Dataset Information

Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants.

ABSTRACT: Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs worldwide. Since CPV-2 emerged as canine pathogen, the original CPV-2 strain has constantly evolved, and its primary variants (CPV-2a, CPV-2b, and CPV-2c) co-circulate to varying extents in canine populations worldwide. Thus, rapid and accurate laboratory diagnoses of CPV-2 variants are crucial to monitor CPV-2 evolution. Conventional methods for CPV-2 genotyping are laborious, time consuming, and determining the genotype of a CPV-2 variant often requires two or more reaction tubes. The present study developed a probe-based fluorescence melting curve analysis (FMCA) for genotyping six different CPV-2 variants (original CPV-2, CPV-2a, CPV-2b, CPV-2c, and vaccine strains of CPVpf and CPVint) in a single reaction tube using only two TaqMan probes. One of the TaqMan probes (FAM labeled) was designed to perfectly match with the target sequence of CPV-2a, this probe allows a 1-bp mismatched hybridization with the CPV-2b VP2 gene region (A4062G), and a 2-bp mismatched hybridization for CPV-2c (A4062G and T4064A); Another TaqMan probe (HEX labeled) was produced to perfectly match with the target sequence of original CPV-2, this probe enables 1-bp mismatched hybridization with the other CPV-2 variants (A3045T). Using the two TaqMan probes, all six CPV-2 variants were readily distinguished by their respective melting temperature values in a single reaction tube. The detection limits of this assay were 1-10 copies per reaction for six CPV-2 construction plasmids and no cross reactions were observed with several other common canine viruses. In this assay, co-infected samples were also directly identified via probe-based FMCA without using a mixing control; only a pure control is required. The clinical evaluation of this assay was demonstrated by analyzing 83 clinical fecal samples, among which 41 (49.39%), 8 (9.63%), and 14 (16.87%) samples were found to be positive for CPV-2a, CPV-2b, and CPV-2c, respectively. The concordance rate between probe-based FMCA and Sanger sequencing was 100%. Thus, the duplex FMCA is effective, rapid, simple, high-throughput, and straightforward for genotyping CPV-2 variants, and is useful to effectively diagnose and monitor CPV-2 epidemiology.

SUBMITTER: Liu Z

PROVIDER: S-EPMC6411689 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants.

Liu Zhicheng Z Bingga Gali G Zhang Chunhong C Shao Junjie J Shen Haiyan H Sun Junying J Zhang Jianfeng J

Frontiers in microbiology 20190305

Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs worldwide. Since CPV-2 emerged as canine pathogen, the original CPV-2 strain has constantly evolved, and its primary variants (CPV-2a, CPV-2b, and CPV-2c) co-circulate to varying extents in canine populations worldwide. Thus, rapid and accurate laboratory diagnoses of CPV-2 variants are crucial to monitor CPV-2 evolution. Conventional methods for CPV-2 genotyping are laborious, time consuming, and determining t ...[more]

PMID: 30891024

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Project description:BackgroundRecently, pseudorabies (PR) outbreaks have been reported in a large number of swine herds vaccinated with the Bartha-K61 vaccine in China, the current pseudorabies virus (PRV) belonging to Genotype II is differential genetically from Bartha-K61 vaccine belonging to Genotype I. Furthermore, it has been proved that the Bartha-K61 vaccine cannot provide sufficient protection against the current PRVs in China. Therefore, the accurate and rapid identification of PRVs is essential. The objective of this study is to develop a duplex fluorescence melting curve analysis (FMCA) capable of rapid, simple, high-throughput differentiation of Chinese, European/American and Bartha-K61 vaccine strains of PRV.ResultsPrimers 6F/6R and probes P1/P2, combined with three recombinant plasmids p-B (Bartha-K61), p-N (Genotype I), and p-H (Genotype II), were used to establish the Bicolor FMCA. FAM Tm values (probe P1) and HEX (probe P2) channels of p-B were used as reference values. Tm differences (ΔTm) between detected samples and reference plasmid p-B were calculated in each channel. Bartha-K61 vaccine samples had ΔTm values of ±1 °C in both FAM and HEX channels, Genotype I samples had ΔTm values of ±1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel, and Genotype II samples had ΔTm values of 6.52 ± 1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel. The minimum detection limit of the duplex FMCA was approximately 1 × 100 copies per reaction for p-B, p-N, and p-H. The duplex FMCA technique was used to detect and different 198 suspected clinical samples, of which 18 (9%) were positive for Genotype II strains and eight (4%) were positive for Bartha-K61 vaccine strains, and the results were compared with sequencing and phylogenetic analyses, which confirmed that the Bicolor FMCA worked correctly for all samples.ConclusionsIn this study, we developed a duplex FMCA of dual-labeled, self-quenched probes that was performed for rapid detection and differentiation of Genotype I, II and Bartha-K61 vaccine strains of PRV. The duplex FMCA was rapid, simple, and high-throughput, and will likely prove useful for molecular epidemiological investigations and pathogen surveillance of PRV.

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

Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants.

Publications

Application of Duplex Fluorescence Melting Curve Analysis (FMCA) to Identify Canine Parvovirus Type 2 Variants.

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