| Schistosomiasis is a serious zoonotic parasitic disease caused by schistosoma infection and is distributed in 78 tropical and subtropical countries and areas.Schistosomiasis japonica is endemic in China,over the past few decades,the prevention and control of schistosomiasis in China has made remarkable achievements.The prevalence of schistosomiasis among humans and livestocks in most regions of China has shown the characteristics of low epidemic rate and low infection.The purpose of this study is to establish a highly sensitive and specific nucleic acid diagnosis method for schistosomiasis japonica.On the basis of previous studies and bioinformatics analysis,G01,F11,E12,H12,G1/3,G7-1,G7-2 and G7-3 were selected as candidate genes for diagnosis in this paper,and primers were designed respectively.Genomic DNA of Schistosoma japonicum,BALB/c mice and New Zealand white rabbits were respectively used as templates for PCR amplification.Genes which could be specifically amplified by genomic DNA of S.japonicum but could not be amplified target fragments from genomic DNA of BALB/c mice and New Zealand white rabbits were selected.At last,G01 was picked out and used as the diagnostic target gene.Three different infection doses(10~,40~,100~)infected 10 mice respectively,and nucleic acid extracted from the plasma on 42 d after infection as the template for PCR amplification.The results showed all of which could be amplified G01 fragment.At the same time,G01 was amplified using the genomic DNA of Toxoplasma Gondii,Fasciola gigantica,Paramphistomum,Sarcocystis cruzi,Trichina and Echinococcus multilocularis as templates,respectively.There was no positive reaction.It was indicated that G01 gene,could be a potential target gene for diagnosis of schistosomiasis japonica.The primers,annealing temperature,template amount,and plasma volumn were optimized,respectively.The G01-4 primers which had a single characteristic peak and small Ct value was selected as the final primers.Finally,the annealing temperature of 58℃,4 μL template amount and the 200 μL plasma volumn were used to establish the Real-time PCR diagnostic method,which can detect only 0.01 fg of the target gene.The sensitivity of Real-time PCR to detecte the mice plasma samples collected at 42 d after infection with S.japonicum cercariae was 99.3 %(152/153).77 negative plasma samples of SPF BALB/c mice without cercariae infection were all detected as negative,which showed the specificity of 100%(77/77).The results indicated that this method possessed the high sensitivity and specificity.Plasma samples of mice infected with 10 and 40 S.japonicum cercariae at different time were detected using the established Real-time PCR diagnostic method.The results showed that the positive rates of mice infected with 10 cercariae were: 40%(8/20),60%(12/20),30%(6/20),40%(8/20),20%(4/20),60%(12/20),60%(12/20)and 95%(19/20)respectively at 7d,10 d,14d,18 d,21d,28 d,35d and 42 d after infection.The positive rates of mice infected with 40 cercariae were 25%(2/8)、80%(4/5)、50%(4/8)、40%(2/5)、100%(8/8)、100%(5/5)at 10 d,14d,18 d,21d,28 d and 35 d after infection.The highest positive rate was 95 % in mice infected with 10 cercariae at 42 d after infection.All mice infected with 40 cercariae were detdcted as positive 28 d after infection.The results showed that the sensitivity of the method was positively correlated with the infection intensitity.The established Real-time PCR diagnostic method was used to weekly monitor the positive rate of mice treated by praziquantel after infected with S.japonicum.The results showed that mice infected with 10 and 40 cercariae were all negative at 6 w after praziquantel treament.It was indicated that this method had a great potential to distinguish current infection and past infection.This study provides a sensitive and specific diagnostic technique for the control of schistosomiasis japonicum. |
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