Abstract
Currently the diagnosis of schistosomiasis is mainly determined by observing the presence of eggs in host stool samples. Because of the overwhelming number of impurities in the stool, eggs are rarely observed. Therefore, the stool hatching method is used to observe the miracidia in the water. However, the miracidia of Schistosoma japonicum are small and difficult to detect, and missed detection is likely to occur when the infection level is low. In this study, recombinant streptococcal protein G-enhanced green fluorescent protein (rSPG-EGFP) was expressed, purified, and used as a fluorescence staining reagent for miracidia. A preliminary miracidium fluorescence staining method based on antigen and antibody bindingwas established by combining recombinant protein staining with the stool hatching method. The stool hatching method was used to collect the miracidia of S. japonicum, and Schistosoma-positive serum and the recombinant protein were mixed to assess the feasibility of fluorescence staining of miracidia. The miracidia of S. japonicum and Schistosoma turkestanicum were incubated with S. japonicum-positive serum and S. turkestanicum-positive serum, respectively, to identify miracidia species. When the fluorescence staining method was used to observe living miracidia, the miracidiawere labelled by the recombinant protein, and their motility status was not affected.
Highlights
The diagnosis of schistosomiasis is mainly determined by observing the presence of eggs in host stool samples
The reconstructed prokaryotic expression plasmid pET28a ( +)-rSPG-enhanced green fluorescent protein (EGFP) was characterized by polymerase chain reaction (PCR) and double digestion (Fig. 2)
The PCR and digestion results were consistent with the expected sizes of the fragments, and the sequencing result was consistent with the designed sequence
Summary
The diagnosis of schistosomiasis is mainly determined by observing the presence of eggs in host stool samples. The main method for diagnosing schistosomiasis is an aetiological diagnosis, which includes a faecal examination and stool hatching and uses the observation of schistosome eggs or miracidia as the basis for the diagnosis of s chistosomiasis[5]. The antigen present on the surface of miracidia can be used to artificially label miracidia with a fluorescent protein through antigen and antibody reactions, increasing the sensitivity of the detection of miracidia in the stool hatching method and facilitating identification. In the present study, a fusion protein consisting of the IgG-binding domains of SPG (C3 domain)and enhanced green fluorescent protein (EGFP) was constructed. RSPG-EGFP retained both activities: the IgG-binding capability of SPG and fluorescence activity of EGFP Using this recombinant protein, specific schistosome antibodies are bound and labelled at the surface of miracidia. The aim of this study was to improve the sensitivity of miracidium observationsin the stool hatching test, reduce the methodological difficulties encountered by researchers, improve the accuracy of diagnosis, and reduce the rate of missed detection
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