natgillin/translations-raw huggingface dataset:
For the purpose of this Annex, ‘transport medium’ means a cell culture medium with 10 % calf serum and with 200 iu penicillin, 200 μg streptomycin, and 200 μg kanamycin per millilitre or with other antibiotics of proven efficacy.
vzorci, shranjeni pri – 20 °C: ni omejeno
PART 1
Združeni vzorci v RNK stabilizacijskem reagentu se obdelajo kot posamezni vzorci v RNK stabilizacijskem reagentu.
DETAILED DIAGNOSTIC METHODS AND PROCEDURES FOR THE SURVEILLANCE AND CONFIRMATION OF IHN AND VHS
V primeru vzorcev, združenih v RNK stabilizacijskem reagentu, količina vzorca ne presega količine, ki jo priporoča proizvajalec pripomočkov za ekstrakcijo RNK, kot so pripomočki RNeasy Mini Kits (Qiagen) ali podobno.
I. Diagnostic methods and procedures for the surveillance of VHS and IHN
Če se združijo večji vzorci, morajo pripomočki ali metode za ekstrakcijo to tudi odražati.
When sampling and laboratory examination for the purpose of obtaining or maintaining disease-free health status with regard to IHN or VHS as set out in Section I of Part 1 of Annex I are carried out, using the diagnostic methods set out in points II.1 and II.2 of Part 1 of that Annex, the detailed diagnostic methods and procedures set out in following points I.1 to I.6 shall apply.
Vzorci, zbrani v RNK stabilizacijskem reagentu, se ne uporabijo za gojenje celic.
Tissues for virological examination on cell culture
Vendar se vsa ribja tkiva zaradi precej manjšega inokuluma za PCR v primerjavi s tistim za gojenje celic previdno homogenizirajo pred zbiranjem organov in tkiv za ekstrakcijo.
Before shipment or transfer to the laboratory, pieces of the organs to be examined shall be removed from the fish with sterile dissection tools and transferred to sterile plastic tubes containing transport medium.
Enako načelo se uporablja tudi, če se vzorci zbirajo v RNK stabilizacijskih reagentih.
The quantity of fish material suitable for virological examination on cell culture and by RT-qPCR is dependent on fish size.
Vendar je v navedenem primeru pogosto težko zbrati reprezentativne organe in tkiva za do 10 rib v eni epruveti, zato se število rib, ki se lahko združijo, zmanjša na 2 do 5.
Thus, whole alevin (body length < 4 cm), viscera including kidney (4 cm < body length < 6 cm) or, for larger size fish, kidney, spleen, heart and/or encephalon, and ovarian fluid from brood fish at the time of spawning shall be the tissues to be sampled.
Virološka preiskava na celični kulturi
Ovarian or seminal fluid or organ pieces from a maximum of 10 fish may be collected in one sterile tube containing at least 4 ml transport medium and represent one pooled sample.
Celična linija -2 ribjih mladic sončnega ostriža (BF-2) ali celična linija gonade šarenke -2 (RTG-2) ter bodisi Epithelioma papulosum cyprini (EPC) bodisi celice črnoglavega pisanca (FHM) se gojijo pri 20 °C do 30 °C na ustreznem gojišču, in sicer na gojišču Eagle's Minimum Essential Medium (EMEM) ali njegovih modifikacijah z dodatkom 10-odstotnega fetalnega govejega seruma in antibiotikov v standardnih koncentracijah.
The virological examination on cell culture shall be started as soon as possible and no later than 48 hours after the collection of the samples.
Z antibiotikom obdelana suspenzija organov se inokulira v celične kulture v dveh razredčitvah, in sicer v primarni razredčitvi in v razredčitvi primarne razredčitve v razmerju 1: 10, tako da sta končni razredčitvi tkivnega materiala v gojišču celične kulture 1: 100 oz. 1: 1000, da se prepreči homologna interferenca.
In exceptional cases, the virological examination may be started at the latest within 72 hours after the collection of the material, provided that the material to be examined is protected by a transport medium and that the temperature requirements during transportation can be fulfilled.
Vsaj dve celični liniji se inokulirata, kot je navedeno v točki I.5.1.
Samples for reversed transcriptase polymerase chain reaction (RT-PCR or RT-qPCR) analysis
Razmerje med velikostjo inokuluma in količino gojišča celične kulture je približno 1: 10.
Samples shall be taken from the fish in accordance with the procedure described in point I.1.1 using a sterile instrument and transferred to a sterile plastic tube containing transport medium.
Za vsako razredčitev in vsako celično linijo se uporabi najmanj približno 2 cm2 celične površine, ki ustreza eni jamici na pladnju s celičnimi kulturami in s 24 jamicami.
Tissue from 10 fish may be collected in one tube and shall represent one pooled sample.
Pladnji s celičnimi kulturami se uporabljajo, kadar je to mogoče.
However, in case the amount of inoculum is small, tissue from up to five fish may be used.
Inokulirane celične kulture se inkubirajo od sedem do deset dni pri 15 °C.
Alternatively, samples may be pooled in RNA stabilization reagents, such as 0,2 g tissue/ml reagent according to the recommendation from the manufacturers, although each fish shall be processed individually and shall not be pooled in the samples because of the small amount of material to be used for extraction.
Če se barva gojišča celične kulture spremeni iz rdeče v rumeno, kar kaže na zakisanje gojišča, se pH prilagodi s sterilno raztopino bikarbonata ali enakovrednih snovi, da se zagotovi dovzetnost celic za okužbo z virusom.
Whole fish may also be sent to the laboratory.
Vsaj vsakih šest mesecev ali v primeru suma zmanjšane dovzetnosti celic se titrirajo zamrznjene zaloge VHSV in IHNV, da se preveri dovzetnost celičnih kultur za okužbo.
However, freezing of the samples shall be avoided.
Če se opazi očiten CPE, se takoj začnejo postopki za določitev virusa v skladu s točko I.6.
The temperature of a sample during transit must never exceed 10 °C and ice must still be present in the transport box at receipt or one or more freeze blocks must still be partly or completely frozen.
Subkultivacija
Whole fish may be sent to the laboratory if the temperature requirements referred to in the first paragraph during transportation can be fulfilled.
Če se po primarni 7- do 10-dnevni inkubaciji CPE ne razvije, se opravi subkultivacija nove celične kulture z uporabo celične površine, podobne površini pri primarni kulturi.
Live fish may also be shipped.
Združeni vzorci se nato inokulirajo v homologne celične kulture, nerazredčene in razredčene v razmerju 1: 10 (kar pomeni končni razredčitvi supernatanta 1: 10 oz. 1: 100), kot je opisano v točki I.5.2.
Collection of supplementary diagnostic material
Druga možnost je, da se alikvoti 10 % gojišča, ki sestavlja primarno kulturo, inokulirajo neposredno v jamico s svežo celično kulturo (tj. subkultivacija iz jamice v jamico).
When approved by the diagnostic laboratory, other fish tissues may be collected as well and prepared for supplementary examinations.
Pred inokulacijo se lahko izvede predinkubacija razredčitev s protiserumom proti virusu IPN pri ustrezni razredčitvi, kot je opisano v točki I.4.3.
Preparation of samples for cell culture examination and RT-qPCR
Inokulirane kulture se nato inkubirajo od 7 do 10 dni pri 15 °C ter se pregledajo v skladu s točko I.5.4.
Freezing in exceptional cases
Če se toksični CPE pojavi v prvih treh dneh inkubacije, se v tej fazi opravi subkultivacija, vendar se celice potem inkubirajo sedem dni in se opravi ponovna subkultivacija z nadaljnjo sedemdnevno inkubacijo.
Where practical difficulties arise, which make it impossible to process the samples within 48 hours after the collection of the fish tissues, it may be acceptable to freeze the tissue specimens in transport medium at – 20 °C or below and to carry out virological examination within 14 days.
Ko se toksični CPE razvije po treh dneh, se celice enkrat presadijo in inkubirajo, da se doseže skupaj 14 dni od primarne inokulacije.
Records shall be kept with details on the reason for each freezing of fish tissue samples.
Če se kljub tretiranju z antibiotiki pojavi bakterijska okužba, se pred subkultivacijo opravi centrifugiranje pri 2000 do 4000 × g za 15 minut do 30 minut pri 2 °C do 5 °C ali filtriranje supernatanta skozi filter 0,45 μm ali oboje (filter, ki veže beljakovine z nizko molekulsko maso).
Homogenisation of organs
Poleg tega subkultivacija poteka po enakih postopkih, kot so opisani za toksični CPE v četrtem odstavku te točke.
In the laboratory, the fish tissue in the tubes shall be completely homogenised, either by stomacher, blender or mortar and pestle with sterile sand, and subsequently suspended in the original transport medium.
Če se CPE ne pojavi, se rezultati testov lahko razglasijo za negativne.
If a sample consists of a whole fish less than 4 cm long, it shall be minced with sterile scissors or scalpel after removal of the body behind the gut opening.
Določanje virusa
If a sample consists of a whole fish with body length between 4 cm and 6 cm, the viscera including kidney shall be collected.
Če je bil v celični kulturi opažen CPE, se gojišče (supernatant) zbere in pregleda po enem ali več od naslednjih postopkov, in sicer z encimsko-imunskim testom (ELISA), indirektno imunofluoerscenco (IF), nevtralizacijo, RT-PCR ali RT-qPCR.
If a sample consists of a whole fish more than 6 cm long, the tissue specimens shall be collected as described in point I.1.
Če ti testi niso omogočili dokončne določitve virusa v enem tednu, se supernatant pošlje nacionalnemu referenčnemu laboratoriju ali referenčnemu laboratoriju EU za bolezni rib iz Priloge VI k Direktivi 2006/88/ES za takojšnjo določitev.
The tissue specimens shall be minced with sterile scissors or scalpel and homogenised as described in the first paragraph of this point and suspended in transport medium.
Za določitev izolata virusa se opravi dvojna sendvič ELISA za dokaz protiteles.
The final ratio between tissue material and transport medium shall be adjusted in the laboratory to 1:10.
Mikrotitrske plošče se premažejo s 50 μl/jamico (0,9 pg) dokazano kakovostnih imunoglobulinov (Ig), očiščenih proteina A, iz kunčjega protiseruma proti IHNV ali VSHV, razredčenega v karbonatnem pufru (pH 9,6), ki vsebuje 15 mM natrijevega azida, in inkubiranega od 18 ur do 2 tedna pri 4 °C.
Centrifugation of homogenate
Na mikrotitrski plošči s pripravljenimi razredčitvami se vsak vzorec, ki vsebuje 1-odstotni Triton X-100 in pozitivne kontrole, razredči s pufrsko raztopino (in sicer s fosfatno pufrano slanico (PBS)-T-BSA, 1 % BSA) v 4-kratni razredčitvi, in sicer nerazredčen ter v razmerjih 1: 4, 1: 16, 1: 64.
If the sample has been shipped in a transport medium, the treatment of the supernatant with antibiotics may be omitted.
Mikrotitrske plošče ELISA se nato inkubirajo 30 minut pri 37 °C.
Where practical difficulties arise, such as incubator breakdown or problems with cell cultures, which make it impossible to inoculate cells within 48 hours after the collection of the fish tissue samples, the supernatant may be frozen at – 80 °C and virological examination may be carried out within 14 days.
Potem se mikrotitrske plošče sperejo in inkubirajo 30 minut pri 37 °C s posebnimi monoklonskimi protitelesi (in sicer MAb IP5B11 za določitev VHSV oz. Hyb 136-3 za določitev IHNV).
If the collected supernatant is stored at – 80 °C within 48 hours after the sampling, it may be reused only once for virological examination.
Na mikrotitrsko ploščo ELISA se prenese 50 μl hrenove peroksidaze (HRP), konjugirane s kunčjimi protimišjimi protitelesi in razredčene v razmerju 1: 1000 v PBS-T-BSA.
Prior to the inoculation of the cells, the supernatant shall be mixed with equal parts of a suitably diluted pool of antisera to the indigenous serotypes of Infectious pancreatic necrosis (IPN) virus and incubated with this for a minimum of one hour at 15 °C or a maximum of 18 hours at 4 °C.
Nazadnje se po ponovnem spiranju razvijejo reakcije z dodajanjem 50 μl/jamico orto-fenilendiamina (OPD).
The titre of the antiserum shall be at least 1/2000 in a 50 % plaque neutralisation test.
Mikrotitrske plošče ELISA se inkubirajo 20 minut pri sobni temperaturi v temi, reakcija pa se ustavi z dodajanjem 100 μl/jamico 0,5 M H2SO4.
Treatment of all inocula with antiserum to IPN virus aims at preventing cytopathic effect (CPE) due to IPN virus from developing in inoculated cell cultures.
Absorbanca se spremlja pri valovnih dolžinah 492 nm in 620 nm na čitalniku testa ELISA.
This will reduce the duration of the virological examinations as well as the number of cases in which occurrence of CPE would have to be considered potentially indicative of VHSV or IHNV.
Vzorci se opredelijo kot pozitivni ali negativni po primerjavi rezultatov testa z vrednostmi absorbance za pozitivne in negativne kontrole.
When samples come from production units, which are considered free from IPN, the treatment of inocula with antiserum to IPN virus may be omitted.
Na splošno se vzorci s kombinirano absorbanco (A) < 0,5 za nerazredčen material štejejo za negativne, vzorci z vrednostmi A med 0,5 in 1,0 se štejejo za sumljive, vzorci z vrednostmi A > 1,0 pa se štejejo za pozitivne.
Sample preparation for RT-PCR and RT-qPCR based surveillance programmes
Namesto testov ELISA iz te točke se lahko uporabijo druge različice testa ELISA z dokazano podobno učinkovitostjo.
If samples were collected in transport medium, the procedure set out in points I.4.2 and I.4.3 shall be carried out.
Patogeni VHSV in IHNV s seznama se določajo z okužbo celic na 96-jamični mikrotitrski plošči „Black“, konvencionalnih 24-jamičnih mikrotitrskih ploščah ali pokrivnih stekelcih na 24-jamičnih mikrotitrskih ploščah.
After centrifugation, supernatant shall be collected and RNA extracted.
Pri določanju IHNV ali VHSV ali obeh virusov z okužbo celic na pokrivnih stekelcih se uporabi naslednji protokol:
If further examination is not to be undertaken directly after centrifugation, the samples shall be immediately frozen at – 20 °C or below.
pokrivna stekelca se nasadijo s celicami z gostoto, ki vodi do 60- do 90-odstotne konfluence po 24 urah gojenja.
samples stored at 37 °C: 1 day;
150 μl supernatanta celične kulture v dveh različnih razredčitvah (1: 10 in 1: 1000) se inokulira v dveh ponovitvah na en dan starih monoslojih in inkubira 24 ur pri 15 °C;
samples stored at 25 °C: 1 week;
nato se gojišče celične kulture odstrani, okuženi celični monosloji pa se fiksirajo z 0,5 ml ledeno mrzle vodne raztopine acetona (80 % vol:vol).
samples stored at 4 °C: 1 month;
Fiksacija poteka 15 minut v komori za odsesavanje dima pri sobni temperaturi, nato se raztopina acetona odstrani, pokrivna stekelca pa se vsaj 30 minut sušijo na zraku.
samples stored at – 20 °C: indefinitely
Na tej stopnji se mikrotitrske plošče bodisi takoj obdelajo bodisi shranijo pri – 20 °C za nadaljnjo uporabo;
Pooled samples in RNA stabilization reagent shall be treated like single samples in RNA stabilization reagent.
specifična monoklonska protitelesa (in sicer MAb IP5B11 za določitev VHSV oz. Hyb 136-3 za določitev IHNV) se razredčijo v 0,01 M PBST, pH 7,2 v razredčitvi, ki jo priporoča proizvajalec monoklonskih protiteles; 50 μl/jamico do 100 μl/jamico se doda fiksiranemu monosloju, mikrotitrske plošče pa se inkubirajo eno uro pri 37 °C v vlažni komori;
If larger samples are pooled, the extraction kits or methods must reflect this pooling.
Celice se nato inkubirajo eno uro pri 37 °C s protitelesi proti mišjim imunoglobulinom, konjugiranimi s fluorescein izotiocianatom (FITC) ali tetrametilrodamin-5 (in -6) izotiocianatom (TRITC), ki se uporablja kot primarno protitelo, nato se razredčijo v skladu z navodili dobavitelja, ponovno sperejo v PBS-T in posušijo.
Samples collected in RNA stabilization reagents shall not be used for cell cultivation.
Obarvane kulture se namestijo na pokrivna stekelca z uporabo solne raztopine glicerola in se pregledajo pod vpadno ultravijolično (UV) svetlobo.
However, in that case it is often difficult to collect representative material from up to 10 fish in one tube, and the number of fish per pool shall therefore be reduced to 2 to 5.
serum, ki vsebuje protitelo, specifično za zadevno skupino, proti VHSV pri razredčitvi 1: 50 (vol:vol);
Virological examination on cell culture
serum, ki vsebuje protitelo, specifično za zadevno skupino, proti IHNV pri razredčitvi 1: 50 (vol:vol);
Cell cultures and media
združeni vzorci protiserumov proti avtohtonim serotipom IPNV pri razredčitvi 1: 50 (vol:vol);
Bluegill fry cell line -2 (BF-2) or Rainbow trout gonad cell line - 2 (RTG-2) and either Epithelioma papulosum cyprini (EPC) or Fathead minnow (FHM) cells shall be grown at 20 to 30 °C in suitable medium, namely Eagle's Minimum essential medium (MEM) or modifications thereof, with a supplement of 10 % foetal bovine serum and antibiotics in standard concentrations.
samo gojišče (pozitivna kontrola).
When the cells are cultivated in closed vials, the medium shall be buffered with bicarbonate.
Iz vsake virusne mešanice supernatanta in seruma se inokulirata vsaj dve celični kulturi, vsaka s 50 μl, nato pa inkubirata pri 15 °C.
The pH must be 7,6 ± 0,2.
Sevi in izolati VHSV, ki pri nevtralizacijskih testih ne reagirajo, se določajo z metodo IF ali ELISA.
The cell cultures to be used for inoculation with fish tissue material shall be young, normally 1 day old cell culture monolayers where possible; however, a range between 4 to 48 hours old may be accepted.
Namesto tega se lahko uporabijo drugi nevtralizacijski testi z dokazano podobno učinkovitostjo.
The cells must be actively growing at inoculation.
Vse delo z RNK se opravlja na ledu z uporabo rokavic.
Inoculation of cell cultures
RNK se ekstrahira z metodo s fenol-kloroformom ali centrifugalnimi kolonami za afiniteto do RNK v skladu z navodili proizvajalca.
At least two cell lines shall be inoculated as referred to in point I.5.1.
RNK se ponovno suspendira v destilirani RNaz prosti vodi (tj. v vodi, tretirani z 0,1-odstotnim dietil pirokarbonatom) ali ustreznem elucijskem pufru.
The ratio between inoculum size and volume of cell culture medium shall be about 1:10.
smiselni začetni oligonukleotid 5′-AGA-GAT-CCC-TAC-ACC-AGA-GAC-3′;
For each dilution and each cell line, a minimum of about 2 cm2 cell area, corresponding to one well in a 24-well cell culture tray, shall be utilised.
protismiselni začetni oligonukleotid 5′-GGT-GGT-GTT-GTT-TCC-GTG-CAA-3′.
Cell culture trays shall be used where possible.
Uporabljajo se naslednji cikli (enostopenjska RT-PCR): 1 cikel: 30 minut pri 50 °C; 1 cikel: 2 minuti pri 95 °C; 30 ciklov: 30 sekund pri 95 °C, 30 sekund pri 50 °C, 60 sekund pri 72 °C; 1 cikel: 7 minut pri 72 °C in namakanje pri 4 °C.
Incubation of cell cultures
Za dokazovanje VHSV se uporabljata naslednja začetna oligonukleotida:
The inoculated cell cultures shall be incubated at 15 °C for seven to 10 days.
Uporabljajo se naslednji cikli (enostopenjska RT-PCR): 30 minut pri 50 °C, 15 minut pri 95 °C, 35 ciklov po 30 sekund pri 94 °C, 30 sekund pri 55 °C in 60 sekund pri 68 °C.
If the colour of the cell culture medium changes from red to yellow indicating medium acidification, pH adjustment with sterile bicarbonate solution or equivalent substances shall be performed to ensure cell susceptibility to virus infection.
Nato se reakcija vzdržuje 7 minut pri 68 °C.
At least every 6 months or if decreased cell susceptibility is suspected, titration of frozen stocks of VHSV and IHNV shall be performed to verify the susceptibility of the cell cultures to infection.
Količina in specifičnost reakcij RT-PCR se ocenjujeta z gelsko elektroforezo v 1,5-odstotnem agaroznem gelu z etidijevim bromidom ter opazujeta z uporabo presvetlitve z UV.
The procedure set out in Section III shall be used, if possible.
Za IHNV je možno opaziti 693 bp PCR amplikon.
Microscopy
Za VHSV je velikost 505 bp.
Inoculated cell cultures shall be inspected regularly, at least three times a week, for the occurrence of CPE at 40 to 150 × magnification.
Rezultati PCR se lahko razlikujejo glede na pogoje, pod katerimi se ta test izvaja, in sicer je morda potrebna optimizacija temperaturnih pogojev reakcije pomnoževanja, odvisno od cikličnega termostata v uporabi.
If obvious CPE is observed, virus identification procedures in accordance with point I.6 shall be initiated immediately.
Poleg tega lahko pride do lažno pozitivnih rezultatov zaradi lažnega prileganja začetnega oligonukleotida ali laboratorijske kontaminacije.
Subcultivation
V izogib morebitnih dvomov se vključijo ustrezne pozitivne in negativne kontrole ter sekvenčni amplikoni.
If no CPE has developed after the primary incubation for 7 to 10 days, subcultivation shall be performed to fresh cell cultures utilising a cell area similar to that of the primary culture.
Za začetne oligonukleotide za VHSV je potrebna posebna pozornost pri uporabi celic BF-2, saj začetni oligonukletoidi lahko reagirajo z DNK/RNK celične linije z lažno pozitivnimi rezultati podobne velikosti.
Aliquots of medium (supernatant) from all cultures or wells constituting the primary culture shall be pooled according to cell line 7 to 10 days after inoculation.
Pri testiranju supernatanta iz celic BF-2 se sekvencirajo amplificirani odseki z metodo PCR.
The pools shall then be inoculated into homologous cell cultures undiluted and diluted 1:10 (resulting in final dilutions of 1:10 and 1:100, respectively, of the supernatant) as described in point I.5.2.
Za VHSV se amplifikacija opravi z uporabo naslednjih začetnih oligonukleotidov in sonde:
Alternatively, aliquots of 10 % of the medium constituting the primary culture shall be inoculated directly into a well with fresh cell culture (namely, well to well subcultivation).
Smiselni začetni oligonukleotid: 5′-AAA-CTC-GCA-GGA-TGT-GTG-CGT-CC-3′;
The inoculation may be preceded by pre-incubation of the dilutions with the antiserum to IPN virus at appropriate dilution as described in point I.4.3.
Protismiselni začetni oligonukleotid: 5′-TCT-GCG-ATC-TCA-GTC-AGG-ATG-AA-3′,
The inoculated cultures shall then be incubated for 7 to 10 days at 15 °C and inspected in accordance with point I.5.4.
in sonda: 5′-FAM-TAG-AGG-GCC-TTG-GTG-ATC-TTC-TG-BHQ1.
If toxic CPE occurs within the first 3 days of incubation, subcultivation shall be performed at that stage, but the cells shall then be incubated for 7 days and sub cultivated again with further 7 days incubation.
Enostopenjska RT-qPCR:
When toxic CPE develops after 3 days, the cells shall be passed once and incubated to achieve the total of 14 days from the primary inoculation.
Negativne kontrole reakcijske mešanice in pozitivne kontrole reakcije se vključijo na vsako serijo mikrotitrskih plošč.
There must be no evidence of toxicity in the final 7 days of incubation.
Pogoji pomnoževanja: 30 minut pri 50 °C, 15 minut pri 95 °C, 40 ciklov po 15 sekund pri 94 °C, 40 sekund pri 60 °C in 20 sekund pri 72 °C; po potrebi se prilagodi.
If bacterial contamination occurs despite treatment with antibiotics, subcultivation shall be preceded by centrifugation at 2000 to 4000 × g for 15 to 30 minutes at 2 to 5 °C, or filtration of the supernatant through a 0,45 μm filter or both (low protein-binding membrane).
Namesto tega se lahko uporabijo druge različice testov RT-PCR ali RT-qPCR z dokazano podobno učinkovitostjo.
In addition to this, subcultivation shall follow the same procedures as described for toxic CPE in the fourth paragraph of this point.
Za IHNV se amplifikacija opravi z uporabo naslednjih začetnih oligonukleotidov in sonde:
If no CPE occurs, the test may be declared negative.
Smiselni začetni oligonukleotid: 5′- AGA-GCC-AAG-GCA-CTG-TGC-G-3′;
Virus identification
Protismiselni začetni oligonukleotid: 5′- TTCTTTGCGGCTTGGTTGA – 3′
If evidence of CPE has been observed in a cell culture, medium (supernatant) shall be collected and examined by one or more of the following techniques: Enzyme-linked immunosorbent essay (ELISA), Immunofluorescence (IF), neutralisation, RT-PCR or RT-qPCR.
in sonda: 5′ 6FAM-TGAGACTGAGCGGGACA-NFQ/MGB.
If these tests have not allowed definitive identification of the virus within 1 week, the supernatant shall be forwarded to the national reference laboratory or to the EU reference laboratory for fish diseases referred to in Annex VI to Directive 2006/88/EC for immediate identification.
Dvostopenjska RT-qPCR:
A double antibody sandwich ELISA shall be performed in order to identify the virus isolate.
Naslednji test temelji na dvostopenjski amplifikaciji, zato je od ene do druge reakcije potrebna posebna pozornost pri ravnanju z epruvetami, da se prepreči kontaminacija.