Bairami Kuzehkanan A, Rezaeian M, Zeraati H, Mohebali M, Meamar A R, Babaei Z, Kashi L, Heydarnezhadi M, Rezaie S
Dept. of Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran.
Iran J Parasitol. 2011 Dec;6(4):1-7.
The main goal of the present study was to develop a new sensitive and specific PCR based method for Identification of Cryptosporidium sp. using novel primers from 18S ribosomal RNA. Cryptosporidiosis in high-risk host groups particularly in neonates and immuno-compromised individuals may result in death. To the best of our knowledge this is the first study regarding develop a new PCR based method to diagnose the cryptosporidiosis in Iran.
A total of 850 human fecal samples from patients clinically suspected to cryptosporidiosis and 100 healthy and diarrheic cattle stool specimens were collected. The simplified formol-ether concentration method was carried out for all samples. They were then examined microscopically by modified Ziehl-Neelsen staining method. Total DNA was extracted by QIA amp DNA stool mini kit. PCR and nested-PCR was carried out by using designed primers.
Twenty nine cases of cryptosporidiosis infection in human and 30 samples from cattle microscopically were positive. The described primary and nested PCR method could detect all Cryptosporidium positive samples from human and cattle. Regards to suspected negative samples in primary PCR examination, the Nested PCR could approve two more positive results. Furthermore, Nested PCR analysis was able to detect one more case which was negative in both microscopically examination and primary PCR. Specificity of the test was 100%. Sensitivity of Nested PCR in comparison to our gold standard; microscopy after Ridley concentration modified ziehl-Neelsen, was 100%.
Our developed PCR based method by using new primers devised from 18S ribosomal RNA revealed the ability for identification of the Cryptosporidium species such as C. parvum and C. huminis with high specificity and sensitivity.
本研究的主要目标是开发一种基于PCR的新方法，该方法灵敏且特异，用于使用来自18S核糖体RNA的新型引物鉴定隐孢子虫属。高危宿主群体尤其是新生儿和免疫功能低下个体中的隐孢子虫病可能导致死亡。据我们所知，这是伊朗第一项关于开发基于PCR的新方法诊断隐孢子虫病的研究。
共收集了850份临床疑似隐孢子虫病患者的人类粪便样本以及100份健康和腹泻牛的粪便标本。对所有样本采用简化的甲醛-乙醚浓缩法。然后通过改良的齐-尼氏染色法进行显微镜检查。使用QIAamp DNA粪便微量提取试剂盒提取总DNA。使用设计的引物进行PCR和巢式PCR。
显微镜检查发现29例人类隐孢子虫病感染病例和30份牛样本呈阳性。所描述的初次PCR和巢式PCR方法能够检测出所有来自人类和牛的隐孢子虫阳性样本。对于初次PCR检查中疑似阴性的样本，巢式PCR又检测出另外两个阳性结果。此外，巢式PCR分析还能检测出1例在显微镜检查和初次PCR中均为阴性的病例。该检测方法的特异性为100%。与我们的金标准（改良齐-尼氏染色法的里德利浓缩后显微镜检查）相比，巢式PCR的灵敏度为100%。
我们开发的基于PCR的方法，通过使用从18S核糖体RNA设计的新引物，显示出能够以高特异性和灵敏度鉴定微小隐孢子虫和人隐孢子虫等隐孢子虫种类的能力。
