Dokaz virusa newcastleske bolesti molekularnim postupkom FTA-PCR

Abstract

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