Dataset Information

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation.

ABSTRACT: Background and Aim:Belonging to the Coronaviridae family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carried out a quantitative real-time polymerase chain reaction followed by high-resolution melting (qRT-PCR/HRM) curve analysis. Materials and Methods:In this study, qRT-PCR was conducted on a partial fragment S1 gene followed by a high resolution melting curve analysis (qRT-PCR/HRM) on 23 IBV-positive samples in Jordan. For this assay, we utilized the most common IBV vaccine strains (Mass and 4/91) as a reference in the HRM assay. To evaluate the discrimination power of the qRT-PCR/HRM, we did the sequencing of the partial S1 gene. Results:It was shown that HRM was able to classify IBV samples into four clusters based on the degree of similarity between their melting points: The first cluster exhibited the highest similarity to the 4/91 strain, while the second was similar to the Mass-related IBV strain. Although the third cluster contained the highest number of samples, it displayed no similarity to any of the reference vaccine strains, and, after comparing them with the sequencing results, we found that the samples in the third cluster were similar to the variant II-like (IS-1494-06) IBV field strain. Finally, the fourth cluster comprised one unique sample that was found to belong to the Q1 IBV strain. Conclusion:Our developed qRT-PCR/HRM curve analysis was able to detect and rapidly identify novel and vaccine-related IBV strains as confirmed by S1 gene nucleotide sequences, making it a rapid and cost-effective tool.

SUBMITTER: Ababneh M

PROVIDER: S-EPMC7183480 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation.

Ababneh Mustafa M Ababneh Ola O Al-Zghoul Mohammad Borhan MB

Veterinary world 20200303 3

<h4>Background and aim</h4>Belonging to the <i>Coronaviridae</i> family, avian infectious bronchitis virus (IBV) causes respiratory, reproductive, and renal diseases in poultry. Preventative measures lie mainly in vaccination, while the gold standard for IBV classification and differentiation is based on the sequence analysis of the spike 1 (S1) gene. In this study, we tested a new assay for IBV strain classification that is less expensive and requires reduced time and effort to perform. We carr ...[more]

PMID: 32367941

Similar Datasets

Project description:With the goal to generate and characterize the phenotypes of null alleles in all genes within an organism and the recent advances in custom nucleases, genome editing limitations have moved from mutation generation to mutation detection. We previously demonstrated that High Resolution Melting (HRM) analysis is a rapid and efficient means of genotyping known zebrafish mutants. Here we establish optimized conditions for HRM based detection of novel mutant alleles. Using these conditions, we demonstrate that HRM is highly efficient at mutation detection across multiple genome editing platforms (ZFNs, TALENs, and CRISPRs); we observed nuclease generated HRM positive targeting in 1 of 6 (16%) open pool derived ZFNs, 14 of 23 (60%) TALENs, and 58 of 77 (75%) CRISPR nucleases. Successful targeting, based on HRM of G0 embryos correlates well with successful germline transmission (46 of 47 nucleases); yet, surprisingly mutations in the somatic tail DNA weakly correlate with mutations in the germline F1 progeny DNA. This suggests that analysis of G0 tail DNA is a good indicator of the efficiency of the nuclease, but not necessarily a good indicator of germline alleles that will be present in the F1s. However, we demonstrate that small amplicon HRM curve profiles of F1 progeny DNA can be used to differentiate between specific mutant alleles, facilitating rare allele identification and isolation; and that HRM is a powerful technique for screening possible off-target mutations that may be generated by the nucleases. Our data suggest that micro-homology based alternative NHEJ repair is primarily utilized in the generation of CRISPR mutant alleles and allows us to predict likelihood of generating a null allele. Lastly, we demonstrate that HRM can be used to quickly distinguish genotype-phenotype correlations within F1 embryos derived from G0 intercrosses. Together these data indicate that custom nucleases, in conjunction with the ease and speed of HRM, will facilitate future high-throughput mutation generation and analysis needed to establish mutants in all genes of an organism.

Project description:Current methods for the detection of single nucleotide polymorphisms (SNPs) associated with aberrant drug-metabolizing enzyme function are hindered by long turnaround times and specialized techniques and instrumentation. In this study, we describe the development and validation of a high-resolution melting (HRM) curve assay for the rapid screening of variant genotypes for targeted genetic polymorphisms in the cytochrome P450 enzymes CYP2C9, CYP2C19, and CYP3A5.Sequence-specific primers were custom-designed to flank nine SNPs within the genetic regions of aforementioned drug metabolizing enzymes. PCR amplification was performed followed by amplicon denaturation by precise temperature ramping in order to distinguish genotypes by melting temperature (Tm). A standardized software algorithm was used to assign amplicons as 'reference' or 'variant' as compared to duplicate reference sequence DNA controls for each SNP.Intra-assay (n=5) precision of Tms for all SNPs was ?0.19%, while inter-assay (n=20) precision ranged from 0.04% to 0.21%. When compared to a reference method of Sanger sequencing, the HRM assay produced no false negative results, and overcall frequency ranged from 0% to 26%, depending on the SNP. Furthermore, HRM genotyping displayed accuracy over input DNA concentrations ranging from 10 to 200 ng/?L.The presented assay provides a rapid method for the screening for genetic variants in targeted CYP450 regions with a result of 'reference' or 'variant' available within 2 h from receipt of extracted DNA. The method can serve as a screening approach to rapidly identify individuals with variant sequences who should be further investigated by reflexed confirmatory testing for aberrant cytochrome P450 enzymatic activity. Rapid knowledge of variant status may aid in the avoidance of adverse clinical events by allowing for dosing of normal metabolizer patients immediately while identifying the need to wait for confirmatory testing in those patients who are likely to possess pharmacogenetically-relevant variants.

Dataset Information

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation.

Publications

High-resolution melting curve analysis for infectious bronchitis virus strain differentiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets