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Genotyping measles virus by real-time amplification refractory mutation system PCR represents a rapid approach for measles outbreak investigations.

ABSTRACT: Real-time PCR has been developed to genotype measles virus (MV) isolates. MV strains circulating in epidemics in Gabon in 1984, Cameroon in 2001, Morocco in 2003, and France in 2004 were investigated. We developed a real-time amplification refractory mutation system PCR (RT-AMRS PCR) using SYBR green fluorescent dye. Six pairs of primers for RT-ARMS PCR were designed to specifically amplify genotypes A, B2, B3.1, B3.2, C2, and D7. Genotypes could be differentiated by melting curve analysis. All strains were also confirmed by direct sequencing. Using the result obtained by direct sequencing and phylogenetic analysis as the reference, the accuracy of MV by RT-ARMS PCR and melting curve analysis was 97%. However, the latter method is more rapid and sensitive than the former method. This method could be a useful tool for molecular epidemiological studies of MV, providing an efficient alternative for large-scale studies.

SUBMITTER: Waku-Kouomou D

PROVIDER: S-EPMC1392642 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Genotyping measles virus by real-time amplification refractory mutation system PCR represents a rapid approach for measles outbreak investigations.

Waku-Kouomou Diane D Alla Amal A Blanquier Bariza B Jeantet Damien D Caidi Hayat H Rguig Ahmed A Freymuth François F Wild Fabian T FT

Journal of clinical microbiology 20060201 2

Real-time PCR has been developed to genotype measles virus (MV) isolates. MV strains circulating in epidemics in Gabon in 1984, Cameroon in 2001, Morocco in 2003, and France in 2004 were investigated. We developed a real-time amplification refractory mutation system PCR (RT-AMRS PCR) using SYBR green fluorescent dye. Six pairs of primers for RT-ARMS PCR were designed to specifically amplify genotypes A, B2, B3.1, B3.2, C2, and D7. Genotypes could be differentiated by melting curve analysis. All ...[more]

PMID: 16455903

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Project description:Infection clusters of multidrug-resistant bacteria increase mortality and entail expensive infection control measures. Whereas whole-genome sequencing (WGS) is the current gold standard to confirm infection clusters, PCR-based assays targeting cluster-specific signatures, such as single nucleotide polymorphisms (SNPs) derived from WGS data, are more suitable to initially screen for cluster isolates within large sample sizes. Here, we evaluated four software tools (SeqSphere+, RUCS, Gegenees, and Find Differential Primers) regarding their efficiency to find SNPs within WGS data sets that were specific for two bacterial monospecies infection clusters but were absent from a WGS reference data set comprising several hundred diverse genotypes of the same bacterial species. Cluster-specific SNPs were subsequently used to establish a probe-based real-time PCR screening assay for in vitro differentiation between cluster and noncluster isolates. SeqSphere+ and RUCS found 2 and 24 SNPs for clusters 1 and 14 and 24 SNPs for cluster 2, respectively. However, some signatures detected by RUCS were not cluster specific. Interestingly, all SNPs identified by SeqSphere+ were also detected by RUCS. In contrast, analyses with the remaining tools either resulted in no SNPs (with Find Differential Primers) or failed (Gegenees). Design of six cluster-specific real-time PCR assays enabled reliable cluster screening in vitro. Our evaluation revealed that SeqSphere+ and RUCS identified cluster-specific SNPs that could be used for large-scale screening in surveillance samples via real-time PCR, thereby complementing WGS efforts. This faster and simplified approach for the surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians. IMPORTANCE Infection clusters of multidrug-resistant bacteria threaten medical facilities worldwide and cause immense health care costs. In recent years, whole-genome sequencing (WGS) has been increasingly applied to detect and to further control bacterial clusters. However, as WGS is still expensive and time-consuming, its exclusive application for screening and confirmation of bacterial infection clusters contributes to high costs and enhanced turnaround times, which many hospitals cannot afford. Therefore, there is need for alternative methods that can enable further surveillance of bacterial clusters that are initially detected by WGS in a faster and more cost-efficient way. Here, we established a system based on real-time PCR that enables rapid large-scale sample screening for bacterial cluster isolates within 7 days after the initial detection of an infection cluster, thereby complementing WGS efforts. This faster and simplified surveillance of bacterial clusters will improve infection control measures and will enhance protection of patients and physicians.

Dataset Information

Genotyping measles virus by real-time amplification refractory mutation system PCR represents a rapid approach for measles outbreak investigations.

Publications

Genotyping measles virus by real-time amplification refractory mutation system PCR represents a rapid approach for measles outbreak investigations.

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