A RAPID DNA ASSAY FOR THE SPECIES-SPECIFIC DETECTION AND QUANTIFICATION OF BRUGIA IN BLOOD-SAMPLES
A RAPID DNA ASSAY FOR THE SPECIES-SPECIFIC DETECTION AND QUANTIFICATION OF BRUGIA IN BLOOD-SAMPLES
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DOI：
10.1016/0166-6851(90)90086-2
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发表时间：
1990-04-01
影响因子：
1.5
通讯作者：
WILLIAMS, SA
中科院分区：
文献类型：
--
作者：
POOLE, CB;WILLIAMS, SA
本报告描述了一种新的检测丝虫属丝虫血液样本中使用标记的DNA探针。这些DNA探针的序列是基于在布氏丝虫属中发现的HhaI重复DNA家族。这些DNA探针是种特异性的，并且可以在杂交测定中检测来自单个微丝蚴的DNA。为了使该测试适用于现场采集的血液样本，消除了从血细胞中分离微丝蚴和纯化寄生虫DNA的复杂步骤。我们发现，最有效的方法是通过5.0 μ m孔径的硝酸纤维素膜过滤血液样品，通过蛋白酶K消化裂解膜上的微丝蚴，用氢氧化钠使寄生虫DNA变性，并与DNA探针杂交。用这种方法，个体微丝蚴可以在放射自显影图上可视化和计数。在模拟现场研究中使用马来丝虫感染的沙鼠进行了评价，并发现该方法是一种有效和准确的方法，用于定量血液样品中的微丝蚴。
This report describes a new assay for detecting filarial parasites of the genus Brugia in blood samples using labeled DNA probes. The sequences of these DNA probes are based on the HhaI repeat DNA family found in the genus Brugia. These DNA probes are species-specific and can detect the DNA from a single microfilaria in hybridization assays. To adapt this test for use on blood samples collected in the field, complex steps to separate microfilariae from blood cells and to purify parasite DNA were eliminated. We found that the most effective method was to filter blood samples through 5.0 .mu.m pore nitrocellulose membranes, lyse the microfilariae on the membranes by proteinase K digestion, denature the parasite DNA with sodium hydroxide, and hybridize with the DNA probe. With this method, individual microfilariae can be visualized and counted on autoradiograms. The assay was evaluated in a mock field study using Brugia malayi-infected jirds and was found to be an efficient and accurate method for quantifying microfilariae in blood samples.