Development of a Loop-Mediated Isothermal Amplification Assay Coupled With a Lateral Flow Dipstick Test for Detection of Myosin Binding Protein C3 A31P Mutation in Maine Coon Cats.

Soontaree Petchdee,Kanokwan Demeekul,Pratch Sukumolanan

doi:10.3389/fvets.2022.819694

Soontaree Petchdee, Kanokwan Demeekul + Show 1 more

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https://doi.org/10.3389/fvets.2022.819694

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Abstract

BackgroundMyosin-binding protein C3 A31P (MYBPC3-A31P) missense mutation is a genetic deviation associated with the development of hypertrophic cardiomyopathy (HCM) in Maine Coon cats. The standard detection of the MYBPC3-A31P 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 MYBPC3-A31P mutations in Maine Coon cats.Materials and MethodsFifty-five Maine Coon cats were enrolled in this study, and blood samples were collected. MYBPC3-A31P 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 A31P-LAMP detection was compared between agarose gel electrophoresis (the standard method) and the LFD test. The A31P-LAMP-LFD test was randomly performed on seven cats (four with the A31P mutation and three wild-type cats).ResultsThe A31P-LAMP procedure was able to distinguish between cats with MYBPC3-A31P wild-type cats and MYBPC3-A31P 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 A31P-LAMP coupled with the LFD test allowed for A31P genotype detection at a lower DNA concentration than agarose gel electrophoresis.DiscussionsThis new A31P-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 MYBPC3-A31P mutations. Thus, this assay has excellent potential and can be recognized as a novel screening test for hypertrophic cardiomyopathy associated with MYBPC3-A31P mutations in felines.

Highlights

Hypertrophic cardiomyopathy (HCM) is the most common hereditary cardiovascular disease in cats [1–4]
This study aimed to develop a specific and rapid diagnostic tool for detecting MYBPC3-A31P mutations in Maine Coon cats using a loop-mediated isothermal amplification assay (LAMP) assay coupled with an lateral flow dipstick (LFD)
The results showed that 0.00001 ng/μl of DNA concentration is the minimal amount that was able to detect A31P mutation with the LFD test in all seven cats (Figure 7)

Summary

Introduction

Hypertrophic cardiomyopathy (HCM) is the most common hereditary cardiovascular disease in cats [1–4]. The HCM phenotype has a prevalence of approximately 15% in cats [5]. The HCM phenotype in cats is related to an increase in age. Specific breeds are highly associated with HCM development, especially the Maine Coon [10] and Ragdoll [11]. Myosin binding protein C3 A31P (MYBPC3-A31P), a single nucleotide polymorphism (SNP), was possibly associated with HCM development in cats, especially Maine Coon cats [12]. Myosin-binding protein C3 A31P (MYBPC3-A31P) missense mutation is a genetic deviation associated with the development of hypertrophic cardiomyopathy (HCM) in Maine Coon cats. This study aimed to develop a loop-mediated isothermal amplification assay (LAMP) coupled with a lateral flow dipstick (LFD) test to detect MYBPC3-A31P mutations in Maine Coon cats

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