Detection of Schistosoma mansoni in Biomphalaria using nested PCR
Detection of Schistosoma mansoni in Biomphalaria using nested PCR
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DOI：
10.2307/3284399
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发表时间：
1997-06-01
影响因子：
1.3
通讯作者：
Loker, ES
中科院分区：
文献类型：
--
作者：
Hanelt, B;Adema, CM;Loker, ES
建立了一种检测曼氏血吸虫孢子囊的套式聚合酶链式反应(PCR)方法。为了实现这一目标，对来自埃及的曼氏丝虫和亚历山大丝虫的18S rRNA编码基因进行了测序，并对光面丝虫的13-16-R1和萨尔瓦多株的18S编码基因进行了测序。在比较宿主和寄生虫序列的基础上，设计了一套嵌套式聚合酶链式反应引物，用于特异扩增曼氏血吸虫18S rDNA的部分片段。这些引物可以检测到稀释到100 ng蜗牛DNA中的10fg曼氏葡萄球菌DNA，而不允许扩增蜗牛18S序列。利用套式聚合酶链式反应，可以在暴露后1天在成年钉螺体内检测到单个曼氏链球菌孢子囊的存在。从74只光滑假单胞菌M系钉螺中提取的DNA样本中，单独使用外部引物对51%的钉螺检测到寄生虫，而连续使用外部和套式引物对92%的钉螺检测到寄生虫。这一方法在确定流行地区的钉螺是否具有潜伏期或非活动性感染以及评估当地钉螺和血吸虫种群之间的相容程度方面具有实用价值。这也将有助于研究蜗牛对感染的抵抗力。
A nested polymerase chain reaction (PCR) protocol was developed for detecting the presence of Schistosoma mansoni sporocysts in intermediate host snails of the genus Biomphalaria. To accomplish this, rDNA genes encoding the 18S rRNA of S. mansoni and Biomphalaria alexandrina from Egypt were sequenced, as were 18S-encoding genes of the 13-16-R1 and Salvador strains of Biomphalaria glabrata. Based on a comparison of host and parasite sequences, a nested set of PCR primers was designed to allow specific amplification of portions of S. mansoni 18S rDNA. These primers allowed detection of as little as 10 fg of S. mansoni DNA diluted in 100 ng of snail DNA and did not allow amplification of snail 18S sequences. Using nested PCR, the presence of a single S. mansoni sporocyst within an adult snail could be detected at 1 day postexposure. In DNA samples extracted from each of 74 snails of the M-line strain of B. glabrata exposed to from 1 to 10 S. mansoni miracidia for intervals ranging from 1 to 44 days, use of the outside primer pair alone detected the parasite's presence in 51% of the snails, whereas the sequential use of outside and nested primer pairs detected parasites in 92% of the snails. This approach has utility in determining if snails in endemic areas bear prepatent or inactive infections and in assessing the degree of compatibility between local snail and schistosome populations. It will also facilitate studies of resistance of snails to infection.