Development of a Loop-Mediated Isothermal Amplification Assay Coupled With a Lateral Flow Dipstick Test for Detection of Myosin Binding Protein C3Mutation in Maine Coon Cats.

Abstract

BACKGROUND: 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 themutation 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 detectmutations in Maine Coon cats. MATERIALS AND METHODS: 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 themutation and three wild-type cats). RESULTS: The-LAMP procedure was able to distinguish between cats withwild-type cats andmutant 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 forgenotype detection at a lower DNA concentration than agarose gel electrophoresis. DISCUSSIONS: 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 ofmutations. Thus, this assay has excellent potential and can be recognized as a novel screening test for hypertrophic cardiomyopathy associated withmutations in felines.