Sulaiman I M, Xiao L, Lal A A
Division of Parasitic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30341, USA.
Appl Environ Microbiol. 1999 Oct;65(10):4431-5. doi: 10.1128/AEM.65.10.4431-4435.1999.
We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.
我们评估了11种先前描述的用于检测隐孢子虫寄生虫的物种鉴别和基因分型PCR方案的特异性和敏感性。使用来自三种隐孢子虫寄生虫（微小隐孢子虫基因型1和基因型2、鼠隐孢子虫和蛇隐孢子虫）、两种艾美耳球虫（内氏艾美耳球虫和乳头艾美耳球虫）以及十二指肠贾第虫的基因组DNA来评估引物的特异性。此外，通过使用从基因型2微小隐孢子虫分离株获得的已知数量卵囊分离的基因组DNA来测试基因分型引物的敏感性。所有引物对均至少重复进行三次PCR扩增。在所研究的11种方案中，有10种扩增出微小隐孢子虫基因型1和2，并获得了预期的片段大小。我们的结果表明，两种物种鉴别方案并非隐孢子虫特异性的，因为这些方案中使用 的引物也能扩增艾美耳球虫物种的DNA。敏感性研究表明，基于小亚基rRNA和二氢叶酸还原酶基因的两种巢式PCR-限制性片段长度多态性（RFLP）方案比单轮PCR或PCR-RFLP方案更敏感。
