Dokaz virusa newcastleske bolesti molekularnim postupkom FTA-PCR

The feasibility of using Flinders Technology Associates (FTA) filter papers to store the Newcastle disease virus (NDV), infected allantoic fluid (AF) and tissue samples, for the molecular detection of NDV by reverse transcriptase - polymerase chain reaction (RT-PCR) - was investigated. An FTA card is a cotton based cellulose membrane, with lyophilized chemicals that lyse the viruses and bacteria. The viral RNA was detectable from FTA cards up to a concentration of 107.6 EID50/100 μL (a 100 times dilution of 109.6 EID50/100 μL of initial stock). The inactivated virus remained stable on the cards for up to 30 days, both at room temperature and 4 ºC. NDV was detected by RT-PCR from all the FTA imprints of the caecal tonsils, kidney, proventriculus, spleen, trachea, faecal swabs and intestinal lesions of NDV-suspected birds. NDV was inactivated upon contact with FTA, as shown by the inability of the virus to propagate in embryonated eggs and its inability to infect chicken embryo fibroblast culture. In conclusion, FTA cards are suitable for collecting and transporting NDV infected samples, without cold storage. The virus inactivated in FTA cards, however, is a suitable source of viral RNA for molecular detection and characterization.Istražena je mogućnost uporabe Flinders tehnologije s filtrirnim papirom za pohranjivanje alantoisne tekućine zaražene virusom newcastleske bolesti i uzoraka tkiva radi molekularnog dokazivanja toga virusa lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Flinders kartica celulozna je membrana pripravljena na osnovi pamuka s liofiliziranim kemikalijama koje liziraju viruse i bakterije. Virusna RNA mogla se na takvoj kartici dokazati u količini od 107,6 EID50/100 μL (100 puta manje razrjeđenje od 109,6 EID50/100 μL osnovne suspenzije). Inaktivirani virus bio je stabilan na karticama tijekom 30 dana pri sobnoj temperaturi i 4 ºC. Virus je bio dokazan RT-PCR-om u svima Flinders tehnologijom pripravljenim otiscima cekalnih tonzila, bubrega, voljke, slezene, dušnika, obriscima fecesa i crijevnih lezija ptica sumnjivih na newcastlesku bolest. Virus je bio inaktiviran nakon dodira s FT fifi ltrirnim papirom što je bilo potvrđeno činjenicom da se nije više mogao uzgojiti u kokošjim embrijima ni na kulturi fibroblasta podrijetlom od kokošjih embrija. Zaključuje se da su FT papirići prikladni za uzimanje i prijenos uzoraka koji sadrže virus newcastleske bolesti bez potrebe pohranjivanja u hladnom prostoru. Virus inaktiviran na FT papiriću (kartici) odgovarajući je izvor virusne RNA za njegov molekularni dokaz i identifikaciju

Dokaz virusa newcastleske bolesti molekularnim postupkom FTA-PCR

The feasibility of using Flinders Technology Associates (FTA) filter papers to store the Newcastle disease virus (NDV), infected allantoic fluid (AF) and tissue samples, for the molecular detection of NDV by reverse transcriptase - polymerase chain reaction (RT-PCR) - was investigated. An FTA card is a cotton based cellulose membrane, with lyophilized chemicals that lyse the viruses and bacteria. The viral RNA was detectable from FTA cards up to a concentration of 107.6 EID50/100 μL (a 100 times dilution of 109.6 EID50/100 μL of initial stock). The inactivated virus remained stable on the cards for up to 30 days, both at room temperature and 4 ºC. NDV was detected by RT-PCR from all the FTA imprints of the caecal tonsils, kidney, proventriculus, spleen, trachea, faecal swabs and intestinal lesions of NDV-suspected birds. NDV was inactivated upon contact with FTA, as shown by the inability of the virus to propagate in embryonated eggs and its inability to infect chicken embryo fibroblast culture. In conclusion, FTA cards are suitable for collecting and transporting NDV infected samples, without cold storage. The virus inactivated in FTA cards, however, is a suitable source of viral RNA for molecular detection and characterization.Istražena je mogućnost uporabe Flinders tehnologije s filtrirnim papirom za pohranjivanje alantoisne tekućine zaražene virusom newcastleske bolesti i uzoraka tkiva radi molekularnog dokazivanja toga virusa lančanom reakcijom polimerazom uz prethodnu reverznu transkripciju (RT-PCR). Flinders kartica celulozna je membrana pripravljena na osnovi pamuka s liofiliziranim kemikalijama koje liziraju viruse i bakterije. Virusna RNA mogla se na takvoj kartici dokazati u količini od 107,6 EID50/100 μL (100 puta manje razrjeđenje od 109,6 EID50/100 μL osnovne suspenzije). Inaktivirani virus bio je stabilan na karticama tijekom 30 dana pri sobnoj temperaturi i 4 ºC. Virus je bio dokazan RT-PCR-om u svima Flinders tehnologijom pripravljenim otiscima cekalnih tonzila, bubrega, voljke, slezene, dušnika, obriscima fecesa i crijevnih lezija ptica sumnjivih na newcastlesku bolest. Virus je bio inaktiviran nakon dodira s FT fifi ltrirnim papirom što je bilo potvrđeno činjenicom da se nije više mogao uzgojiti u kokošjim embrijima ni na kulturi fibroblasta podrijetlom od kokošjih embrija. Zaključuje se da su FT papirići prikladni za uzimanje i prijenos uzoraka koji sadrže virus newcastleske bolesti bez potrebe pohranjivanja u hladnom prostoru. Virus inaktiviran na FT papiriću (kartici) odgovarajući je izvor virusne RNA za njegov molekularni dokaz i identifikaciju

Brzi lateks aglutinacijski test za serološki dokaz serotipa 4 ptičjeg adenovirusa upotrebom rekombinantnog antigena

Hydropericardium hepatitis syndrome (HPS) is a newly emerging disease of poultry, which is caused by fowl adenovirus serotype 4. The virus was propagated in a primary chicken embryo liver culture. The cytopathic effect was observed from the third passage onwards. DNA was isolated from the infected culture and used as a template for amplification of a partial hexon gene (700 bp) using hexon gene specific primers. The amplified product was cloned into pProEX HT b vector and the ligated product was transformed into DH5α cells. The recombinant clones were analyzed by colony PCR and plasmid isolation, followed by restriction digestion to check the insert release. The positive clones were induced by IPTG. The induced culture fractions were checked at different hours and the induction was high at the 4th hour onwards. The expressed proteins were purified and confirmed by using hyperimmune serum against FAV4 by western blot analysis and the protein size of 50kda was obtained. The purified recombinant FAV4 protein was used as a serodiagnostic agent using enzyme linked immunosorbent assay and latex agglutination test.Sindrom hidroperikarda i hepatitisa nova je bolest peradi uzrokovana serotipom 4 ptičjeg adenovirusa. Virus je bio umnožen u primarnoj staničnoj kulturi podrijetlom od jetrenoga tkiva pilećega zametka. Citopatski učinak javio se nakon treće pasaže. DNK je bila izdvojena iz zaražene kulture i rabljena kao kalup za umnožavanje dijela gena heksona (700 bp) uz upotrebu specifičnih početnica. Umnoženi odsječak bio je kloniran u vektoru pProEX HT b, a proizašli proizvod prebačen u DH5α stanice. Rekombinantni klonovi bili su analizirani lančanom reakcijom polimerazom i izdvajanjem plazmida nakon čega je pomoću restrikcijske digestije provjerena uspješnost postupka. Pozitivni klonovi bili su inducirani pomoću IPTG-a. Frakcije induciranih stanica bile su provjeravane svakog sata, a indukcija je bila velika nakon četiri sata. Proizvedene bjelančevine bile su pročišćene i identificirane uporabom hiperimunoga seruma za FAV4 Western blot analizom te se pokazalo da je proizvedena bjelančevina veličine 50 kda. Pročišćena rekombinantna bjelančevina FAV4 bila je rabljena kao antigen u imunoenzimnom testu i lateks aglutinacijskom testu

Brzi lateks aglutinacijski test za serološki dokaz serotipa 4 ptičjeg adenovirusa upotrebom rekombinantnog antigena

Hydropericardium hepatitis syndrome (HPS) is a newly emerging disease of poultry, which is caused by fowl adenovirus serotype 4. The virus was propagated in a primary chicken embryo liver culture. The cytopathic effect was observed from the third passage onwards. DNA was isolated from the infected culture and used as a template for amplification of a partial hexon gene (700 bp) using hexon gene specific primers. The amplified product was cloned into pProEX HT b vector and the ligated product was transformed into DH5α cells. The recombinant clones were analyzed by colony PCR and plasmid isolation, followed by restriction digestion to check the insert release. The positive clones were induced by IPTG. The induced culture fractions were checked at different hours and the induction was high at the 4th hour onwards. The expressed proteins were purified and confirmed by using hyperimmune serum against FAV4 by western blot analysis and the protein size of 50kda was obtained. The purified recombinant FAV4 protein was used as a serodiagnostic agent using enzyme linked immunosorbent assay and latex agglutination test.Sindrom hidroperikarda i hepatitisa nova je bolest peradi uzrokovana serotipom 4 ptičjeg adenovirusa. Virus je bio umnožen u primarnoj staničnoj kulturi podrijetlom od jetrenoga tkiva pilećega zametka. Citopatski učinak javio se nakon treće pasaže. DNK je bila izdvojena iz zaražene kulture i rabljena kao kalup za umnožavanje dijela gena heksona (700 bp) uz upotrebu specifičnih početnica. Umnoženi odsječak bio je kloniran u vektoru pProEX HT b, a proizašli proizvod prebačen u DH5α stanice. Rekombinantni klonovi bili su analizirani lančanom reakcijom polimerazom i izdvajanjem plazmida nakon čega je pomoću restrikcijske digestije provjerena uspješnost postupka. Pozitivni klonovi bili su inducirani pomoću IPTG-a. Frakcije induciranih stanica bile su provjeravane svakog sata, a indukcija je bila velika nakon četiri sata. Proizvedene bjelančevine bile su pročišćene i identificirane uporabom hiperimunoga seruma za FAV4 Western blot analizom te se pokazalo da je proizvedena bjelančevina veličine 50 kda. Pročišćena rekombinantna bjelančevina FAV4 bila je rabljena kao antigen u imunoenzimnom testu i lateks aglutinacijskom testu