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首页> 外文期刊>Nematropica >DIFFERENTIATION OF MELOIDOGYNE FLORIDENSIS FROM M. ARENARIA USING HIGH-FIDELITY PCR AMPLIFIED MITOCHONDRIAL AT-RICH SEQUENCES
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DIFFERENTIATION OF MELOIDOGYNE FLORIDENSIS FROM M. ARENARIA USING HIGH-FIDELITY PCR AMPLIFIED MITOCHONDRIAL AT-RICH SEQUENCES

机译：高分辨率PCR扩增的线粒体at富集序列从无性疟原虫中分离出扇叶锦鸡儿

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Standard polymerase chain reaction (PCR) and high-fidelity PCR procedures were compared for amplifying DNA sequences from five root-knot nematode species. High-fidelity PCR was found to be at least four orders of magnitude more efficient than standard PCR. High-fidelity PCR was used to amplify mitochondrial DNA sequences located between the cytochome oxidase subunit II (COII) and 16S rRNA genes from the following root-knot nematode species; Meloidogyne mayaguensis (0.7 kb), M. floridensis (1.1 kb), M. arenaria (1.1 kb), M. incognita (1.5 kb), and M. javanica (1.6 kb). This region includes the non-coding "control" region, which is also referred to as AT-rich region and tRNA-His gene. The high-fidelity PCR products were cloned and sequenced. The COII 3' partial, tRNA-His, and 16S rRNA 5' partial sequences were found to display few nucleotide differences, but the AT-rich region displayed extensive nucleotide and length variations in all five species; M. mayaguensis (167 bp), M. arenaria (573 bp), M. floridensis (603 bp), M. incognita (963 bp), and M. javanica (1110 bp). A stem and loop structure, which has been characterized from insects and other higher organisms, was detected in a conserved block of AT-rich sequences from M. floridensis, M. arenaria, M. incognita, and M. javanica. Similar stem and loop structures were also detected from the AT-rich sequences previously reported from Caenorhabditis elegans and Ascaris suum and compared to the M. floridensis stem and loop structure. Even though the high-fidelity PCR products amplified from M. floridensis and M. arenaria were about the same size (1.1 kb), their AT-rich sequences were found to contain nucleotide differences at several locations. Hence, digesting the 1.1 kb high-fidelity PCR products from these species with the restriction enzyme Ssp I produced two distinct banding patterns that distinguished M. floridensis from M. arenaria.
机译：比较了标准聚合酶链反应（PCR）和高保真PCR程序，以扩增5种根结线虫物种的DNA序列。发现高保真PCR的效率比标准PCR高至少四个数量级。使用高保真PCR扩增线粒体DNA序列，该线粒体DNA序列位于以下根结线虫物种的胞嘧啶氧化酶亚基II（COII）和16S rRNA基因之间;梅格亚种（Meloidogyne mayaguensis）（0.7 kb），弗洛里德木霉（M. floridensis）（1.1 kb），沙蒿（M. arenaria）（1.1 kb），隐身木（M. incognita）（1.5 kb）和爪哇木兰（M. javanica）（1.6 kb）。该区域包括非编码“对照”区域，也称为富AT区域和tRNA-His基因。高保真PCR产物被克隆并测序。发现COII 3'部分，tRNA-His和16S rRNA 5'部分序列显示出极少的核苷酸差异，但富含AT的区域在所有五个物种中均显示出广泛的核苷酸和长度变化。 M.mayaguensis（167 bp），M。arenaria（573 bp），florida。M. floridensis（603 bp），M。incognita（963 bp）和Java.M. javanica（1110 bp）。在昆虫分支和其他高等生物的特征下，发现了茎和环结构的保守结构，该保守结构来自弗洛里德氏菌，砂仁分支杆菌，隐身分支杆菌和爪哇分支杆菌富含AT的序列。还从先前从秀丽隐杆线虫和猪A虫（Ascaris suum）报道的富含AT的序列中检测到相似的茎和stem结构，并将其与弗洛里德氏菌的茎和loop结构进行比较。即使从弗洛雷德氏菌和沙蒿分枝杆菌扩增的高保真PCR产物大小大约相同（1.1 kb），但发现它们富含AT的序列在几个位置上都含有核苷酸差异。因此，用限制酶Ssp I消化来自这些物种的1.1 kb高保真PCR产物，产生了两种不同的条带模式，从而将弗洛里德氏菌与沙雷氏菌区分了。

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