Goudarzi R, Mortazavi M M
Department of Agriculture, Damghan Islamic Azad University, Damghan, Iran.
Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran.
J Genet Eng Biotechnol. 2020 Oct 2;18(1):55. doi: 10.1186/s43141-020-00062-6.
Pathogenic bacteria cause significant economic damages in agriculture. The detection of such bacteria is considered as a continual interest for plant pathologists to prevent disease dissemination. Pseudomonas syringae pv. syringae is one of the most important bacterial pathogens infecting yield and quality of stone fruits throughout the world. Biochemical assays such as a LOPAT and GATTa are common methods to detect this pathogen. Serological tests and culturing on King's B selective medium also used to isolate this bacterium. Selective media is composed of specific and effective ingredients to inhibit the growth of certain species of microbes in a mixed culture while allowing others to grow. These are used for the growth of only selected microorganisms. King's B medium can be used as a general medium for the non-selective isolation cultivation and pigment production of Pseudomonas species from foods, cosmetic samples, plants, etc. Nevertheless, the mentioned methods are not enough accurate to differentiate the strains. On the other hand, PCR-based techniques are sensitive and efficient in detecting plant diseases. However, these techniques are not practicable for those researchers who do not have access to a thermal cycler. We have used loop-mediated isothermal amplification to couple with a target. The amplification of syrD gene using loop and bumper primers can be used to prevent disease dissemination.
The outcome of this investigation indicated more sensitivity of LAMP in comparison to PCR. The direct addition of SYBR Gold in microtube is more sensitive than gel in both LAMP and PCR byproducts so we can eliminate gel electrophoresis, while the LAMP showed high sensitivity and high specificity in comparison to results obtained by cultivation. The described molecular test could detect Pseudomonas syringae pv. syringae type in nearly 1 h, and this is the first time that Lamp molecular detection of Pseudomonas syringae pv. syringae particularly on stone fruits is described and introduced.
The obtained data confirmed that LAMP is a fast, cheap, and high specific method for the rapid detection of Pseudomonas syringae pv. syringae to the comparison of PCR and culture.
病原菌给农业造成了巨大的经济损失。植物病理学家一直关注此类细菌的检测，以防止病害传播。丁香假单胞菌丁香致病变种是影响全球核果产量和品质的最重要细菌病原体之一。诸如LOPAT和GATTa等生化检测是检测该病原体的常用方法。血清学检测以及在金氏B选择性培养基上培养也用于分离这种细菌。选择性培养基由特定且有效的成分组成，可在混合培养物中抑制某些微生物种类的生长，同时允许其他微生物生长。这些仅用于特定微生物的生长。金氏B培养基可作为从食品、化妆品样品、植物等中进行假单胞菌属非选择性分离培养和色素产生的通用培养基。然而，上述方法在区分菌株方面不够准确。另一方面，基于聚合酶链反应（PCR）的技术在检测植物病害方面灵敏且高效。然而，对于那些无法使用热循环仪的研究人员来说，这些技术并不实用。我们使用了环介导等温扩增技术并结合一个靶标。使用环引物和保险杠引物对syrD基因进行扩增可用于防止病害传播。
本研究结果表明，与PCR相比，环介导等温扩增（LAMP）更灵敏。在微管中直接添加SYBR Gold在LAMP和PCR副产物中都比凝胶更灵敏，因此我们可以省去凝胶电泳，而与培养结果相比，LAMP显示出高灵敏度和高特异性。所描述的分子检测方法可在近1小时内检测出丁香假单胞菌丁香致病变种，这是首次描述并介绍LAMP分子检测丁香假单胞菌丁香致病变种，特别是在核果上的检测。
获得的数据证实，与PCR和培养相比，LAMP是一种快速、廉价且特异性高的方法，可用于快速检测丁香假单胞菌丁香致病变种。
