Abstract

The detection of Acidovorax citrulli requires new highly specific methods based on qPCR. The goal of the study was to develop a new qPCR-based test for the identification of A. citrulli. By clustering the protein sequences of nine A. citrulli genomes and nine A. avenae genomes, using the CD-HIT program, 153 protein clusters present in all nine A. citrulli strains and absent in all A. avenae strains were identified. The resulting clusters were compared with known protein sequences (nr) using the BLASTp program to determine their specificity for A. citrulli. Coding sequences of A. citrulli-specific proteins were used for the selection of primers and the probe. The specificity of qPCR was tested by using the DNA of six A. citrulli strains and 188 nontarget bacteria strains. As a result of the search for A. citrulli-specific protein sequences, the S-box PAS domain protein was identified. The performed qPCR tests showed 100% specificity both when testing target strains (all reactions were positive) and for nontarget strains (all reactions were negative). A new qPCR test for the identification of A. citrulli, based on the sequence encoding the S-box protein of the PAS domain was developed.

中文翻译：

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基于 PAS 结构域 S-Box 蛋白基因的基于 qPCR 的瓜酸食酸新检测系统

摘要

Acidovorax citrulli的检测需要基于 qPCR 的新的高度特异性方法。该研究的目标是开发一种新的基于 qPCR 的测试，用于鉴定A. citrulli。通过群集的九个蛋白质序列A. citrulli基因组和9个A. 麦长管蚜的基因组，利用CD-HIT程序，153个蛋白簇存在于所有9个A. citrulli菌株和不存在于所有A. 蚜菌株进行了鉴定。使用 BLASTp 程序将得到的簇与已知的蛋白质序列 (nr) 进行比较，以确定它们对A. citrulli的特异性。编码序列A. citrulli特异性蛋白质用于选择引物和探针。qPCR 的特异性通过使用 6 个A. citrulli菌株和 188 个非目标细菌菌株的 DNA 进行测试。作为寻找的结果A. citrulli特异性蛋白序列，S盒PAS结构域蛋白被鉴定。进行的 qPCR 测试在测试目标菌株（所有反应均为阳性）和非目标菌株（所有反应均为阴性）时均显示出 100% 的特异性。一种新的qPCR试验的鉴定A.  citrulli，基于编码PAS域的S-box蛋白序列被开发。