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

Procjena osjetljivosti lančane reakcije polimerazom za dokaz virusa anemije u pilića uporabom različitih početnica za tri gena.

Chicken Anaemia Virus (CAV) genes cloned plasmid DNA templates (pCR4-CAV-VP1 and pCR4-CAVVP2& 3) were used in this study to develop a sensitive polymerase chain reaction (PCR) for CAV gene detection. A total of nine sets of primers, which include one set of published primers and two sets of designed primers for each gene of CAV, viz. VP1, VP2 and VP3, were used to assess the sensitivity of PCR. The PCR cycle conditions were standardized with the designed primers to get a single, specific sized amplicon for each gene separately. PCR sensitivity assessment was done by making serial 10 fold dilutions of the cloned CAV plasmid templates and subjected to PCR with each set of primers for each gene of CAV. The highest dilution of the CAV plasmid DNA showing a visible PCR amplicon was taken as the detection limit. The results showed that the designed primer VP1.2 was found to be more sensitive for the VP1 gene and the concentration of the plasmid DNA was 0.05 fg/μL or 8.6 ×103 molecules/mL and VP 2.2 was found to be more sensitive for the VP2 gene and the concentration of the plasmid was 5 ag/μL or 9.9 ×102 molecules/mL. In the case of VP3, the published primer VP 3.1 was found to be more sensitive for the VP3 gene and the concentration of the plasmid was 5 ×10-4 ag/μL or 10.5 ×10-2 molecules/mL. The findings of this study may be very useful for diagnostic, sequencing, cloning and expression purposes.Matrice gena virusa anemije pilića klonirane u plazmidnoj DNA (pCR4 CAV-VP1 te pCR4-CAV-VP2 i 3) bile su rabljene radi razvijanja osjetljive lančane reakcije polimerazom (PCR) za njihovo dokazivanje. Za procjenu osjetljivosti PCR-a bilo je rabljeno devet kompleta početnica među kojima je bio jedan komplet već objavljenih i dva kompleta sintetiziranih početnica za svaki gen virusa anemije pilića, odnosno za VP1, VP2 i VP3. Uvjeti PCR-a za umnožavanje bili su standardizirani sa sintetiziranim početnicama kako bi se dobio pojedinačni specifični umnožak za svaki gen. Osjetljivost PCR-a bila je procijenjena na osnovi deseterostrukih serijskih razrjeđenja kloniranih plazmidnih matrica u reakciji rabljenih u svakom kompletu početnica za svaki gen virusa. Najveće razrjeđenje plazmidne DNA virusa koje je pokazivalo jasno vidljiv umnožak bilo je uzeto kao krajnja mogućnost dokaza. Rezultati su pokazali da je proizvedena početnica VP1.2 bila osjetljivija za gen VP1, a koncentracija plazmidne DNA iznosila je 0,05 fg/μL ili 8,6 ×103 molekula/mL. Početnica VP2.2 bila je osjetljivija za gen VP2, a koncentracija plazmida bila je 5 ag/μL ili 9,9 ×102 molekula/mL. U slučaju VP3, objavljena početnica VP3.1 pokazala se osjetljivijom za gen VP3, a koncentracija plazmida iznosila je 5 ×10-4 ag/μL ili 10,5 ×10-2 molekula/mL. Nalazi ovog istraživanja mogu biti korisni u dijagnostici, sekvencioniranju, kloniranju i ekspresiji

A study on Methicillin resistant Staphylococcus aureus mastitis in dairy cows

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious problem in dairy animals suffering from mastitis. The study was carried out to evaluate the incidence of Methicillin resistant S. aureus from clinical mastitis milk samples and their antibiotic resistance profile and characterised with respect to the molecular features that contributed to the resistance in these pathogens. Isolation and identification of Methicillin resistant S. aureus were performed from acute clinical mastitis samples. The isolates were tested using agar disc diffusion method for their antimicrobial susceptibility and modified resazurin assay micro dilution technique for MIC to 8 different antimicrobial drugs. A total of 235 clinical mastitis milk samples from dairy cows were cultured for incidence of S. aureus. Methicillin resistant S. aureus was isolated from a total of 12 (44.25%) of the 116 S. aureus samples. Based on the antimicrobial sensitivity and MIC results, MRSA isolates were found sensitive to gentamicin, enrofloxcain, amoxicillin+sulbactam, ceftriaxone and resistant to amoxicillin, oxytetracycline, penicillin G and oxacillin. Most of MRSA isolates were found to be multi-drug resistant. MRSA alert kit test and mecA and blaZ target gene PCR were found to be useful in the confirmation of MRSA

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

Are rhetorical commitments to adolescents refected in planning documents? An exploratory content analysis of adolescent sexual and reproductive health in Global Financing Facility country plans

The Global Financing Facility (GFF) offers an opportunity to close the financing gap that holds back gains in women, children's and adolescent health. However, very little work exists examining GFF practice, particularly for adolescent health. As momentum builds for the GFF, we examine initial GFF planning documents to inform future national and multi-lateral efforts to advance adolescent sexual and reproductive health.We undertook a content analysis of the first 11 GFF Investment Cases and Project Appraisal Documents available on the GFF website. The countries involved include Bangladesh, Cameroon, Democratic Republic of Congo, Ethiopia, Guatemala, Kenya, Liberia, Mozambique, Nigeria, Tanzania and Uganda

Does a perception of increased blood safety mean increased blood transfusion? An assessment of the risk compensation theory in Canada

BACKGROUND: The risk compensation theory is a widely used concept in transport economics to analyze driver risk behaviour. This article explores the feasibility of applying the theory in blood transfusion to raise important questions regarding the increased blood safety measures and their possible effects on blood usage (e.g., the appropriateness in transfusion). Further, it presents the findings of a pilot survey of physicians in Canada. DISCUSSION: While studies have attempted to define transfusion appropriateness, this article argues that if the risk compensation theory holds true for transfusion practice, physicians may actually be transfusing more. This may increase the possibility of contracting other unknown risks, such as the variant Creutzfeldt-Jakob Disease (vCJD), as well as increasing the risk of non-infectious transfusion risks, such as transfusion reactions. SUMMARY: A much larger study involving psychosocial assessment of physician decision making process to fully assess physician behaviour within the context of risk compensation theory and transfusion practice in Canada is needed to further explore this area