A cleaved amplified polymorphic sequence (CAPS) method for the identification of geographical isolates of Schistosoma japonicum in China.
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ABSTRACT:
SUBMITTER: Li J
PROVIDER: S-EPMC4100313 | biostudies-literature | 2011 Oct
REPOSITORIES: biostudies-literature
Publications
A cleaved amplified polymorphic sequence (CAPS) method for the identification of geographical isolates of Schistosoma japonicum in China.
Li J J Zhao G H GH Chen F F Song H Q HQ Zhu X Q XQ Zhao G H GH Li J J Chen F F Lin R Q RQ Weng Y B YB Mahmoud M S MS Zou F C FC
Annals of tropical medicine and parasitology 20111001 7
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Project description:We examined the utility of a PCR-based method termed cleaved amplified polymorphic sequences (CAPS) to type 35 well-characterized isolates of Staphylococcus epidermidis. The results were compared with detailed epidemiologic information and typing obtained by using pulsed-field gel electrophoresis (PFGE). To identify CAPS markers for this study, eight pairs of oligonucleotide primers corresponding to five previously sequenced S. epidermidis genes were synthesized and then used to amplify DNA sequences from the S. epidermidis strains by using PCR. Amplified products were reproducibly obtained for seven of eight primer pairs from chromosomal DNA of 33 of the 35 isolates. Seven restriction site polymorphisms were found in five of the amplified products when they were subjected to digestion with a panel of restriction endonucleases. Each fragment-enzyme combination that was polymorphic demonstrated only two alleles in the 33 S. epidermidis isolates analyzed, corresponding to the presence or absence of a single restriction site. Overall, five distinct combinations of alleles were detected and were designated CAPS types A through E. There was a close correlation between the CAPS grouping, the epidemiologic information for the strains, and grouping by PFGE following SmaI digestion of chromosomal DNA. Although PFGE analysis was more discriminatory than typing based on the limited number of CAPS markers used in this study (isolates from the same CAPS group were sometimes distributed into more than one PFGE group), no isolates from the same PFGE group were found in more than one CAPS group. The CAPS procedure was highly reproducible, in contrast to published experience with arbitrarily primed PCR. These preliminary data suggest that CAPS represents a PCR-based technique for strain typing that is highly reproducible, rapid, utilizes widely available technologies, and provides results that are relatively easy to interpret and express.
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Project description: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.
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2012-07-13 | E-GEOD-39329 | biostudies-arrayexpress