Parr Jonathan B, Sevilleja Jesus Emmanuel, Samie Amidou, Alcantara Cirle, Stroup Suzanne E, Kohli Anita, Fayer Ron, Lima Aldo A M, Houpt Eric R, Guerrant Richard L
University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Am J Trop Med Hyg. 2007 May;76(5):938-42.
Cryptosporidium is a significant cause of diarrheal illness worldwide, especially among children and immunocompromised patients. Currently used diagnostic techniques are time-consuming, require skilled technicians, and are not useful for quantification of oocysts in fecal and environmental samples. In this study, we examined the use of a real-time polymerase chain reaction (PCR) assay for detecting and quantifying Cryptosporidium parvum in three distinct and progressively more complex matrices: phosphate-buffered saline (PBS), HCT-8 cells (human ileocecal carcinoma), and human fecal specimens. A reliable standard curve was generated using the PBS samples spiked with pure oocysts, and oocyst starting quantities were calculated for the infected HCT-8 cell and spiked fecal samples. The assay detected Cryptosporidium in samples infected/spiked with > or =10(3) oocysts/sample and detected both C. hominis and C. parvum in clinical specimens. This assay is useful in a variety of samples in the research laboratory and will likely prove to be a useful tool in the clinical laboratory.
隐孢子虫是全球腹泻疾病的一个重要病因，尤其在儿童和免疫功能低下的患者中。目前使用的诊断技术耗时，需要技术熟练的技术人员，并且对于粪便和环境样本中卵囊的定量检测并无帮助。在本研究中，我们检测了实时聚合酶链反应（PCR）测定法在三种不同且复杂度逐渐增加的基质中检测和定量微小隐孢子虫的应用：磷酸盐缓冲盐水（PBS）、HCT-8细胞（人回盲部癌细胞）和人类粪便标本。使用添加了纯卵囊的PBS样本生成了可靠的标准曲线，并计算了感染的HCT-8细胞和添加卵囊的粪便样本中的卵囊起始数量。该测定法在感染/添加了≥10³个卵囊/样本的样本中检测到了隐孢子虫，并在临床标本中检测到了人隐孢子虫和微小隐孢子虫。该测定法在研究实验室的各种样本中都很有用，并且可能会被证明是临床实验室中的一个有用工具。
