Sequence characterized amplified region (SCAR) markers are one of the most effective and accurate tools for microbial identification. In this study, we applied SCAR markers for the rapid and accurate detection of Phytophthora katsurae, the casual agent of chestnut ink disease in Korea. In this study, we developed seven SCAR markers specific to P. katsurae using random amplified polymorphic DNA (RAPD), and assessed the potential of the SCAR markers to serve as tools for identifying P. katsurae. Seven primer pairs (SOPC 1F/SOPC 1R, SOPC 1-1F/SOPC 1-1R, SOPC 3F/SOPC 3R, SOPC 4F/SOPC 4R, SOPC 4F/SOPC 4-1R, SOPD 9F/SOPD 9R, and SOPD 10F/SOPD 10R) from a sequence derived from RAPD fragments were designed for the analysis of the SCAR markers. To evaluate the specificity and sensitivity of the SCAR markers, the genomic DNA of P. katsurae was serially diluted 10-fold to final concentrations from 1 mg/mL to 1 pg/mL. The limit of detection using the SCAR markers ranged from 100 µg/mL to 100 ng/mL. To identify the limit for detecting P. katsurae zoospores, each suspension of zoospores was serially diluted 10-fold to final concentrations from 10 × 10(5) to 10 × 10(1) zoospores/mL, and then extracted. The limit of detection by SCAR markers was approximately 10 × 10(1) zoospores/mL. PCR detection with SCAR markers was specific for P. katsurae, and did not produce any P. katsurae-specific PCR amplicons from 16 other Phytophthora species used as controls. This study shows that SCAR markers are a useful tool for the rapid and effective detection of P. katsurae.

译文

:序列鉴定的扩增区(SCAR)标记是微生物鉴定最有效,最准确的工具之一。在这项研究中,我们将SCAR标记物用于快速准确地检测韩国板栗油墨病的临时病原疫霉。在这项研究中,我们使用随机扩增的多态性DNA(RAPD)开发了7种特异于胜克假单胞菌的SCAR标记,并评估了SCAR标记物作为识别胜克假单胞菌的工具的潜力。七个引物对(SOPC 1F / SOPC 1R,SOPC 1-1F / SOPC 1-1R,SOPC 3F / SOPC 3R,SOPC 4F / SOPC 4R,SOPC 4F / SOPC 4-1R,SOPD 9F / SOPD 9R和SOPD 10F /设计了来自RAPD片段的序列的SOPD 10R)用于SCAR标记的分析。为了评估SCAR标记物的特异性和敏感性,将克雷伯氏疟原虫的基因组DNA连续稀释10倍至终浓度(从1 mg / mL到1 pg / mL)。使用SCAR标记的检测限范围为100 µg / mL至100 ng / mL。为了确定检测克雷伯氏菌游动孢子的极限,将每个游动孢子悬浮液从10×10(5)到10×10(1)游动孢子/ mL连续稀释10倍至最终浓度,然后提取。 SCAR标记的检测极限约为10×10(1)游动孢子/ mL。用SCAR标记进行的PCR检测对白克雷伯氏菌具有特异性,并且没有从用作对照的其他16个疫霉菌中产生任何对克雷伯氏菌的特异性PCR扩增子。这项研究表明,SCAR标记物是快速有效检测片状疟原虫的有用工具。

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