water before counterstaining with Harris haematoxylin and mounted in aqueous mounting medium.
Vendar pa se parazit lahko pojavi tudi zunaj celice.
Interpretation of the result of IHC
Opazijo se lahko celice z več jedri.
The interpretation of the result of the IHC test shall be as set out in points (a) and (b):
Rezine tkiv, ki vključujejo škrge in prebavno žlezo, se vsaj 24 ur fiksirajo v Davidsonovem fiksativu, čemur sledi običajni postopek za parafinsko histologijo in barvanje, na primer s hematoksilinom in eozinom.
control sections shall be considered as positive, when it is observed that the control sections have clearly visible red-coloured (reddish) cytoplasmic and intranuclear staining of endothelia cells in blood vessels of endocardium.
Rezultat je pozitiven, če so paraziti prisotni kot zelo majhne, od 2 μm do 5 μm široke celice v hemocitih ali proste v vezivnem tkivu ali sinusih škrg, drobovju in epitelu plašča, kar je pogosto povezano z intenzivno vnetno reakcijo.
A test sample section shall only be regarded as positive if such clear, intranuclear red staining of endothelial cells is found;
V izogib kakršnim koli dvomom je diagnoza pozitivna, če se opazi parazit v hemocitu.
control sections shall be considered as negative if they don't have any significant colour reaction.
Lahko se uporabijo kompleti za ekstrakcijo DNK, ki so na voljo komercialno in običajno zagotavljajo visokokakovostno DNK, primerno za uporabo s protokoli PCR, kot je opisano v nadaljnjem besedilu.
Endothelial staining reactions within very extensive haemorrhagic lesions may be slight or absent, possibly because of lysis of infected endothelial cells.
Vzorci se pet minut denaturirajo v cikličnem termostatu pri 94 °C, nato pa se na njih opravi 30 ciklov (eno minuto pri 94 °C, eno minuto pri 55 °C, eno minuto pri 72 °C), ki jim sledi dokončna ekstenzija 10 minut pri 72 °C.
DETAILED DIAGNOSTIC METHODS AND PROCEDURES FOR THE SURVEILLANCE AND CONFIRMATION OF INFECTION WITH MARTEILIA REFRINGENS
Pozitiven rezultat je pozitivna amplifikacija PCR ob pričakovani velikosti (in sicer 300 bp), pri čemer so vse negativne kontrole negativne, vse pozitivne kontrole pa pozitivne;
I. Detailed diagnostic methods and procedures for the diagnosis of infection with Marteilia refringens
drugi protokol PCR je PCR v realnem času z nespecifičnim načinom detekcije z barvilom SYBR® Green.
When sampling and laboratory examination are carried out for the purpose of obtaining or maintaining a health status with regard to infection with Marteilia refringens as set out in Section I of Part 4 of Annex I or to confirm or rule out the presence of that listed disease in accordance with Article 57(b) of Directive 2006/88/EC, using the diagnostic methods set out in Section II of Part 4 of Annex I, the detailed diagnostic methods and procedures set out in points I.1, I.2 and I.3 of this Part shall apply.
Omogoča specifično dokazovanje Bonamia ostreae (kot je opisano v nadaljnjem besedilu) in se lahko kombinira s PCR v realnem času z nespecifičnim načinom detekcije z barvilom SYBR® Green, kar omogoča specifično dokazovanje Bonamia exitiosa (Ramilo et al.
Sampling procedure
2013).
Gaping or freshly dead individual molluscs shall be sampled by priority, to increase the chances of finding infected animals.
Začetna oligonukleotida BOSTRE-F (5′- TTACGTCCCTGCCCTTTGTA-3′) in BOSTRE-R (5′- TCGCGGTTGAATTTTATCGT-3′) amplificirata produkt 208 bp.
Once sampled, oysters or mussels shall be maintained at 4 °C or on refrigerated ice for no more than 24 hours if samples include gaping molluscs and no more than 72 hours if not, in a plastic bag including a label with details related to the nature and the origin of the oysters or mussels.
Mešanice PCR vsebujejo osnovno zmes SYBR® Green Master Mix (1X), 0,3 μM smiselnega in protismiselnega začetnega oligonukleotida ter 200 ng ekstrahirane DNK.
Gaping or freshly dead molluscs shall be maintained separately from other molluscs.
Vzorci se 10 minut denaturirajo v sistemu za dokazovanje v realnem času pri 95 °C, nato pa se na njih opravi 35 ciklov (30 sekund pri 95 °C, 45 sekund pri 55 °C in eno minuto pri 72 °C).
A 3 to 5 mm thick section of tissues including gills and heart tissue shall be used for the diagnostic of Marteilia refringens by histology.
Krivulja tališča se analizira s povečevanjem temperature po 0,5 °C/s od 55 °C do 95 °C, pri vsaki spremembi temperature pa se evidentira fluorescenca.
A piece of digestive gland shall be used for some tests, including imprints and polymerase chain reaction (PCR).
Pozitivne kontrole sestojijo iz genomske DNK iz močno okužene živali gostiteljice ali plazmidne DNK, vključno s ciljno regijo.
Microscopic techniques
Negativne kontrole sestojijo iz genomske DNK iz neokuženih živali gostiteljic in reagentov PCR brez ciljne DNK.
Cytology (imprint cytology)
Pozitiven rezultat je pozitivna amplifikacija PCR z enotnim tališčem (78,25 °C ± 0,25 °C pod pogoji, objavljenimi v Ramilo et al.
After drying digestive gland tissues on absorbent paper, several imprints shall be made on a glass slide.
2013), pri čemer so vse negativne kontrole negativne, vse pozitivne kontrole pa pozitivne.
Slides shall be air-dried, fixed in methanol or in absolute ethanol and stained using a commercially available blood-staining kit such as Diff-Quik®/Hemacolor®, in accordance with the manufacturer's instructions.
Uporabi se sonda, ki je ciljno usmerjena v malo enoto genskega kompleksa rDNK, čeprav se je izkazalo, da navzkrižno reagirajo z nekaterimi drugimi predstavniki rodu Haplosporidia.
After rinsing in tap water and drying, the slides shall be mounted with a cover-slip using an appropriate synthetic resin.
Rezine tkiv, ki vključujejo škrge in prebavno žlezo, se vsaj 24 ur fiksirajo v Davidsonovem fiksativu, čemur sledi običajni postopek za parafinsko histologijo.
The slides shall be observed first at × 200 magnification and then under oil immersion at × 1000 magnification.
Rezine se inkubirajo s 100 μl hibridizacijskega pufra (4 × SSC [standardna raztopina slanice in citrata], 50-odstotni formamid, 1 × Denhardtova raztopina, 250 μg ml– 1 tRNK iz kvasovk, 10-odstotni dekstran sulfat), ki vsebuje 20 ng (2 μl reakcije PCR, pripravljenih v skladu s točko I.3.2 z uporabo začetnih oligonukleotidov Bo in Boas) sonde, označene z digoksigeninom.
A positive result shall be the observation of cells ranging in size up to between 30 to 40 μm.
Rezine se spirajo dvakrat po pet minut v 2 × SSC pri sobni temperaturi in enkrat po 10 minut v 0,4 × SSC na 42 °C.
Pale halos around large, strongly stained (refringent) granules and, in larger cells, cell within cell arrangements are observed.
Ciljni regiji sta mala podenota rDNK in ITS1.
Sections of tissue that include gills, digestive gland, mantle, and gonad shall be fixed for at least 24 hours in Davidson's fixative followed by normal processing for paraffin histology and staining, for example with haematoxylin and eosin.
Za namene nadzora in potrditve prisotnosti ali izključitve suma na okužbo z Bonamia ostreae v skladu z zahtevami iz oddelka II dela 5 Priloge I se uporabijo diagnostične metode in ustrezni postopki v skladu s smernicami iz tabele 5.1.
Observations shall be made at increasing magnifications up to × 1000.
Tabela 5.1
A positive result shall be the observation of cells ranging in size from 4 up to 40 μm.
Smernice za uporabo diagnostičnih metod za programe nadzora in za izključitev ali potrditev okužbe z Bonamia ostreae
Early stages consist of multinucleate, spheric to elongated cells.
Odtisi srca ali škrg
These are mainly found in the epithelium of oesophagus and stomach and sometimes of labial palps.
NATANČNE DIAGNOSTIČNE METODE IN POSTOPKI ZA NADZOR IN POTRDITEV BOLEZNI BELIH PIK (WSD)
Sporulation involves divisions of cells within cells and takes place in the digestive gland tubules and ducts.
Diagnostični postopki za dokazovanje WSSV
Refringent granules appear in the course of sporulation, but are not observed in early stages.
Pri vzorčenju in laboratorijski preiskavi za namene programov nadzora in izkoreninjenja iz oddelka I dela 6 Priloge I ter za potrditev prisotnosti ali izključitev suma na okužbo z WSSV v skladu s členom 57(b) Direktive 2006/88/ES z uporabo diagnostičnih metod iz oddelka II dela 6 Priloge I se uporabijo natančne diagnostične metode in postopki iz točk 2 do 7 tega dela.
In late phases of infection, sporangia are observed free in the lumen of the digestive tract.
Metode in postopki, opisani v tem delu Priloge II, so prilagojeni glede na akreditacijski test ISO 17025, ki se uporablja v referenčnem laboratoriju Evropske unije za bolezni rakov.
The granules can range from deep orange to deep red.
Lahko se uporabljajo tudi alternativni pristopi, pri katerih se uporabljajo enaki pogoji ali kompleti, ki jih proizvajajo različni proizvajalci, toda ki zagotavljajo enakovredno občutljivost in specifičnost kot kompleti, opisani v tem delu.
Molecular techniques
V vseh primerih se amplificirani pridelek reakcije PCR sekvencira, da se potrdi določitev virusa kot virusa sindroma belih pik (WSSV).
Polymerase chain reaction (PCR)
Faze, ki se zahtevajo za določitev WSSV iz vzorcev tkiv, so homogenizacija tkiva, ekstrakcija DNK, specifična amplifikacija DNK WSSV z uporabo PCR, vizualizacija amplificiranega pridelka na gelu, prečiščenje DNK in sekvenciranje za potrditev določitve patogena.
Several PCR protocols have been developed and published.
Homogenizacija tkiva
PCR primers that target the internal transcribed spacer (ITS1) region shall be used as they are able to amplify only M. refringens.
Tkivna disrupcija in priprava homogenata v ustreznem pufru se izvajata z uporabo tkivnega homogenizatorja Fast Prep in epruvet Lysing Matrix A (MP Biomedicals).
PCR shall be carried out in a 50 μl volume.
Tkivo se stehta, vstavi v epruvete Lysing Matrix A, razredči v razmerju 1 v 10 w/v ali v skladu z navodili proizvajalca v ustreznem pufru (G2 in 10 μl proteinaze K za uporabo s kompletom DNA Tissue (Qiagen)) in dve minuti homogenizira z uporabo tkivnega homogenizatorja Fast Prep 24.
PCR mixtures shall contain buffer (500 mM KCl, 100 mM Tris/HCl [pH 9,0 at 25 °C] and 1 % Triton® X-100), 2,5 mM MgCl2, 0,2 mM dNTP mix, 1 μM forward and reverse primers, 0,02 units μl– 1 Taq DNA polymerase, and 10 to100 ng of extracted DNA.
Homogenizirani vzorci se najmanj štiri ure ali čez noč inkubirajo pri 56 °C.
After denaturation of DNA at 94 °C for five minutes, 30 cycles shall be run as follows: denaturation at 94 °C for one minute, annealing at 55 °C for one minute, and elongation at 72 °C for one minute per kilo-base pair.
Vzorci se premešajo v vorteks mešalniku, dve minuti centrifugirajo pri 9000 vrtljajev na minuto, v epruveto z vzorcem se nato doda 50 μl supernatanta ali količina, ki ustreza 5 mg tkiva (teža tkiva, optimalna za komplet za ekstrakcijo), za ekstrakcijo DNK, s pufrom G2 pa se količina nato poveča do 200 μl.
A final elongation step of 10 minutes at 72 °C shall be performed.
Skupna DNK se ekstrahira z uporabo kompleta za ekstrahiranje DNK tkiva in stroja EZ1 Advanced XL Biorobot (Qiagen) po navodilih proizvajalca.
For the detection of M. refringens, PCR shall be performed with primers that target the ITS1 region (5′-CCG-CAC-ACG-TTC-TTC-ACT-CC-3′ and 5′-CTC-GCG-AGT-TTC-GAC-AGA-CG-3′).
Z vsako serijo vzorcev se opravita kontrola ekstrakcije (DNK iz telečjega timusa) in negativna kontrola (G2).
Positive controls shall consist of genomic DNA from a highly infected host or plasmidic DNA including the target region.
DNK se eluira v količino 50 μl.
Negative controls shall consist of genomic DNA from non-infected hosts and PCR reactives without target DNA.
Za zagotovitev, da je bila ekstrakcija uspešna, se koncentracija DNK za vse vzorce in kontrole določi s strojem Nano Drop.
A positive result shall be positive PCR amplification at the expected size (412 bp), with all negative controls being negative and all positive controls being positive.
Ekstrahirana DNK se zamrzne pri – 20 °C, če se ne uporabi takoj.
In-situ hybridisation (ISH)
Verižna reakcija s polimerazo (PCR) za WVVS
Several ISH protocols have been developed and published.
Metoda za dokazovanje WSSV je protokol za dokazovanje WSSV z ugnezdeno PCR, določen v naslednjih odstavkih, ki amplificira amplikona 1447 bp in 848 bp iz gena 18s rRNK v prvem oz. drugem krogu PCR.
Probe that targets the SSU of the rRNA gene complex shall be used because it has been validated against histology.
Prvi krog reakcije PCR se vzpostavi v količini 50 μl, ki vsebuje končne koncentracije pufra GoTaq Buffer (Promega), 5 mM MgCl2, 1 pmol/μl začetnega oligonukleotida WSSV 146 F1, 1 pmol/μl začetnega oligonukleotida WSSV 146 R1 (tabela 1), 0,25 mM dNTPs, 1,25 U Taq polimeraze in 2,5 μl DNK.
Sections of tissue that include gills, digestive gland, shall be fixed for at least 24 hours in Davidson's fixative followed by normal processing for paraffin histology.
Vsak vzorec se analizira v dveh izvodih skupaj z negativno kontrolo ekstrakcije, negativno kontrolo PCR (namesto DNK se doda 2,5 μl H2O) in pozitivno kontrolo.
Sections of 5 μm shall be cut and placed on aminoalkylsilane-coated slides, which shall then be baked overnight in an oven at 40 °C.
Pozitivna kontrola je razredčen plazmid WSSV, pripravljen in potrjen za interno uporabo (ki ga da na voljo referenčni laboratorij EU).
The sections shall be dewaxed by immersing in xylene for 10 minutes.
Drugi krog reakcije PCR za WVVS se vzpostavi na enak način kot prvi krog, vendar z uporabo kompleta začetnih oligonukleotidov WSSV 146 F2/R2 in z drugo pozitivno kontrolo, da se preveri, ali je ta faza PCR delovala.
This step shall be repeated once and then the solvent shall be eliminated by immersion in two successive absolute ethanol baths for 10 minutes each.
Začetni oligonukleotid
The sections shall then be dehydrated by immersion in a graded ethanol series.
Tako prvi kot drugi krog PCR se izvedeta pod naslednjimi pogoji pomnoževanja na cikličnem termostatu DNA Engine Tetrad 2 Peltier Thermal Cycler (ali enakovrednem termostatu).
The sections shall be treated with proteinase K (100 μg ml– 1) in TE buffer (Tris [50 mM], EDTA [10 mM]), at 37 °C for 30 minutes.
Na začetku se izvede denaturiranje za dve minuti pri 94 °C, ki mu sledi 30 sekund pri 94 °C, 30 sekund pri 62 °C, 30 sekund pri 72 °C, kar se ponovi za 30 ciklov, nato pa sledita ekstenzija za 2 minuti pri 72 °C in zadržanje pri 4 °C.
Slides shall be dehydrated by immersion in a graded ethanol series and then air dried.
Gelska elektroforeza
Sections shall be incubated with 100 μl of hybridisation buffer (4 × SSC [standard saline citrate], 50 % formamide, 1 × Denhardt's solution, 250 μg ml– 1 yeast tRNA, 10 % dextran sulphate) containing 10 ng (1 μl of the PCR reactants prepared as described in point I.3.2 using primers CCG-GTG-CCA-GGT-ATA-TCT-CG and TTC-GGG-TGG-TCT-TGA-AAG-GC) of the digoxigenin-labelled probe.
Amplificirani pridelki reakcije PCR iz prvega in drugega kroga PCR se vizualizirajo na 2-odstotnem agaroznem gelu, pripravljenem s pufrom TAE.
Sections shall be covered with in-situ plastic cover-slips and placed on a heating block at 95 °C for five minutes.
15 μl vsakega vzorca se približno 20 minut analizira pri 120 voltih in opazuje pod UV svetlobo.
Slides shall then be cooled on ice for one minute before overnight hybridisation at 42 °C in a humid chamber.
Pri pozitivnih vzorcih se bo v prvem krogu PCR pojavila proga v velikosti 1447 bp, v drugem krogu pa v velikosti 848 bp.
Sections shall be washed twice for five minutes in 2 × SSC at room temperature, and once for 10 minutes in 0,4 × SSC at 42 °C.
Vzorci navedenih velikosti se izrežejo in vstavijo v 1,5-mililitrsko mikrocentrifugirko.
The detection steps shall be performed according to the manufacturer's instructions.
DNK, ki jo vsebujejo rezine gela, se prečisti z uporabo gela Promega Wizard® SV Gel in sistemom PCR Clean-Up System v skladu z navodili proizvajalca.
The slides shall then be rinsed in sterile distilled water (dH2O).
Koncentracija DNK se oceni s strojem Nano Drop.