APPLICATION OF HIGH-RESOLUTION MELTING (HRM) ASSAY WITH EVAGREEN DYE AS A RAPID SCREENING OF CYTOMEGALOVIRUS (CMV) DRUG RESISTANCE MUTATIONS AND ITS CHALLENGES

Abstract

Intro: HRM is being utilized for identification of bacteria and viruses harbouring gene mutations that affect pathogenicity and/or drug susceptibility. We have used the HRM technique to detect the two common UL97 protein mutations in CMV; M460V/M460I and H520Q. These mutations confer resistant to the first line antiviral, ganciclovir, and associated with high mortality infection. Methods: Clinical samples of HIV patients with known CMV infection were extracted and subjected to CMV drug-resistant testing. HRM was developed using specific primer targeting the UL97 gene with EvaGreen dye. DNA plasmid carrying gene sequence for wild-type CMV and UL97 M460V mutation were constructed, where they were distinguished by the differences in the melting peak temperature (Tm), 89.49°C and 89.71°C, respectively. Findings: 40 clinical samples with satisfactory PCR amplification were proceeded directly with HRM. Out of 40 samples, 36 samples were wild type, 2 samples were inconclusive, and 2 samples were suggestive of UL97 mutants. The suggestive mutant samples were subjected to Sanger sequencing analysis. The analysis showed no M460V/I or H520Q mutation detected, but new mutations, N407H and T409K identified in the mutant samples. Discussion: A small Tm variance (0.22°C) in between wild-type and M460V mutant virus could be contributed by the large amplicon size. It affected the HRM assay's capability to detect single nucleotide polymorphism (SNP), especially when non-specific mutations co-existed. A smaller amplicon provides a discrete Tm difference, but could miss to detect other drug-resistant mutations. The newly discovered N407H and T409K mutation in the UL97 mutant samples proves the assay's sensitivity to detect genetic variance. Both mutations have yet to be reported to associate with CMV drug resistance. Conclusion: A direct HRM PCR using EvaGreen dye can potentially screen genetic variations in CMV. However, the usage is limited by the size of the amplicon and the distribution of the mutations of interest.