Gobet P, Buisson J C, Vagner O, Naciri M, Grappin M, Comparot S, Harly G, Aubert D, Varga I, Camerlynck P, Bonnin A
Laboratoire de Parasitologie et Mycologie, Hôpital du Bocage, Dijon, France.
J Clin Microbiol. 1997 Jan;35(1):254-6. doi: 10.1128/jcm.35.1.254-256.1997.
We developed a PCR-based method that can be used to identify Cryptosporidium parvum in human feces. Fecal oocysts were concentrated by centrifugation on a sodium chloride gradient and filtration on a nitrocellulose filter prior to DNA extraction and PCR amplification of a 452-bp C. parvum-specific DNA sequence with a protocol including dUTP and uracil-N-glycosylase. All samples obtained from naturally infected humans (n = 10), calves (n = 4), and goats (n = 2) were positive. A 100% detection rate was achieved with both formed and solid stools (n = 10) seeded with 1,000 C. parvum oocysts per g. Procedures based on stool concentration by a modified Ritchie method and subsequent oocyst identification by immunofluorescent labeling or acid-fast staining require concentrations of 50,000 to 500,000 oocysts per g to achieve a 100% detection rate with formed stools. The described PCR-based assay thus has a 50- to 500-fold increase in sensitivity compared to those of the methods commonly used to analyze formed feces.
我们开发了一种基于聚合酶链反应（PCR）的方法，可用于鉴定人粪便中的微小隐孢子虫。在提取DNA并使用包含脱氧尿苷三磷酸（dUTP）和尿嘧啶-N-糖基化酶的方案对微小隐孢子虫特异性的452碱基对DNA序列进行PCR扩增之前，通过在氯化钠梯度上离心和在硝酸纤维素滤膜上过滤来浓缩粪便中的卵囊。从自然感染的人类（n = 10）、小牛（n = 4）和山羊（n = 2）获得的所有样本均为阳性。每克接种1000个微小隐孢子虫卵囊的成形粪便和固体粪便（n = 10）的检测率均达到100%。基于改良的Ritchie方法进行粪便浓缩，随后通过免疫荧光标记或抗酸染色进行卵囊鉴定的程序，对于成形粪便要达到100%的检测率需要每克50000至500000个卵囊的浓度。因此，所描述的基于PCR的检测方法与通常用于分析成形粪便的方法相比，灵敏度提高了50至500倍。
