United States Department of Agriculture, National Animal Disease Center, Ruminant Diseases and Immunology Research Unit, Agricultural Research Service, Ames, IA, 50010, USA.
United States Department of Agriculture, Agricultural Research Service, U.S. Meat Animal Research Center, Animal Health Genomic Research Unit, Clay Center, NE, 68933, USA.
BMC Res Notes. 2023 Jan 19;16(1):4. doi: 10.1186/s13104-023-06272-8.
Mannheimia haemolytica is the primary bacterial pathogen associated with bovine respiratory disease complex (BRDC). While M. haemolytica has been subdivided into 12 capsular serotypes (ST), ST1, ST2 and ST6 are commonly isolated from cattle. More recently, M. haemolytica strains isolated from North American cattle have been classified into genotypes 1 (ST2) and 2 (ST1 and ST6). Of the two genotypes, genotype 1 strains are frequently isolated from healthy animals whereas, genotype 2 strains are predominantly isolated from BRDC animals. However, isolation of both genotypes from pneumonic lung samples can complicate diagnosis. Therefore, the aim of this study was to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay to differentiate M. haemolytica genotypes.
The genotype specificity of the LAMP was tested using purified genomic DNA from 22 M. haemolytica strains (10 genotype 1, 12 genotype 2) and strains from four related Pasteurellaceae species; Bibersteinia trehalosi, Mannheimia glucosida, Pasteurella multocida, and Histophilus somni. Genotype 1 (adhesin pseudogene B1) specific-LAMP reactions amplified DNA only from genotype 1 strains while genotype 2 (adhesin G) reactions amplified DNA only from genotype 2 strains. The overall detection sensitivity and specificity of the newly developed colorimetric LAMP assay for each genotype were 100%. The limits of detection of two LAMP assays were 1-100 target gene copies per reaction. LAMP primers designed in this study may help the differential identification of M. haemolytica genotypes 1 and 2.
溶血曼海姆菌是牛呼吸道疾病复合症（BRDC）的主要细菌性病原体。虽然溶血曼海姆菌已被细分为 12 个荚膜血清型（ST），但 ST1、ST2 和 ST6 通常从牛中分离出来。最近，从北美牛中分离出的溶血曼海姆菌菌株已被分为 1 型（ST2）和 2 型（ST1 和 ST6）基因型。在这两种基因型中，1 型菌株常从健康动物中分离出来，而 2 型菌株主要从 BRDC 动物中分离出来。然而，从肺炎肺部样本中同时分离出这两种基因型会使诊断变得复杂。因此，本研究的目的是开发一种基于比色环介导等温扩增（LAMP）的检测方法来区分溶血曼海姆菌的基因型。
使用来自 22 株溶血曼海姆菌（10 株 1 型，12 株 2 型）和来自 4 种相关巴斯德氏菌科菌株的纯化基因组 DNA 测试了 LAMP 的基因型特异性；比伯氏菌trehalosi、曼海姆氏菌 glucosida、多杀巴斯德氏菌和豪氏霍尔氏菌。1 型（粘附素假基因 B1）特异性-LAMP 反应仅从 1 型菌株扩增 DNA，而 2 型（粘附素 G）反应仅从 2 型菌株扩增 DNA。新开发的比色 LAMP 检测方法对每个基因型的总检测灵敏度和特异性均为 100%。两种 LAMP 检测方法的检测限分别为每个反应 1-100 个靶基因拷贝。本研究设计的 LAMP 引物可能有助于溶血曼海姆菌 1 型和 2 型的差异鉴定。
