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

本研究将PCR-RFLP（PCR-Restriction Fragment Length Polymorphism）和PCR-SSCP（PCR-Single Strand Conformational Polymorphism）技术相结合，建立了微小隐孢子虫种内多态性检测方法。从不同的地理来源（日本、意大利和尼泊尔）和宿主（人、小牛和山羊）收集了41个分离株。C.通过DNA序列测定、PCR-RFLP和RFLP-SSCP等方法对分离株的Cpgp 40/15进行了鉴定。序列分析表明，基因型I和基因型II之间以及基因型I分离株内部存在DNA多态性。由于PCR-RFLP和RFLP-SSCP的高分辨率，基因型I的分离物可分为基因型Ia1、Ia2、Ib和Ie。基因型II的分离株可分为IIa、IIb和IIc基因型。从小牛，山羊和日本人的分离物被确定为基因型II。在基因型内 ...更多信息 II，日本分离株为基因型IIa，意大利犊牛分离株为基因型IIb，山羊分离株为基因型IIc。所有基因型I分离株均来自人类。日本分离株（HJ 3）和所有尼泊尔分离株分别被鉴定为基因型Ia1和Ia2。意大利分离株为基因型Ib，日本分离株（HJ 2）为基因型Ie.因此，该糖蛋白基因Cpgp 40/15的PCR-RFLP-SSCP分析产生了高分辨率，这是以前的方法在C. parvum的3个新基因。克隆了一个编码甲硫氨酸氨基肽酶的基因、一个编码含TCP-1 δ亚基的伴侣蛋白的基因和一个功能未知的基因。根据这3个基因的序列，3对C.构建了小孢子虫特异性引物。这些引物在检测C.小的DNA序列分析表明，这些基因是相当保守的，但有相同的碱基对之间的差异，基因型I和基因型II分离物。对来自不同寄主和地理来源的41个菌株的3个基因进行PCR-RFLP分析，证实了这些差异。本研究结果为检测和分型C.小孢子分离物少

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