Sukumolanan Pratch, Demeekul Kanokwan, Petchdee Soontaree
Program of Veterinary Clinical Studies, Graduate School, Kasetsart University, Nakorn Pathom, Thailand.
Department of Cardio-Thoracic Technology, Faculty of Allied Health Sciences, Naresuan University, Phitsanulok, Thailand.
Front Vet Sci. 2022 Mar 7;9:819694. doi: 10.3389/fvets.2022.819694. eCollection 2022.
Myosin-binding protein C3 () missense mutation is a genetic deviation associated with the development of hypertrophic cardiomyopathy (HCM) in Maine Coon cats. The standard detection of the mutation is complicated, time-consuming, and expensive. Currently, there has been a focus on the speed and reliability of diagnostic tools. Therefore, this study aimed to develop a loop-mediated isothermal amplification assay (LAMP) coupled with a lateral flow dipstick (LFD) test to detect mutations in Maine Coon cats.
Fifty-five Maine Coon cats were enrolled in this study, and blood samples were collected. was genotyped by DNA sequencing. Primers for LAMP with a LFD test were designed. The optimal conditions were determined, including temperature and time to completion for the reaction. The sensitivity of -LAMP detection was compared between agarose gel electrophoresis (the standard method) and the LFD test. The -LAMP-LFD test was randomly performed on seven cats (four with the mutation and three wild-type cats).
The -LAMP procedure was able to distinguish between cats with wild-type cats and mutant cats. The LAMP reactions were able to be completed in 60 min at a single temperature of 64◦C. Moreover, this study demonstrated that -LAMP coupled with the LFD test allowed for genotype detection at a lower DNA concentration than agarose gel electrophoresis.
This new -LAMP with a LFD test is a successful and reliable assay with a rapid method, cost-effectiveness, and low requirements for sophisticated equipment for the detection of mutations. Thus, this assay has excellent potential and can be recognized as a novel screening test for hypertrophic cardiomyopathy associated with mutations in felines.
肌球蛋白结合蛋白C3（）错义突变是一种与缅因猫肥厚型心肌病（HCM）发生相关的基因变异。该突变的标准检测方法复杂、耗时且昂贵。目前，诊断工具的速度和可靠性备受关注。因此，本研究旨在开发一种环介导等温扩增法（LAMP）结合侧向流动试纸条（LFD）检测法，用于检测缅因猫中的突变。
本研究纳入了55只缅因猫，并采集了血液样本。通过DNA测序对进行基因分型。设计了用于LAMP结合LFD检测的引物。确定了最佳条件，包括反应的温度和完成时间。比较了琼脂糖凝胶电泳（标准方法）和LFD检测法对-LAMP检测的灵敏度。对7只猫（4只携带突变和3只野生型猫）随机进行-LAMP-LFD检测。
-LAMP程序能够区分野生型猫和突变型猫。LAMP反应能够在64℃的单一温度下60分钟内完成。此外，本研究表明，与琼脂糖凝胶电泳相比，-LAMP结合LFD检测能够在更低的DNA浓度下进行基因型检测。
这种新的-LAMP结合LFD检测是一种成功且可靠的检测方法，具有快速、成本效益高以及对复杂设备要求低的特点，用于检测突变。因此，该检测方法具有巨大潜力，可被视为一种针对猫肥厚型心肌病相关突变的新型筛查检测方法。
