不同熔解曲线分析技术在犬细小病毒基因分型中的比较

Comparison of Different Melting Curve Analysis for Genotyping Canine Parvovirus Variants

本试验旨在建立基于聚合酶链式反应(PCR)技术的犬细小病毒2型(CPV-2)2种不同熔解曲线技术基因分型方法,并通过比较其熔解温度差异(△Tm)评价不同方法对犬细小病毒单核苷酸多态性(SNP)位点的分型鉴别能力。结果显示,探针熔解曲线聚合酶链式反应(FMCA-PCR)方法中不同SNP位点之间熔解温度差异(△Tm)均大于3℃,而在高分辨率熔解曲线聚合酶链式反应(HRM-PCR)方法中熔解温度差异(△Tm)只有0.2~0.6℃。临床30份CPV-2核酸样本基因分型结果显示,FMCA-PCR方法的准确率为100%;而HRM-PCR方法准确率为96.6%(未形成杂合子前)。虽然HRM-PCR方法中不同SNP位点之间熔解温度差异较小,影响其基因分型结果,但通过杂合子HRM分析后可提高该方法的准确率。本试验结果表明,基于PCR方法的2种不同熔解曲线基因分型技术均具有较好的SNP位点分型鉴别能力,为熔解曲线基因分型技术在兽医临床中的应用提供实际参考依据。

This study aimed to establish two kinds of melting curve analysis in conjunction with polymerase chain reaction(PCR)for genotyping of canine parvovirus type 2(CPV-2)variants and to assess the discriminatory power of genotyping for identifying canine parvovirus single nucleotide polymorphism(SNP)sites by melting temperature difference(△Tm).The results showed that the melting temperature differences(△Tm)between the different SNP sites was greater than 3℃ in the FMCA-PCR method,while it was only 0.2-0.6℃ in the HRM-PCR method.The genotyping results of 30 clinical CPV-2 nucleic acid samples showed that the accuracy of the FMCA-PCR method was 100%;while it was 96.6% in the HRM-PCR method before forming a heterozygote.The difference(△Tm)between SNP sites of CPV-2 was too small to clearly distinguish the genotype in the HRM-PCR method,however,genotyping accuracy can be improved by forming heteroduplexes.This study confirmed that different SNP loci of canine parvovirus are identifiable by both of two melting curve analysis,which can be readily adopted in veterinary clinic.