Optimization Of Dyeing Methods Of Eimeria Oocysts And Single Oocyst PCR Amplification

Reports about applications of dyeing in coccidia have been deficient.In this assay we developed dyeings used frequently in parasites in EmeriaAenella in the avian faeces,which included Wright Stain,Gimesa Stain, Ziehl-Neelson Stain, Acid-Fast Stain and Auramine-Phone Stain.And it was identified by the experiment that oocysts of E. tenella could be stained easier than the others by using Ziehl-Neelson Stain, Acid-Fast Stain and Auramine-Phone Stain.And when there were 130 oocysts in the faeces per gram ,they can be found by the three methods infered. So we found dyeings were more insensitive the standard flotation techniques when there were a few oocysts in the faeces.The essay refered to these measures used in animals, plants, fungus and bacterias, and the feature of the structure of the Eimeria,and attempted to develop seven methods to extract DNA repeatively of Eimeria to find the most effective and practical one. We designed 5 different numbers of oocysts,which were 10⁵·mL^-1, 10⁴·mL^-1, 10³Â·mL^-1, 10²Â·mL^-1, the lowest one was 50·mL^-1, but it only can be identified by 10²Â·mL^-1. We analysed the datas using the softwairs of SPSS, and found using CTAB, Lysozyme and Glass-Beads being the betters in all the methods when oocysts reaching 10²-10⁵, but when number going beyond it, using improved commerce kits being better.Based on 18S rDNA gene, we developed the primers and nested primers which were special with Eimeria. spp. Then the templates we had got from different DNA extract methods were amplified by PCR. PCR products were analyzed on 1% agarose gel and visualized by ethidium bromide staining.One of the positive PCR which was selected randomly was purified, then cloned. The cloned product was sent to be sequen ced.The sequence then was aligned with Eimeria tenella 18S gene sequences which were loaded down from Gene Bank, and identified by inheritance distance analysis and homology analysis. At last, we found it just was Eimeria tenella that had little variation with submissioned sequences.We evaluated the effect setting PCR facilitate factors just like BSA with deep concentration.It was identified BSA with deep concentration can improve quality of PCR product. As to single Eimeria tenella oocyst, we used frequent freezing and melting, CTAB, single-cell- alkali-lysising, single-cell-protease. We used Glass paper-Transfer to isolate Eimeria tenella. When simultaneously 30 oocysts were amplified after different DNA extraction method, none of target products were got by frequent freezing and melting method and CTAB method, but 6 specimens were got by single-cell-alkali-lysising, 2 specimens were got by single-cell-protease. And we found single-cell-alkali-lysising being better than others obviously by using one-way-AVONA analysis with SPSS. Then the templates we had got from different DNA extract methods were amplified by PCR. PCR products were analyzed on 1% agarose gel and visualized by ethidium bromide staining.One of the positive PCR which was selected randomly was purified, then cloned. The cloned product was sent to be sequenced.The sequence then was aligned with Eimeria tenella 18S gene sequences which were loaded down from Gene Bank, and identified by inheritance distance analysis and homology analysis. At last, we found it just was Eimeria tenella that had little variation with submissioned sequences.At the same time, we amplied oocysts that unsporulated, but we found there were not any remarkable diversity between diferent development stages of Eimeria. We developed single oocyst PCR predure, offered pratical tools for further study about Eimeria and other Coccidia.