Study on detection method of intraspecies polymorphism of Cryptosporidium parvum and its application

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In the present research project, we developed the method to detect intraspecies polymorphism of Cryptosporidium parvum by combining the techniques of PCR-RFLP (PCR-Restriction Fragment Length Polymorphism) and PCR-SSCP (PCR-Single Strand Conformational Polymorphism). Forty-one isolates were collected from different geographical origins (Japan, Italy and Nepal) and hosts (humans, calves and a goat). A glycoprotein gene of C. parvum (Cpgp40/15) of these isolates were characterized by means of DNA sequencing, PCR-RFLP and RFLP-SSCP. The sequence analysis indicated that there was DNA polymorphism between genotype I and genotype II as well as within genotype I isolates. Because of high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotype Ia1, Ia2, Ib and Ie. The isolates of genotype II could be subtyped as genotype IIa, IIb and IIc. The isolates from calves, a goat and one Japanese human were identified as genotype II. Within the genotype … More II, the isolates from Japan were identified as genotype IIa, those from Italy calves as genotype IIb, and the goat isolate as IIc. All of the genotype I isolates were from humans. The Japanese isolate (HJ 3) and all Nepal isolates were identified as genotype Ia1 and Ia2, respectively. The Italy isolates were identified as genotype Ib and the Japanese isolate (HJ 2) as genotype Ie. Thus the PCR-RFLP-SSCP analysis of this glycoprotein gene Cpgp40/15 generated a high resolution, which has not been achieved by previous methods in genotypic differentiation of C. parvum.Meanwhile, 3 new genes of C. parvum were cloned, including a gene encoding methionine aminopeptidase, one encoding chaperonin containing TCP-1 delta subunit and one with unknown function. Based on the sequence of these 3 genes, 3 pairs of C. parvum specific primers were constructed. All of these primers exhibit extra high sensitivity in detecting DNA of C. parvum. DNA sequence analysis indicates that these genes are quite conserved, but there are identical base pair differences between genotype I and genotype II isolates. These difference were confirmed by PCR-RFLP analysis of the 3 genes from 41 isolates collected from different host and geographical origins. The results provide new genes and tools for detecting and genotyping C. parvum isolates. Less