Purpose

Bacterial quantification by qPCR is considered the gold standard for microbial molecular diagnosis. However, a fundamental pre-requisite in this methodology is the designing of specific primers for the bacterium of interest. With the increase in bacterial genome sequencing data in the recent years, it has become possible to design specific primers that can be used to quantify different strains of the same bacterial species.

Methods

To develop a real-time PCR (qPCR) protocol for the specific quantification of Azospirillum baldaniorum Sp245 strain (old Azospirillum brasilense), the Sp245 genome sequence was fragmented into small contigs with 500 base pairs each, and analyzed for similarity against the NCBI non-redundant database. A. baldaniorum-specific contigs were used to design the primers. The best pair of primers was used to quantify these bacteria after inoculation in different cultivars of Brachiaria, grown under field conditions.

Results

Our results showed that the primer pair Sp245p10 was highly specific for the Sp245 strain in the Brachiaria root and shoot field under different conditions. The qPCR assay using these primers showed differences among cultivars in the number of bacteria detected in plants after inoculation. Additionally, the number of bacteria observed in the roots was higher than that in the shoots.

Conclusion

The qPCR methodology using a Sp245 strain-specific primer may be used to monitor A. baldaniorum inoculated into other plants and may find potential application in field experiments.

中文翻译：

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田间条件下臂形草上固氮螺菌 Sp245 菌株的实时 PCR 定量

目的

qPCR 的细菌定量被认为是微生物分子诊断的金标准。然而，这种方法的一个基本先决条件是为感兴趣的细菌设计特定的引物。随着近年来细菌基因组测序数据的增加，设计可用于量化同一细菌物种不同菌株的特异性引物已成为可能。

方法

为了开发实时 PCR (qPCR) 协议以特定定量Azospirillum baldaniorum Sp245 菌株（旧Azospirillum brasilense），Sp245 基因组序列被分割成每个具有 500 个碱基对的小重叠群，并分析与 NCBI 非-冗余数据库。A. baldaniorum特异性重叠群用于设计引物。在田间条件下生长的臂形属不同品种中接种后，使用最佳引物对对这些细菌进行定量。

结果

我们的结果表明，引物对 Sp245p10在不同条件下对臂根和地上部中的 Sp245 菌株具有高度特异性。使用这些引物的 qPCR 分析显示，在接种后植物中检测到的细菌数量在栽培品种之间存在差异。此外，在根中观察到的细菌数量高于在芽中观察到的细菌数量。

结论

使用 Sp245 菌株特异性引物的 qPCR 方法可用于监测接种到其他植物中的A. baldaniorum，并可能在田间实验中找到潜在的应用。