DIRECT AND SENSITIVE DETECTION OF A PATHOGENIC PROTOZOAN, TOXOPLASMA-GONDII, BY POLYMERASE CHAIN-REACTION
DIRECT AND SENSITIVE DETECTION OF A PATHOGENIC PROTOZOAN, TOXOPLASMA-GONDII, BY POLYMERASE CHAIN-REACTION
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DOI：
10.1128/jcm.27.8.1787-1792.1989
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发表时间：
1989-08-01
影响因子：
9.4
通讯作者：
BOOTHROYD, JC
中科院分区：
文献类型：
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作者：
BURG, JL;GROVER, CM;BOOTHROYD, JC
我们应用聚合酶链反应检测的基础上，我们确定了一个35倍重复基因（B1基因）作为目标的病原性原虫刚地弓形虫。使用这种方法，我们能够直接从粗细胞裂解物中扩增和检测单个生物体的DNA。这种灵敏度水平也使我们能够在10万人白细胞存在的情况下，从含有10种寄生虫的纯化DNA样本中检测出B1基因。这代表了从弓形体脑炎患者获得的1 ml脑脊液中的最大细胞浸润（105/ml）。B1基因在所有6个T.迄今为止检测的弓形虫菌株，包括从获得性免疫缺陷综合征患者分离的两个菌株。通过使用该测定和来自各种其他生物体的DNA，包括可能在免疫受损宿主的中枢神经系统中发现的几种DNA，未检测到信号。这种结合的敏感性和特异性，使检测的B1基因的基础上聚合酶链反应扩增的一个非常有用的方法诊断弓形虫病的免疫功能低下的主机和先天性感染的胎儿。
We applied the polymerase chain reaction to detection of the pathogenic protozoan Toxoplasma gondii based on our identification of a 35-fold-repetitive gene (the B1 gene) as a target. Using this procedure, we were able to amplify and detect the DNA of a single organism directly from a crude cell lysate. This level of sensitivity also allowed us to detect the B1 gene from purified DNA samples containing as few as 10 parasites in the presence of 100,000 human leukocytes. This is representative of the maximal cellular infiltration (105/ml) in 1 ml of cerebrospinal fluid obtained from patients with toxoplasmic encephalitis. The B1 gene is present and conserved in all six T. gondii strains tested to date, including two isolates from patients with acquired immunodeficiency syndrome. No signal was detected by using this assay and DNAs from a variety of other organisms, including several which might be found in the central nervous system of an immunocompromised host. This combination of sensitivity and specificity should make detection of the B1 gene based on polymerase chain reaction amplification a very useful method for diagnosis of toxoplasmosis both in immunocompromised hosts and in congenitally infected fetuses.