Detection of Cryptosporidium parvum DNA in human feces by nested PCR
Detection of Cryptosporidium parvum DNA in human feces by nested PCR
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DOI：
10.1128/jcm.34.7.1769-1772.1996
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发表时间：
1996-07-01
影响因子：
9.4
通讯作者：
Silva, J
中科院分区：
文献类型：
--
作者：
Balatbat, AB;Jordan, GW;Silva, J
小隐孢子虫是一种球虫原生动物，可引起人类腹泻，对于艾滋病患者来说通常是慢性且严重的腹泻。传统上，诊断是通过粪便浓缩，然后进行抗酸染色（AF）或免疫荧光染色来进行的。通过这些方法检测人类粪便样本的阈值可能需要每克粪便中存在 50,000 个（免疫荧光染色）至 500,000 个（AF）卵囊。在本研究中，开发了一种巢式 PCR 检测方法，可直接从粪便样本中检测微小念珠菌 DNA。从福尔马林粪便中提取 DNA 后，用两个 26 聚体外部引物扩增微小念珠菌 DNA 的 400 bp 片段。用第二对引物对来自该反应的扩增子进行扩增。利用这些嵌套引物，扩增出 194 bp DNA 片段，并通过酶联化学发光系统进行内部探测，确认为微小念珠菌 DNA。这种基于 PCR 的测试可以检测到每克粪便 500 个卵囊或 100 ng 微小念珠菌 DNA。研究表明，所使用的引物对微小隐孢子虫的 DNA 具有特异性。之前报告 AF 呈阴性的 28 名患者中，有 4 名患者的粪便标本中也检测到了 DNA 序列。总之，已经开发出一种快速、灵敏且特异的直接从粪便样本中检测微小梭菌的方法。该测试具有检测无症状感染、监测治疗反应以及检测环境来源中的微生物的潜力。
Cryptosporidium parvum is a coccidian protozoan that causes diarrhea in humans, often chronic and severe in patients with AIDS. Conventionally, diagnosis is made by concentration of stools followed by acid-fast staining (AF) or immunofluorescent staining. The threshold of detection in human stool specimens by these methods may require the presence of 50,000 (immunofluorescent staining) to 500,000 (AF) oocysts per g of stool. In this study, a nested PCR assay was developed to detect C, parvum DNA directly from stool specimens. After extraction of DNA from formalinized stool, a 400-bp fragment of C, parvum DNA was amplified with two 26-mer outer primers. The amplicon from this reaction was amplified with a second primer pair. With these nested primers, a 194-bp DNA fragment was amplified and confirmed as C, parvum DNA by internal probing with an enzyme-linked chemiluminescence system. This PCR-based test allowed the detection of 500 oocysts per g of stool or 100 ng of C, parvum DNA. Studies indicate that the primers utilized are specific for the DNA of C, parvum. DNA sequences were also detected in stool specimens from 4 of 28 patients previously reported negative by AF. In summary, a rapid, sensitive, and specific assay for the detection of C, parvum directly from stool specimens has been developed. This test has the potential for detecting asymptomatic infection, monitoring the response to therapy, and detecting the organism in environmental sources.