full issue - Association of Biotechnology and Pharmacy

Current Trends in Biotechnology and PharmacyVol. 5 (2) 1123-1129 April 2011. ISSN 0973-8916 (Print), 2230-7303 (Online)1125monolayers were frozen at -20 o C, then thawedat 37 o C, and the inclusions were disrupted byshaking with glass beads for 1 min beforecentrifugation at 5000x g for 10 min at 4 o C. Thesupernatant was collected and the pellets wereresuspended in sucrose phosphate glucose (SPG). The suspension was centrifuged again afterbrief sonication. The supernatant was pooled andcentrifuged at 30,000 g for 30 min at 4 o C (Sorvallinstrument, Dupoint, USA). The pellet obtainedafter ultracentrifugation was used to inoculatefresh McCoy cell monolayers. After threeinoculation cycles in fresh cells, the media andcell pellet were tested for chlamydia-specificlipopolysaccharide (LPS) by using the ClearviewChlamydiae test kit (Unipath, inc., Nepean,Ontario, Canada). Direct faecal samples smearswere examined for the presence of MZN-positiveelementary by Modified Zeihl Nelson (MZN)stain.Immunological TechniqueEnzyme linked immunosorbent assay(ELISA): The ELISA test was performed onserum samples collected before insertion ofprogesterone sponge insertion and 13 days later(sponge removal and PMSG injection).Polymers Chain Reaction (PCR)DNA extraction of vaginal and faecal swabs:The swabs collected in Chlamydia transportmedium (CTM) were centrifuged at 2000 rpmfor 10 minutes. About 400 µl of the supernatantwas then pelleted by centrifugation for 10 minuteat 1200xg. After resuspending the pellet in 180µl of ATL solution (supplied in the QIAamp DNAmini kit) and adding 20 µl of proteinase K, thesamples were incubated at 65 o C overnight. Themicro-centrifuge tube was centrifuged beforeadding 200 µl buffer AL to the sample, then vortexmixed for 15 sec and incubated at 70 o C for 10min. The tube was centrifuged and 200 µl ofethanol (96-100%) was added and mixed. Themixture was then carefully poured in the QIAampspin column . The cap was then closed andcentrifuged at 6000 x g for 1 min. The QIAampspin column was placed in a clean 2 ml collectiontube and the other tube containing the filtratediscarded. 500 µl of buffer AW1 was added andcentrifuged at 6000 x g for 1 min and the tubecontaining the filtrate was discarded. Then, 500µl of Buffer AW2 was added and centrifuged at20,000 x g for 3 minute and repeated as above.200 µl of Buffer AE or distilled water was added.After incubation at room temperature for 1-5 min,the mixture was centrifuged at 6000 x g for 1 minand the DNA extract was stored at -20 o C untilused.DNA amplification: DNA was amplified asdescribed by Creelan and McCullough (2000),using Clone 8 forward primer, 5’ TGG TAT TCTTGC CGA TGA C 3’; and clone 8 reverse primer,5’ GAT CGT AAC TGC TTA ATA AAC CG 3’for the omp gene of Chlamydophila abortus..3 µl of the DNA sample was added to 47 µl of areaction mixture containing 1xPCR super mix(Life Technologies (20 mM Tris-HCl pH 8.4, 5mM KCl, 1.5mM MgCl 2), 200 µMdeoxynucleoside triphosphates, 2 Units Taq DNApolymerase and 1 mM of each primer. Thereaction mixture was overlaid with 30 µl of mineraloil and initial denaturation at 95 o C for 5 minutesfollowed by 40 cycles of; 1 minute of at 94 o C,1min of at 45 o C, 2 minutes at 72 o C and final 7min extension at 72 o C.The amplified product was analysed bysubjecting 5 of PCR products to electrophoresisfor 45 minute at 100 V on a 1 percent agarosegel. The gel was then stained with ethidiumbromide and illuminated by ultraviolet light.Molecular weight markers X 174(Sigma) wereincluded in every gel.Studies on carrier state of Chlamydophila abortus