natgillin/translations-raw huggingface dataset:
The assessment, which was presented to the Commission in May 2015, concluded that the competitive enzyme-linked immunosorbent assay (ELISA) is no longer available, indirect ELISA is not in common use but could be provided in 4-6 months after the request and that the blocking ELISA is commercially available and commonly used for analysis of samples during the Proficiency Test exercises organised by the European Union Reference Laboratory for African horse sickness.
Procjenom, koja je predstavljena Komisiji u svibnju 2015., zaključeno je da kompetitivni imunoenzimni test (ELISA) više nije dostupan, indirektni test ELISA obično se ne upotrebljava, ali se može osigurati u roku od 4 – 6 mjeseci nakon zahtjeva te da je blokirajući test ELISA dostupan na tržištu i da se obično upotrebljava za analizu uzoraka tijekom ispitivanjâ osposobljenosti koja organizira Referentni laboratorij Europske unije za konjsku kugu.
In addition, the report points out that the nucleic acid recognition by reverse-transcription polymerase chain reaction (RT-PCR) methods have advantages over serological diagnostic methods, because they allow for the detection of the disease at an early stage of infection.
Osim toga, iz izvješća se može vidjeti da metode prepoznavanja nukleinske kiseline obrnutom transkripcijom lančane reakcije polimerazom (RT-PCR) imaju prednost pred serološkim dijagnostičkim metodama, jer se njima omogućuje otkrivanje bolesti u ranom stadiju infekcije.
On the 24-25 November 2015, the Joined Workshop of African Horse Sickness/Bluetongue European Union Reference Laboratories together with national reference laboratories held in Ascot, United Kingdom, recommended the inclusion in Annex IV to Directive 2009/156/EC of real time reverse transcription (RRT)-polymerase chain reaction (PCR) methods for the detection of the African horse sickness virus.
Na Zajedničkoj radionici za konjsku kugu/bolest plavog jezika koja se održala 24. i 25. studenoga 2015. u Ascotu u Ujedinjenoj Kraljevini, Referentni laboratorij Europske unije za konjsku kugu zajedno s nacionalnim referentnim laboratorijima preporučio je uključivanje u Prilog IV. Direktivi 2009/156/EZ metode obrnute transkripcije lančane reakcije polimerazom u stvarnom vremenu radi otkrivanja virusa konjske kuge.
Although all available real-time RT-PCR methods for the African horse sickness genome detection are sufficiently sensitive, the procedure described by Agüero et al.
Iako su sve dostupne metode RT-PCR-a u stvarnom vremenu dovoljno osjetljive za otkrivanje genoma konjske kuge, laboratoriji najčešće upotrebljavaju postupak koji su opisali Agüero et al. 2008.
was specifically designed to ensure that horses from areas at risk of African horse sickness can be transported safely after the minimum quarantine period required in accordance with the Terrestrial Animal Health Code [6] of the OIE.
specifično je osmišljena kako bi se omogućilo sigurno premještanje konja iz područja koja su ugrožena konjskom kugom nakon minimalnog razdoblja karantene koje je potrebno u skladu s Kodeksom o zdravlju kopnenih životinja OIE-a [6].
It is therefore appropriate to incorporate in Annex IV to Directive 2009/156/EC methods for agent identification and for the detection of antibody as complementary methods for a rapid diagnosis of African horse sickness.
Stoga je primjereno Prilogom IV. Direktivi 2009/156/EZ obuhvatiti metode za identifikaciju uzročnika bolesti i za otkrivanje protutijela kao komplementarne metode za brzo dijagnosticiranje konjske kuge.
Annex IV to Directive 2009/156/EC should be therefore amended by deletion of the competitive ELISA test and by an updating the procedures for the indirect and blocking ELISA tests in accordance with Chapter 2.5.1. of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals of the OIE, Edition 2016 based on the Version adopted by the World Assembly of Delegates of the OIE in May 2012 [7].
Prilog IV. Direktivi 2009/156/EZ potrebno je stoga izmijeniti brisanjem kompetitivnog testa ELISA i ažuriranjem postupaka za neizravni test ELISA i blokirajući test ELISA u skladu s poglavljem 2.5.1.
At the same time, real-time RT-PCR procedures as described by Agüero et al.
Priručnika o dijagnostičkim testovima i cjepivima za kopnene životinje OIE-a, izdanje 2016., na temelju verzije koju je u svibnju 2012. donijela opća skupština delegata OIE-a [7].
Directive 2009/156/EC should therefore be amended accordingly.
Direktivu 2009/156/EZ trebalo bi stoga na odgovarajući način izmijeniti.
Annex IV to Directive 2009/156/EC is replaced by the text set out in the Annex to this Decision.
Prilog IV. Direktivi 2009/156/EZ zamjenjuje se tekstom utvrđenim u Prilogu ovoj Uredbi.
Invest., 20, 325-328.
Invest., 20, 325.–328.
2013;189(1):30-35.
2013;189(1):30.-35.
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahc/current/chapitre_ahs.pdf
http://www.oie.int/fileadmin/Home/eng/Health_standards/tahc/current/chapitre_ahs.pdf.
AFRICAN HORSE SICKNESS
KONJSKA KUGA
DIAGNOSIS
DIJAGNOSTIKA
The serological method described hereinafter are enzyme-linked immunosorbent assays (ELISA) based on point 2 of Section B in Chapter 2.5.1 of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Edition 2016 as adopted by the World Assembly of Delegates of the OIE in May 2012.
Serološka metoda koja se opisuje dalje u tekstu jesu imunoenzimni testovi (ELISA) koji se temelje na poglavlju 2.5.1. odjeljku B, točki 2. Priručnika o dijagnostičkim testovima i cjepivima za kopnene životinje, izdanje 2016., koji je donijela opća skupština delegata OIE-a u svibnju 2012.
The VP7 viral protein is an immuno-dominant major antigen of the African horse sickness virus (AHSV) and is conserved across the nine AHSV serotypes.
Virusni protein VP7 glavni je imunološki dominantan antigen virusa konjske kuge (VKK) i sačuvan je u devet VKK serotipova.
Recombinant AHSV-VP7 proteins have been shown to be stable and innocuous and suitable to be used as antigens in ELISA procedures for determination of AHSV antibodies with a high degree of sensitivity and specificity (Laviada et al., 1992b [1]; Maree and Paweska, 2005).
Rekombinirani proteini VKK-VP7 pokazali su se stabilnima, neškodljivima i prikladnima za upotrebu kao antigeni u postupcima ELISA za otkrivanje protutijela VKK-a s velikim stupnjem osjetljivosti i specifičnosti (Laviada et al., 1992b [1]; Maree i Paweska, 2005.).
The indirect ELISA and the blocking ELISA are the two AHS-VP7 ELISA tests suitable for serological diagnosis of African horse sickness (AHS).
Indirektni test ELISA i blokirajući test ELISA dva su KK-VP7 testa ELISA koja su primjerena za serološku dijagnostiku konjske kuge (KK).
Indirect ELISA for the detection of antibodies to African horse sickness virus (AHSV)
Indirektni test ELISA za otkrivanje protutijela za virus konjske kuge (VKK)
The conjugate used in this method is a horseradish peroxidase anti-horse gamma-globulin reacting with the serum of horses, mules and donkeys.
Konjugat koji se upotrebljava u ovoj metodi jest peroksidaza iz hrena anti-konjskog gama-globulina koja reagira sa serumom konja, mula i magaraca.
The method described by Maree & Paweska (2005) [2] uses protein G as conjugate that also reacts with zebra serum.
Metoda koju su opisali Maree i Paweska 2005. [2] kao konjugat upotrebljava protein G koji reagira i sa serumom zebri.
The antigen may be provided by the Centro de Investigación en Sanidad Animal (CISA), Spain, within 4 to 6 months of request.
Centar Centro de Investigación en Sanidad Animal (CISA) iz Španjolske može osigurati antigen u roku od 4 do 6 mjeseci od podnošenja zahtjeva.
Solid phase
Čvrsta faza
Coat ELISA plates with recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6.
Na ELISA plitice nanijeti rekombinantni VKK-4 VP7 razrijeđen u karbonatnom/bikarbonatnom puferu, pH 9,6.
Incubate plates overnight at 4 °C.
Inkubirati plitice preko noći na 4 °C.
Wash the plates five times with distilled water containing 0,01 % (v/v) Tween 20 (washing solution).
Plitice isprati pet puta destiliranom vodom koja sadrži 0,01 % (v/v) Tween 20 (otopina za ispiranje).
Gently tap the plates onto absorbent material to remove any residual wash.
Lagano pritisnuti plitice na upijajući materijal kako bi se uklonili ostaci ispiranja.
Block the plates with phosphate buffered saline (PBS) pH 7,2 + 5 % (w/v) skimmed milk (Nestlé Dry Skim MilkTM), 200 μl/well, for 1 hour at 37 °C.
Plitice blokirati stavljajući u svaku jažicu 200 μl fosfatnog pufera (PBS- phosphate buffered saline) pH 7,2 + 5 % (w/v) obranog mlijeka (Nestlé Dry Skim MilkTM) i ostaviti 1 sat na 37 °C.
Remove the blocking solution and gently tap the plates onto absorbent material.
Ukloniti blokirajuću otopinu i lagano pritisnuti plitice na upijajući materijal.
Test samples
Ispitni uzorci
Serum samples to be tested, and positive and negative control sera, are diluted 1 in 25 in PBS + 5 % (w/v) skimmed milk + 0,05 % (v/v) Tween 20, 100 μl per well.
Uzorci seruma koji se pretražuju, te pozitivni i negativni kontrolni serumi, razrijede se u omjeru 1: 25 u PBS + 5 % (w/v) obranog mlijeka + 0,05 % (v/v) Tween 20, 100 μl po jažici.
Incubate for 1 hour at 37 °C.
Inkubirati 1 sat na 37 °C.
For titration, make a twofold dilution series from 1 in 25 (100 μl/well), one serum per plate column, and do the same with positive and negative controls.
Za titraciju napraviti serije dvostrukog razrjeđenja od 1: 25 (100 μl po jažici), koristeći jedan stupac plitice za svaki serum, te isto učiniti s pozitivnim i negativnim kontrolama.
Conjugate
Konjugat
Dispense 100 μl/well of horseradish-peroxidase (HRP) -conjugated anti-horse gamma-globulin diluted in PBS + 5 % milk + 0,05 % Tween 20, pH 7,2.
U svaku jažicu staviti pipetom po 100 μl anti-konjskih gamaglobulina konjugiranih peroksidazom iz hrena razrijeđenih u PBS-u + 5 % mlijeka + 0,05 % Tween 20, pH 7,2.
Chromogen/Substrate
Kromogen/Supstrat
Add 200 μl/well of chromogen/substrate solution (10 ml of 80,6 mM DMAB (dimethyl aminobenzaldehyde) + 10 ml of 1,56 mM MBTH (3-methyl-2-benzo-thiazoline hydrazone hydrochlorid) + 5 μl H2O2).
U svaku jažicu dodati 200 μl otopine kromogena/supstrata (10 ml 80,6 mM DMAB (dimetil aminobenzaldehid) + 10 ml 1,56 mM MBTH (3-metil-2-benzo-tiazolin hidrazon hidroklorid) + 5 μl H2O2).
Colour development is stopped by adding 50 μl of 3N H2SO4 after approximately 5 to 10 minutes (before the negative control begins to be coloured).
Razvijanje boje zaustaviti dodavanjem 50 μl 3N H2SO4 nakon približno 5 do 10 minuta (prije nego se počne bojiti negativna kontrola).
Other chromogens such as ABTS (2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]), TMB (tetramethyl benzidine), or OPD (ortho-phenyldiamine) can also be used.
Mogu se upotrebljavati i drugi kromogeni kao što su ABTS (2,2′-azino-bis-[3-etilbenzotiazolin-6-sulfonska kiselina]), TMB (tetrametil benzidin) ili OPD (orto-fenildiamin).
Read the plates at 600 nm (or 620 nm).
Očitati plitice na 600 nm (ili 620 nm).
Calculate the cut-off value by adding 0,06 to the value of the negative control (0,06 is the standard deviation derived with a group of 30 negative sera).
Izračunati graničnu vrijednost tako da se vrijednosti negativne kontrole doda 0,06 (0,06 je standardna devijacija dobivena sa skupinom od 30 negativnih seruma).
Test samples giving absorbance values lower than the cut-off are regarded as negative.
Pretraživani uzorci koji daju vrijednost apsorbancije nižu od granične vrijednosti smatraju se negativnima.
Test samples giving absorbance values greater than the cut-off + 0,15 are regarded as positive.
Pretraživani uzorci koji daju vrijednost apsorbancije veću od granične vrijednosti + 0,15 smatraju se pozitivnima.
Test samples giving intermediate absorbance values are considered to be inconclusive and a second technique must be employed to confirm the result.
Pretraživane uzorke koji daju vrijednost apsorbancije između pozitivnih i negativnih smatra se dvojbenima i mora se primijeniti druga metoda kako bi se rezultat potvrdio.
Blocking ELISA for the detection of antibodies to African horse sickness virus (AHSV)
Blokirajući test ELISA za otkrivanje protutijela za virus konjske kuge (VKK)
The competitive blocking ELISA is designed to detect specific AHSV antibodies in sera from animals of any equine species, i.e. horses, donkeys, zebra and their crosses, preventing the problem of specificity experienced occasionally using the indirect ELISAs.
Kompetitivni blokirajući test ELISA namijenjen je otkrivanju specifičnih protutijela za VKK u serumima životinja svih vrsta kopitara, tj. konja, magaraca, zebra i njihovih križanaca čime se sprečava problem specifičnosti do kojeg povremeno dolazi pri upotrebi indirektnih testova ELISA.
The principle of the test is the blocking of the reaction between the recombinant VP7 protein absorbed to the ELISA plate and a conjugated AHS-VP7 specific monoclonal antibody (Mab).
Test se temelji na prekidanju reakcije između rekombinantnog proteina VP7 koji se apsobrira na ELISA pliticu i konjugiranog monoklonskog protutijela (Mpt) specifičnog za KK-VP7.
Antibody in the test sera will block the reaction between the antigen and the Mab resulting in a reduction in colour.
Protutijela u pretraživanim serumima blokirat će reakciju između antigena i monoklonskog protutijela što će dovesti do smanjenja obojenja.
Because the Mab is directed against the VP7, the assay will give a high level of sensitivity and specificity.
Budući da je monoklonsko protutijelo usmjereno i protiv VP7, test pruža visoku razinu osjetljivosti i specifičnosti.
The competitive blocking ELISA is commercially available.
Kompetitivni blokirajući test ELISA dostupan je na tržištu.
Solid Phase
Čvrsta faza
Coat ELISA plates with 50-100 ng of recombinant AHSV-4 VP7 diluted in carbonate/bicarbonate buffer, pH 9,6.
Na ELISA plitice nanijeti 50-100 ng rekombinantnog VKK-4 VP7 razrijeđenog u karbonatnom/bikarbonatnom puferu, pH 9,6.
Incubate overnight at 4 °C.
Inkubirati preko noći na 4 °C.
Wash the plates three times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST).
Plitice isprati tri puta fosfatnim puferom (PBS) 0,1x koji sadrži 0,135 M NaCl i 0,05 % (v/v) Tween 20 (PBST).
Gently tap the plates on to absorbent material to remove any residual wash.
Lagano pritisnuti plitice na upijajući materijal kako bi se uklonili ostaci ispiranja.
Test samples and controls
Pretraživani uzorci i kontrole
Serum samples to be tested, and positive and negative control sera, are diluted 1 in 5 in diluent containing 0,35 M NaCl, 0,05 % (v/v) Tween 20 and 0,1 % Kathon, 100 μl per well.
Uzorci seruma koji se pretražuju, te pozitivni i negativni kontrolni serumi, razrijede se u omjeru 1: 5 u sredstvu za razrjeđivanje koje sadržava 0,35 M NaCl, 0,05 % (v/v) Tween 20 i 0,1 % Kathona, 100 μl po jažici.
For titration, make a twofold dilution series of the test sera from 1 in 10 to 1 in 280 across 8 wells (100 μl/well), one serum per plate column, and do the same with positive and negative controls.
Za titraciju napraviti serije dvostrukog razrjeđenja seruma koje se pretražuju od 1: 10 do 1: 280 u osam jažica (100 μl po jažici), koristeći jedan stupac plitice za svaki serum, te isto učiniti s pozitivnim i negativnim kontrolama.
Wash the plates five times with phosphate buffered saline (PBS) 0,1× containing 0,135 M NaCl and 0,05 % (v/v) Tween 20 (PBST).
Plitice isprati pet puta fosfatnim puferom (PBS) 0,1× koji sadržava 0,135 M NaCl i 0,05 % (v/v) Tween 20 (PBST).
Dispense 100 μl/well of horseradish peroxidase-conjugated Mab anti-VP7.
U svaku jažicu staviti pipetom po 100 μl monoklonskih protutijela protu-VP7 konjugiranih peroksidazom iz hrena.
In advance, this Mab has been diluted 1/5000-1/15000 in a 1/1 solution of StabiliZyme Select® Stabilizer (SurModics. Reference: SZ03) in distilled water.
Prije toga su ta monoklonska protutijela razrijeđena 1/5000 – 1/15000 u otopini 1/1 StabiliZyme Select® Stabilizer (SurModics. Uputa: SZ03) u destiliranoj vodi.
Incubate for 30 minutes at 37 °C.
Inkubirati 30 minuta na 37 °C.
Add 100 μl/well chromogen/substrate solution, i.e. 1 ml of ABTS (2,2′-Azino-bis-[3-ethylbenzothiazoline-6-sulphonic acid]) 5 mg/ml + 9 ml of substrate buffer (0,1 M Phosphate-Citrate buffer of pH 4 containing 0,03 % H2O2), and incubate for 10 minutes at room temperature.
U svaku jažicu dodati 100 μl otopine kromogena/supstrata tj. 1 ml ABTS (2,2′-azino-bis[3-etilbemzotiazolin-6-sulfonska kiselina]) 5 mg/ml + 9 ml supstratnog pufera (0,1 M fosfat-citratni pufer s pH 4 koji sadržava 0,03 % H2O2) i inkubirati 10 minuta na sobnoj temperaturi.
Colour development is stopped by adding 100 μl/well of 2 % (w/v) SDS (sodium dodecyl sulphate).
Razvijanje boje zaustavlja se tako da se u svaku jažicu doda 100 μl 2 % (w/v) SDS-a (natrij dodecil sulfat).
Read at 405 nm in an ELISA reader.
Očitati na 405 nm u ELISA čitaču.
Determine the blocking percentage (BP) of each sample by applying the following formula, where “Abs” stands for antibodies:
Odredite blokirajući postotak (BP) svakog uzorka primjenom sljedeće formule u kojoj ‘Abs’ znači protutijela:
Abscontrol
Abskontrolna
Abssample
Absuzorak
Samples showing a BP value higher than 50 % should be considered as positive for AHSV antibodies.
Uzorke čija je vrijednost blokirajućeg postotka veća od 50 % treba smatrati pozitivnima na protutijela za VKK.
Samples showing a BP value lower than 45 % should be considered as negative for AHSV antibodies.
Uzorke čija je vrijednost blokirajućeg postotka niža od 45 % treba smatrati negativnima na protutijela za VKK.
Samples showing a BP value between 45 % and 50 % should be considered as inconclusive and must be retested.
Uzorke čija je vrijednost blokirajućeg postotka između 45 % i 50 % treba smatrati dvojbenima i za njih se ispitivanje mora ponoviti.
If the result is again inconclusive, the animals should be retested on samples taken not earlier than two weeks after the sample which was considered to be inconclusive was taken.
Ako je rezultat ponovno dvojben, životinje bi trebalo ponovno testirati na uzorcima koji bi se uzeli najranije dva tjedna nakon uzimanja uzorka koji se smatralo dvojbenim.
Identification of the agent
Identifikacija uzročnika bolesti
Real-time Reverse-Transcription Polymerase Chain Reaction (rRT-PCR)
Obrnuta transkripcija lančane reakcije polimerazom u stvarnom vremenu (rRT-PCR)
Agent identification tests based on nucleic acid methods must detect reference strains from the nine virus serotypes of the AHSV.
Testovi za identifikaciju uzročnika bolesti utemeljeni na metodama nukleinske kiseline moraju među devet serotipova virusa VKK-a otkriti referentne sojeve.
The method described in point 2.1 is based on point 1.2 of Section B in Chapter 2.5.1 of the Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Edition 2016 as adopted by the World Assembly of Delegates of the OIE in May 2012.
Metoda opisana u točki 2.1. utemeljena je na poglavlju 2.5.1. odjeljku B, točki 1.2. Priručnika o dijagnostičkim testovima i cjepivima za kopnene životinje, izdanje 2016., koji je donijela opća skupština delegata OIE-a u svibnju 2012.
Any RT-PCR detection method used for the testing of samples, either blood or spleen, in the context of Directive 2009/156/EC must perform equal to or exceed the sensitivity of the methodologies described in point 2.
Svaka metoda otkrivanja transkripcijom lančane reakcije polimerazom koja se upotrebljava za ispitivanje uzoraka krvi ili slezene u kontekstu Direktive 2009/156/EZ mora dati rezultate koji su po osjetljivosti jednaki rezultatima metodologija opisanih u točki 2. ili bolji od njih.
Inactivated virus of serotypes 1 to 9 reference strains may be obtained from the European Union Reference Laboratory or the OIE Reference Laboratory for African horse sickness, Algete, Spain.
Referentni sojevi inaktiviranog virusa serotipa 1 do 9 mogu se nabaviti od Referentnog laboratorija Europske unije ili Referentnih laboratorija OIE-a za bolest konjske kuge u Algeteu, Španjolska.
Extraction of viral RNA
Ekstrakcija virusne RNK
To assure a good reaction it is necessary to extract from the sample an AHSV RNA of high quality.
Kako bi se osigurala dobra reakcija, potrebno je iz uzorka VKK-a ekstrahirati visokokvalitetnu RNK.
The extraction of nucleic acids from clinical samples can be performed by a variety of in-house and commercially available methods.
Ekstrakcija nukleinskih kiselina iz kliničkih uzoraka može se izvesti nizom internih metoda i metoda dostupnih na tržištu.
Commercial kits use different approaches for RNA isolation.
Komercijalna oprema upotrebljava različite pristupe izolaciji RNK.
Most are based on one of the following procedures:
Većina ih se temelji na jednom od sljedećih postupaka:
Phenol-chloroform extraction of nucleic acids;
ekstrakcija nukleinske kiseline fenol-kloroformom,
Adsorption of nucleic acids to filter system;
adsorpcija nukleinskih kiselina filtarskim sustavom,
Adsorption of nucleic acids to magnetic beads system.
adsorpcija nukleinskih kiselina sustavom magnetskih kuglica.
An example of an in-house RNA extraction is given below:
Primjer interne ekstrakcije RNK naveden je u nastavku:
1 g of tissue sample is homogenised in 1 ml of denaturing solution (4 M guanidium thiocyanate, 25 mM sodium citrate, 0,1 M 2-mercaptoethanol, 0,5 % sarcosyl).
1 g uzorka tkiva homogenizira se u 1 ml otopine za denaturiranje (4 M gvanidin tiocijanata, 25 mM natrijeva citrata, 0,1 M 2-merkaptoetanola, 0,5 % sarkozila).
After centrifugation, 1 μg of yeast RNA, 0,1 ml of 2 M sodium acetate pH 4, 1 ml of phenol and 0,2 ml of chloroform/isoamyl alcohol mixture (49/1) are added to the supernatant.
Nakon centrifugiranja supernatantu se dodaju 1 μg kvaščane RNK, 0,1 ml 2 M natrijeva acetata pH 4, 1 ml fenola i 0,2 ml alkoholne mješavine kloroform/izoamila (49/1).
The suspension is vigorously shaken and cooled on ice for 15 minutes.
Otopina se jako protrese i 15 minuta hladi na ledu.
After centrifugation, the RNA present in the aqueous phase is phenol extracted, ethanol precipitated and resuspended in sterile water.
Nakon centrifugiranja, iz RNK prisutne u vodenoj fazi ekstrahira se fenol, taloži etanol i ponovno je se suspendira u sterilnoj vodi.
Real-time RT-PCR Procedure
Postupak RT-PCR-a u stvarnom vremenu
Group-specific real-time RT-PCR by Agüero et al., 2008 [3]
Grupno specifični RT-PCR u stvarnom vremenu, Agüero et al., 2008. [3]
This group-specific real-time RT-PCR targets VP7 of the AHSV and is able to detect all known AHSV serotypes and strains currently circulating.
Ovaj grupno specifični RT-PCR u stvarnom vremenu usredotočen je na VP7 VKK-a te se njime može otkriti sve poznate serotipe i sojeve VKK-a koji trenutačno kruže.
Primer and probe sequences for the detection of AHSV species viruses:
Nizovi početnica i sondi za otkrivanje virusa vrste VKK:
forward Primer 5′-CCA-GTA-GGC-CAG-ATC-AAC-AG-3′
uzvodna početnica 5′-CCA-GTA-GGC-CAG-ATC-AAC-AG-3′
reverse Primer 5′-CTA-ATG-AAA-GCG-GTG-ACC-GT-3′
nizvodna početnica 5′-CTA-ATG-AAA-GCG-GTG-ACC-GT-3′
MGB-TaqMan probe 5′-FAM-GCT-AGC-AGC-CTA-CCA-CTA-MGB-3′
sonda MGB-TaqMan 5′-FAM-GCT-AGC-AGC-CTA-CCA-CTA-MGB-3′
Primer stock concentration is diluted to a working concentration of 8 μM (“primer working stock 8 μM”) whereas probe is diluted to a working concentration of 50 μM (“probe working stock 50 μM”).
Radna koncentracija početnica razrijeđena je na radnu koncentraciju od 8 μM (‘radna zaliha početnica 8 μM’), a sonda je razrijeđena na radnu koncentraciju od 50 μM (‘radna zaliha sonde 50 μM’).
A test plate layout should be designed and loaded into the real time PCR machine software.
Raspored ispitne plitice potrebno je osmisliti i učitati u softver stroja za PCR u stvarnom vremenu.
Using the layout as a guide, 2,5 μl of each primer working stock 8 μM is added to each well that will contain RNA samples, positive and/or negative controls (final concentration of the primer will be 1 μM in the 20 μl RT-PCR mix).
Upotrebljavajući raspored kao vodilju 2,5 μl svake radne zalihe početnica 8 μM dodaje se svakoj jažici koja će sadržavati uzorke RNK, pozitivne i/ili negativne kontrole (konačna koncentracija početnice bit će 1 μM u 20 μl mješavine RT-PCR-a).
The plate is held on ice.
Plitica se čuva na ledu.