Establishment Of Single-tube Nested PCR As Well As ITS1and529bp Sequence Analysis Of Toxoplasma Gondii In Dairy Goat

Toxoplasmosis is a disease caused by Toxoplasma gondii. T. gondii can infect virtuallyall warm-blooded animals. Actually, it is one of the most common parasites. The protozoanparasite Toxoplasma gondii is a serious cause of fetal mortality in goats. Oocysts, the parasitestage responsible for initiating infection, arc produced following a primary infection in cats. Aprimary infection in pregnant sheep and goats can establish a placental and fetal infectionwhich may result in fetal death and resorption, abortion or stillbirth. Thus it is essential todiagnose toxoplasmosis timely and accurately.Etiological detection method is both time-consuming and low sensitivity, and serologicaldetection may results false negative or false positive. In molecular biology diagnosis,conventional PCR has lower sensitivity, by contrast, nested PCR has both higher sensitivityand specificity, but the operation is complex and more likely to contaminate. For the sake ofreducing the likelihood of contamination and simplifying the test procedure, single tubenested PCR targeting529bp repetitive sequence was established and applied to the bloodsample of dairy goat in this study. The results are as follows:PCR successfully amplified a single band of the expected size (216bp) from positivecontrol. No amplicon was generated from Amoebida, Giardia lamblia, Cryptosporidiaparvum or negative control. Analysis of samples from serial dilutions of T. gondii RH puregenomic DNA established a detection limit of the PCR assay of1fg.A total of137samples of dairy goat blood were analyzed by single-tube nested PCR, ofwhich38samples were positive.ITS1and529bp repetitive sequence was analyzed by sequence alignment and the resultsuggested that the T. gondii in dairy goat was close related to RH strain.