Long PCR improves Wolbachia DNA amplification:: wsp sequences found in 76% of sixty-three arthropod species
Long PCR improves Wolbachia DNA amplification:: wsp sequences found in 76% of sixty-three arthropod species
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DOI：
10.1046/j.1365-2583.2000.00203.x
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发表时间：
2000-08-01
影响因子：
2.6
通讯作者：
Hoy, MA
中科院分区：
文献类型：
--
作者：
Jeyaprakash, A;Hoy, MA
沃尔巴克氏体属细菌与节肢动物的多种生殖异常有关。等位基因特异性聚合酶链反应（= 标准 PCR）通常用于扩增节肢动物的沃尔巴克氏体 DNA。在测试二斑叶螨和其他已知感染沃尔巴克氏体的节肢动物时，标准 PCR 经常产生假阴性，这可能是因为来自节肢动物宿主的 DNA 干扰了 Taq DNA 聚合酶的扩增。使用两种酶（Taq 和 Pwo）的长 PCR 能够一致地扩增沃尔巴克氏体 DNA，敏感性分析表明，在扩增掺入昆虫基因组 DNA 中的质粒 DNA 时，长 PCR 的敏感性比标准 PCR 大约高出六个数量级。一项调查表明，62 种节肢动物物种和 13 个目的 2 个亚种中，76% 通过长 PCR 检测出 Wolbachia wsp 序列呈阳性，这大大高于以前使用标准 PCR 获得的 ftsZ 序列的 16.9% 的阳性率（Werren, J.H.、Windsor, D. 和 Gau, L. (1995a) Proc R Soc Lond B 262： 197-204）。对来自 14 个节肢动物物种和两个亚种的 Long PCR 产物的子样本进行了直接测序和克隆后测序。检测到 2 个 A-沃尔巴克氏体菌株和 11 个 B-沃尔巴克氏体菌株，它们的 wsp 序列在该位点显示最大 23.7% 的序列分歧。除了早先报告的 19 个之外，还鉴定了两个新组（名为 Fus 和 Ten）（Zhou, W.、Rousset, F. 和 O'Neill, S.L. (1998) Proc R Soc Lond B 265: 1-7；van Meer, M.M.M.、Witteveldt, J. 和 Stouthamer, R. (1999) Insect Mol Biol 8： 399-408)，因为它们与其他沃尔巴克氏体 wsp 序列显示出超过 2.5% 的序列差异。由于明显受到多种沃尔巴克氏体菌株的感染，所分析的二十九种节肢动物中的十七种 (59%) 的 PCR 产物无法直接测序。从两个此类物种（小菜蛾和粉纹夜蛾）克隆的 wsp 序列表明，A-沃尔巴克氏体和 B-沃尔巴克氏体同时存在于单个个体中。因此，重复感染的发生率也可能比之前估计的 1.2% 更为广泛。
Bacteria belonging to the genus Wolbachia are associated with a variety of reproductive anomalies in arthropods. Allele-specific polymerase chain reaction (= Standard PCR) routinely has been used to amplify Wolbachia DNA from arthropods. While testing the two-spotted spider mite Tetranychus urticae and other arthropods known to be infected with Wolbachia, Standard PCR frequently produced false negatives, perhaps because the DNA from the arthropod host interfered with amplification by Taq DNA polymerase. Long PCR, which uses two enzymes (Taq and Pwo), consistently amplified Wolbachia DNA and a sensitivity analysis indicated that Long PCR was approximately six orders of magnitude more sensitive than Standard PCR in amplifying plasmid DNA spiked into insect genomic DNA. A survey indicated that 76% of sixty-two arthropod species and two subspecies in thirteen orders tested positive for the Wolbachia wsp sequence by Long PCR, which is considerably higher than the rate of 16.9% obtained previously for the ftsZ sequence using Standard PCR (Werren, J.H., Windsor, D. and Gao, L. (1995a) Proc R Soc Lond B 262: 197-204). A subsample of Long PCR products from fourteen arthropod species and two subspecies were sequenced, both directly and after cloning. Two A- and eleven B-Wolbachia strains were detected and their wsp sequences displayed a maximum of 23.7% sequence divergence at this locus. Two new groups (named Fus and Ten) were identified in addition to nineteen reported earlier (Zhou, W., Rousset, F. and O'Neill, S.L. (1998) Proc R Soc Lond B 265: 1-7; van Meer, M.M.M., Witteveldt, J. and Stouthamer, R. (1999) Insect Mol Biol 8: 399-408), because they displayed more than 2.5% sequence divergence from other Wolbachia wsp sequences. PCR products from seventeen of twenty-nine (59%) arthropod species analysed could not be sequenced directly due to apparent infection by multiple Wolbachia strains. The wsp sequences cloned from two such species (Plutella xylostella and Trichoplusia ni) indicated both A- and B-Wolbachia were present in a single individual. Hence, superinfection also may be more widespread than the 1.2% incidence previously estimated.