Dataset Information

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities.

ABSTRACT: Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and "Heyl", Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.

SUBMITTER: Miller M

PROVIDER: S-EPMC4640598 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities.

Miller Manuel M Zorn Julia J Brielmeier Markus M

PloS one 20151110 11

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes "Jawetz" and ...[more]

PMID: 26556281

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Project description:BackgroundEarthworm communities are an important component of soil biodiversity and contribute to a number of ecosystem functions such as soil-nutrient cycling. Taxonomic identification is an essential requirement to assess earthworm biodiversity and functionality. Although morphological identification of species is labour-intensive, it is the most commonly used method due to a lack of cost-efficient alternatives. Molecular approaches to identify earthworms at species and haplotype level such as DNA barcoding are gaining popularity in science but are rarely applied in practice. In contrast to barcoding, the differentiation of PCR products based on their thermal denaturation properties using high-resolution melting (HRM) curve analysis is a fast and cost-efficient molecular closed-tube, post-PCR tool that allows identification of taxa.MethodsWe developed a HRM curve assay to identify eight earthworm species common to agricultural soils in Central Europe (Allolobophora chlorotica, Aporrectodea caliginosa, Apo. limicola, Apo. longa, Apo. rosea, Lumbricus castaneus, L. rubellus, and L. terrestris). For this, a new primer pair targeting a 158-bp long subregion of the cytochrome c oxidase I (COI) gene was designed. Our HRM assay was further tested for the differentiation of COI haplotypes using 28 individuals of the earthworm species Allo. chlorotica. Furthermore, we developed a novel extraction method for DNA from earthworm tissue that is fast and requires minimal consumables and laboratory equipment.ResultsThe developed HRM curve assay allowed identifying all eight earthworm species. Performing the assay on 28 individuals of the earthworm species Allo. chlorotica enabled the distinction among different COI haplotypes. Furthermore, we successfully developed a rapid, robust, scalable, and inexpensive method for the extraction of earthworm DNA from fresh or frozen tissue.ConclusionsHRM curve analysis of COI genes has the potential to identify earthworm species and haplotypes and could complement morphological identification, especially for juvenile or damaged individuals. Our rapid and inexpensive DNA extraction method from earthworm tissue helps to reduce the costs of molecular analyses and thereby promote their application in practice.

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:Diagnosis of enterotoxigenic E. coli (ETEC) associated diarrhea is complicated by the diversity of E.coli virulence factors. This study developed a multiplex quantitative PCR assay based on high-resolution melting curves analysis (HRM-qPCR) to identify and quantify genes encoding five ETEC fimbriae related to diarrhea in swine, i.e. K99, F41, F18, F6 and K88.Five fimbriae expressed by ETEC were amplified in multiple HRM-qPCR reactions to allow simultaneous identification and quantification of five target genes. The assay was calibrated to allow quantification of the most abundant target gene, and validated by analysis of 30 samples obtained from piglets with diarrhea and healthy controls, and comparison to standard qPCR detection.The five amplicons with melting temperatures (Tm) ranging from 74.7 ± 0.06 to 80.5 ± 0.15 °C were well-separated by HRM-qPCR. The area of amplicons under the melting peak correlated linearly to the proportion of the template in the calibration mixture if the proportion exceeded 4.8% (K88) or <1% (all other amplicons). The suitability of the method was evaluated using 30 samples from weaned pigs aged 6-7 weeks; 14 of these animals suffered from diarrhea in consequence of poor sanitary conditions. Genes encoding fimbriae and enterotoxins were quantified by HRM-qPCR and/or qPCR. The multiplex HRM-qPCR allowed accurate analysis when the total gene copy number of targets was more than 1 × 105 / g wet feces and the HRM curves were able to simultaneously distinguish fimbriae genes in the fecal samples. The relative quantification of the most abundant F18 based on melting peak area was highly correlated (P < 0.001; r2 = 0.956) with that of individual qPCR result but the correlation for less abundant fimbriae was much lower.The multiplex HRM assay identifies ETEC virulence factors specifically and efficiently. It correctly indicated the predominant fimbriae type and additionally provides information of presence/ absence of other fimbriae types and it could find broad applications for pathogen diagnosis.

Dataset Information

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities.

Publications

High-Resolution Melting Curve Analysis for Identification of Pasteurellaceae Species in Experimental Animal Facilities.

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