利用计算机设计并在体外开发一种高准确度的布鲁氏菌属检测方法。

In silico design and in vitro development of a highly accurate test to detect Brucella species.

出版信息

Turk J Med Sci. 2019 Feb 11;49(1):368-374. doi: 10.3906/sag-1807-20.

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC7350864/

Abstract

BACKGROUND

Conventionalmethods of detecting Brucella spp. suffer from technical and biological complications. Besides, newly characterized species of the genus Brucella could be neglected by previously designed polymerase chain reaction (PCR) tests. Therefore, a more accurate PCR-based test seems to be imminently needed

MATERIALS AND METHODS

Blood samples were collected from 39 patients diagnosed with brucellosis and 25 healthy controls. Multiple sequence alignments (MSA) were performed on 500 Omp2-related protein and gene sequences. Thereafter, specific primers were designed and synthesized for the regions with highest conservancy. The collected samples were assessed by PCR test. To overcome the cross-reactivity issue, PCR thermal program was optimized regarding annealing time and temperature.

RESULTS

The MSA results indicated that the N terminus region of the Omp2 protein (DNA 5’ end) is associated with highest conservancy. Primers with highest specificity were designed and synthesized. A two-step PCR reaction was successfully designed and optimized. The desirable bands were observed in clinical samples with high accuracy.

CONCLUSION

It should be pointed out that using a precisely designed primer pair would bring about early infection detection, more success to detect all natural variants and higher cost-to-efficacy ratio in comparison to other detection methods

摘要

背景

传统的布鲁氏菌属检测方法存在技术和生物学方面的复杂性。此外，先前设计的聚合酶链反应（PCR）检测方法可能会忽略新鉴定的布鲁氏菌属物种。因此，似乎迫切需要一种更准确的基于 PCR 的检测方法。

材料与方法

采集了 39 例确诊为布鲁氏菌病的患者和 25 例健康对照者的血液样本。对 500 个 Omp2 相关蛋白和基因序列进行多重序列比对（MSA）。然后，针对具有最高保守性的区域设计并合成了特异性引物。通过 PCR 检测评估采集的样本。为了克服交叉反应问题，优化了 PCR 热循环程序，包括退火时间和温度。