Nucleic Acid Assay For Early Diagnosis And Therapy Evaluation Of S.japonicum Infection In Hosts

In order to observe the stability of PCR assay for early diagnosis and therapy evaluation in S. japonicum infection, PCR assay was repeatedly used to detect different samples from rabbit infected with S. japonicum, including adult worms, liver homogenate, eggs, feces and sera. The results showed that a specific DNA fragment of S. japonicum was amplified by PCR when the different samples DNA was used as templates, and had no cross-reactivity between S. mansoni and C. sinensis. The minimum amounts of DNA detectable using PCR assay were 800fg/μl. As to the early diagnosis and therapy evaluation, PCR assay could detect the specific DNA at the 1st week post-infection, and then the DNA in serum became negative at the 12th week post-treatment. These data were consistent with the former findings.On the basis of PCR assay, a new nucleic acid assay was developed and evaluated in this research, which was called Loop-mediated isothermal amplification (LAMP). It had provided higher sensitivity and specificity than PCR assay. The minimum amounts of DNA detectable using LAMP assay was 0.08fg/μl, which was 104 times more sensitive than PCR assay. Similarly, no cross-reactivity was observed between S. mansoni and C. sinensis. And the results of LAMP detection, in the sera of rabbit infected with S. japonicum from 1w to 30w, were compared with PCR assay. LAMP could also amplify the specific DNA fragment in sera at the 1st week post-infection for the early diagnosis, and the DNA detection became negative at the 13th week post-treatment for the therapy evaluation. One week later than PCR assay, due to its higher sensitivity. However, LAMP assay was easy to perform, reacted rapidly and was inexpensive, it may therefore be applied in the routine diagnosis in human.In order to investigate the source of nucleic acid in the sera of host infected with S. japonicum, single or dual sex cercariae of S. Japonicum infected mice models were set up for further research. And the results showed that in the early infectious period, the nucleic acids may mainly come from schistosomula and the epidermis tissue of worm, however, in the acute and chronic stages, they may essentially come from tissues with broken or dead eggs. It concluded that nucleic acid assays were useful methods for early detection, and had potential value for therapy evaluation.At the base of the encouraging results from animal model, both methods were used to detect 30 cases sera of patients, and the results showed that PCR assay could detect 18 positive cases(positive rate was 60.0%), while LAMP could detect 29 positive cases(positive rate was 96.7%). All these data suggested that LAMP assay was more sensitive than PCR assay, meanwhile it was easy to perform, and the results were intuitionistic, therefore it is a potential tool for early diagnosis and therapy evaluation in S. japonicum infection.