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Randomly amplified polymorphic DNA (RAPD) has been used for rapid typing of Lactobacillus plantarum strains. RAPD was used with either purified chromosomal DNA serving as template in the polymerase chain reaction, or with crude cell extracts, and using a 9-mer primer with 80% G+C content. Amplified DNA was visualized by ethidium bromide staining after separation on agarose gels. Patterns from 20 Lact. plantarum strains and two Lact. pentosus strains were analysed using the Pearson products moment correlation coefficient (r) and the unweighted pair group method with arithmetic averages (UPGMA). With some exceptions, the two sources of template DNA gave the same clusters and subclusters of strains at the similarity level of 50%. About 50% of the strains could be individually separated from all the other tested strains. The buffer brand, the amount of primer and crude cell extract used in the PCR-step were crucial for the final pattern.
The opportunistic human pathogen Propionibacterium acnes is composed of a number of distinct phylogroups, designated types IA1, IA2, IB, IC, II, and III, which vary in their production of putative virulence factors, their inflammatory potential, and their biochemical, aggregative, and morphological characteristics. Although multilocus sequence typing (MLST) currently represents the gold standard for unambiguous phylogroup classification and individual strain identification, it is a labor-intensive and time-consuming technique. As a consequence, we developed a multiplex touchdown PCR assay that in a single reaction can confirm the species identity and phylogeny of an isolate based on its pattern of reaction with six primer sets that target the 16S rRNA gene (all isolates), ATPase (types IA1, IA2, and IC), sodA (types IA2 and IB), atpD (type II), and recA (type III) housekeeping genes, as well as a Fic family toxin gene (type IC). When applied to 312 P. acnes isolates previously characterized by MLST and representing types IA1 (n=145), IA2 (n=20), IB (n=65), IC (n=7), II (n=45), and III (n=30), the multiplex displayed 100% sensitivity and 100% specificity for detecting isolates within each targeted phylogroup. No cross-reactivity with isolates from other bacterial species was observed. This multiplex assay will provide researchers with a rapid, high-throughput, and technically undemanding typing method for epidemiological and phylogenetic investigations. It will facilitate studies investigating the association of lineages with various infections and clinical conditions, and it will serve as a prescreening tool to maximize the number of genetically diverse isolates selected for downstream higher-resolution sequence-based analyses.
Genotype 2M. haemolytica predominantly associate over genotype 1 with the lungs of cattle with respiratory disease and ICEs containing antimicrobial resistance genes. Distinct protein masses were detected by MALDI-TOF MS between genotype 1 and 2 strains. MALDI-TOF MS could rapidly differentiate genotype 2 strains in veterinary diagnostic laboratories.
Coxiella burnetii has the potential to cause serious disease and is highly prevalent in the environment. Despite this, epidemiological data are sparse and isolate collections are typically small, rare, and difficult to share among laboratories as this pathogen is governed by select agent rules and fastidious to culture. With the advent of whole genome sequencing, some of this knowledge gap has been overcome by the development of genotyping schemes, however many of these methods are cumbersome and not readily transferable between institutions. As comparisons of the few existing collections can dramatically increase our knowledge of the evolution and phylogeography of the species, we aimed to facilitate such comparisons by extracting SNP signatures from past genotyping efforts and then incorporated these signatures into assays that quickly and easily define genotypes and phylogenetic groups. We found 91 polymorphisms (SNPs and indels) among multispacer sequence typing (MST) loci and designed 14 SNP-based assays that could be used to type samples based on previously established phylogenetic groups. These assays are rapid, inexpensive, real-time PCR assays whose results are unambiguous. Data from these assays allowed us to assign 43 previously untyped isolates to established genotypes and genomic groups. Furthermore, genotyping results based on assays from the signatures provided here are easily transferred between institutions, readily interpreted phylogenetically and simple to adapt to new genotyping technologies.
Sequence based DNA signatures are widely used for molecular typing as they provide unambiguous results that are easily transferred and compared between labs. In this era of rapid and inexpensive sequencing, whole genome sequence comparisons often reveal many polymorphisms that can be used to develop new assays for increased discrimination among samples and to better define phylogenetic relatedness. Despite drastic reductions in cost, whole genome sequencing is still expensive relative to other typing technologies. As well, the data handling, processing, and interpretation required for whole genome sequence analyses make sub-genome typing methods more viable when many samples need processing. For phylogenetic and population genetic inferences, a large sample size is also important as samples are compared to each other and accuracy of conclusions is directly tied to comprehensive sampling. Unfortunately, switching to new typing methods often results in lost information between old and new systems as data cannot be directly compared. As such, past data and efforts may be simply discarded or, when possible, old samples may be re-analyzed with the new typing scheme (for example, see [1]). Ideally, new signatures or assays should not only be transferrable between labs, but also enable newly typed samples to be directly compared to existing collections. For Coxiella burnetii, it is particularly important to compare typing results to other collections as C. burnetii collections are rare, sparse and not easily transferred due to select agent regulations and biosecurity concerns. In order to better understand epidemiological patterns we have therefore built upon an existing sequence based typing scheme to produce a few simple and rapid assays whose results are unambiguous, easily transferrable, and can be directly compared to the largest characterized collection of C. burnetii in the world.
Despite the serious nature of Q fever, little is known about the prevalence and dissemination patterns of C. burnetii. Most genotyping methods are cumbersome and require relatively large quantities of DNA. Before the very recent development of a cell-free growth procedure [6], propagation required cell tissue culture or proliferation in embryonated eggs. Even with this significant improvement, culturing still requires a select agent facility, considerable expertise, and is a slow process. Thus, in the rare instances where a case of Q fever is identified, it is not likely that a sample will be successfully cultured and genotyped. Therefore, tools that facilitate the comparison of isolates or field-collected strains are particularly important.
We developed genotyping assays based on 14 SNPs. Twelve SNPs that define the major clades were used to develop Melt-MAMA assays (Table 1) as described by Vogler et al. [14]. Briefly, the melt-MAMA design utilizes allele-specific mismatch amplification mutation assay primers [15] coupled with GC- or T-rich primer tails. These tails force allele specific melt properties for PCR amplicons, allowing allelic differentiation via melt curve analysis. Two other SNPs from MST allele comparisons were used to develop TaqMan minor groove binding dual-probe assays according to Easterday et al. [16] (Table 2) and to illustrate that multiple SNP-interrogation methods can be used to assay SNP signatures.
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