Strangles: The Molecular Identification and Epidemiology of Streptococcus equi subsp. equi

A conventional PCR diagnostic test was established to confirm the microbiological isolation of Streptococcus equi subsp. equi (S. equi), the causative agent of strangles. This test was based on the amplification of the seeI gene, which is species-specific for S. equi. Further, a multiplex PCR was developed using species-specific primers; to identify the presence of S. equi and two other streptococci know complicate the diagnosis of strangles in horses, Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subsp. equisimilis (S. equisimilis). A total of 18 clinical isolates of S. equi plus the Pinnacle IN vaccine isolate, two isolates of S. zooepidemicus and four isolates of S. equisimilis were obtained via culture and used in the development of the multiplex diagnostic tool. Two multiplex tests were trialed; a conventional multiplex PCR and a real-time multiplex PCR. Both the conventional and real-time multiplex PCR’s were able to distinguish between the streptococci and accurately identified all isolates. However, further testing on 26 field specimens revealed that the real-time multiplex PCR had lower specificity, sensitivity and diagnostic accuracy as compared to the conventional multiplex PCR. This was theorised to be the result of the PEG/KOH solution used in the DNA extraction, possibly interfering with the intercalating dye in the real-time reaction. Based on these preliminary results, the conventional multiplex PCR diagnostic test developed here is recommended for further trials to determine its robustness. The 19 S. equi isolates obtained, including the vaccine, were further subjected to epidemiological studies. These included sequencing of the variable N-terminal region of the antiphagocytic M-protein SeM to determine SeM allele subtypes and a Sau-PCR amplification method, which previously has not been trialled on S. equi isolates. Sau-PCR involves digestion of genomic DNA and subsequent amplification. Two novel strains of S. equi were found within NZ based on the variable region of the seM gene, SeM alleles 99 and 100. SeM allele 100 had a higher pervalance over allele 99 as it was isolated in 6 out of 9 outbreaks and was found to occur on both the North and South Islands of New Zealand. SeM allele 99 was only found to occur on the North Island. Further to this study, the Pinnacle IN vaccine strain, SeM 2 was isolated from lymph node abcesses of two horses. It was unclear as to whether this ‘vaccine breakdown’ was just a severe adverse reaction to the vaccine or if the vaccine reverted to a more virulent type. The Sau-PCR was able to differentiate between the field isolates of S. equi and the vaccine strain but was unable to further differentiate between the field isolates and was therefore determined not as valuable for S. equi epidemiological studies

Multiplex Detection of Aspergillus fumigatus Mycoviruses.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).Mycoviruses are viruses that naturally infect and replicate in fungi. They are widespread in all major fungal groups including plant and animal pathogenic fungi. Several dsRNA mycoviruses have been reported in Aspergillus fumigatus. Multiplex polymerase chain reaction (PCR) amplification is a version of PCR that enables amplification of different targets simultaneously. This technique has been widely used for detection and differentiation of viruses especially plant viruses such as those which infect tobacco, potato and garlic. For rapid detection, multiplex RT-PCR was developed to screen new isolates for the presence of A. fumigatus mycoviruses. Aspergillus fumigatus chrysovirus (AfuCV), Aspergillus fumigatus partitivirus (AfuPV-1), and Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) dsRNAs were amplified in separate reactions using a mixture of multiplex primer pairs. It was demonstrated that in the presence of a single infection, primer pair mixtures only amplify the corresponding single virus infection. Mixed infections using dual or triple combinations of dsRNA viruses were also amplified simultaneously using multiplex RT-PCR. Up until now, methods for the rapid detection of Aspergillus mycoviruses have been restricted to small scale dsRNA extraction approaches which are laborious and for large numbers of samples not as sensitive as RT-PCR. The multiplex RT-PCR assay developed here will be useful for studies on determining the incidence of A. fumigatus mycoviruses. This is the first report on multiplex detection of A. fumigatus mycovirusesPeer reviewe

Evaluation of a Multiplex PCR Assay for the Identification of Campylobacter jejuni and Campylobacter coli

Background: Campylobacter jejuni and Campylobacter coli are identified as the major causes of acute gastroenteritis in humans. Because of the fastidious nature of Campylobacters, many clinical laboratories fail to routinely culture them. The detection of Campylobacter spp. using molecular-based techniques can be useful for diagnostic and epidemiological applications. This study aimed to develop a multiplex PCR assay for the simultaneous detection of C. jejuni and C. coli strains from clinical specimens. Materials and Methods: During a 19-month period, stool samples were collected from 980 children admitted to a hospital in Tehran, Iran and then examined. The samples were cultured on both Brucella agar and Modified Charcoal-Cefoperazone-Deoxycholate agar (mCCDA) media at 42\ub0C for 48 h. To confirm suspected bacteria, Gram staining and other biochemical tests were carried out. Finally, after extracting DNA from pure cultures using the boiling method, the multiplex PCR assay was performed. Results: The multiplex PCR assay showed that Campylobacter spp. can be detected using 400 bp target product of cadF. It can also accurately distinguish between C. jejuni and C. coli species with different bands of 735 bp and 500 bp using hipO and asp genes, respectively. Conclusions: Results showed that the multiplex PCR assay can replace the biochemical assays for differentiating between C. jejuni and C. coli strains in a single-step PCR test

Use of Whole Genome Phylogeny and Comparisons in the Development of a Multiplex-PCR Assay to Identify Sequence Type 36 Vibrio parahaemolyticus

Vibrio parahaemolyticus sequence type (ST) 36 strains that are native to the Pacific Ocean have recently caused multi-state outbreaks of gastroenteritis linked to shellfish harvested from the Atlantic Ocean. Whole genome comparisons of 295 genomes of V. parahaemolyticus, including several traced to northeastern US sources, were used to identify diagnostic loci: one putatively encoding an endonuclease (prp), and two others potentially conferring O-antigenic properties (cps and flp). The combination of all three loci was present only in one clade of closely-related strains, of ST36, ST59 and one additional unknown sequence type. However, each locus was also identified outside this clade, with prp and flp occurring in only two non-clade isolates, and cps in four. Based on the distribution of these loci in sequenced genomes, prp could identify clade strains with \u3e99% accuracy, but the addition of one more locus would increase accuracy to 100%. Oligonucleotide primers targeting prp and cps were combined in a multiplex PCR method that defines species using the tlh locus, and determines presence of both the tdh and trh hemolysin-encoding genes which are also present in ST36. Application of the method in vitro to a collection of 94 clinical isolates collected over a four year period in three Northeastern US, and 87 environmental isolates, revealed the prp and cps amplicons were only detected in clinical isolates identified as belonging to the ST36-clade, and in no environmental isolates from the region. The assay should improve detection and surveillance, thereby reducing infections

Mutation screening of the medium-chain acyl-CoA dehydrogenase (MCAD) and the ornithine transcarbamylase (OTC) genes by multiplex PCR amplification and sequencing

Background: Sequencing based mutation screening assays of genes encompassing large numbers of exons could be substantially optimized by multiplex PCR, which enables simultaneous amplification of many targets in one reaction. In the present study, a multiplex PCR protocol originally developed for fragment analysis was evaluated for sequencing based mutation screening of the ornithine transcarbamylase (OTC) and the medium-chain acyl-CoA dehydrogenase (MCAD) genes. Methods: Single exon and multiplex PCR protocols were applied to generate PCR templates for subsequent DNA sequencing of all exons of the OTC and the MCAD genes. For each PCR protocol and using the same DNA samples, 66 OTC and 98 MCAD sequence reads were generated. The sequences derived from the two different PCR methods were compared at the level of individual signal-to-noise ratios of the four bases and the proportion of high-quality base-signals. Results: The single exon and the multiplex PCR protocol gave qualitatively comparable results for the two genes. Conclusions: Many existing sequencing based mutation analysis protocols may be easily optimized with the proposed method, since the multiplex PCR protocol was successfully applied without any re-design of the PCR primers and other optimization steps for generating sequencing templates for the OTC and MCAD genes, respectively. Clin Chem Lab Med 2009;47:56-

PlantID – DNA-based identification of multiple medicinal plants in complex mixtures

Background An efficient method for the identification of medicinal plant products is now a priority as the global demand increases. This study aims to develop a DNA-based method for the identification and authentication of plant species that can be implemented in the industry to aid compliance with regulations, based upon the economically important Hypericum perforatum L. (St John’s Wort or Guan ye Lian Qiao). Methods The ITS regions of several Hypericum species were analysed to identify the most divergent regions and PCR primers were designed to anneal specifically to these regions in the different Hypericum species. Candidate primers were selected such that the amplicon produced by each species-specific reaction differed in size. The use of fluorescently labelled primers enabled these products to be resolved by capillary electrophoresis. Results Four closely related Hypericum species were detected simultaneously and independently in one reaction. Each species could be identified individually and in any combination. The introduction of three more closely related species to the test had no effect on the results. Highly processed commercial plant material was identified, despite the potential complications of DNA degradation in such samples. Conclusion This technique can detect the presence of an expected plant material and adulterant materials in one reaction. The method could be simply applied to other medicinal plants and their problem adulterants

Multiplex quantitative PCR for single-reaction genetically modified (GM) plant detection and identification of false-positive GM plants linked to Cauliflower mosaic virus (CaMV) infection.

BACKGROUND:Most genetically modified (GM) plants contain a promoter, P35S, from the plant virus, Cauliflower mosaic virus (CaMV), and many have a terminator, TNOS, derived from the bacterium, Agrobacterium tumefaciens. Assays designed to detect GM plants often target the P35S and/or TNOS DNA sequences. However, because the P35S promoter is derived from CaMV, these detection assays can yield false-positives from non-GM plants infected by this naturally-occurring virus. RESULTS:Here we report the development of an assay designed to distinguish CaMV-infected plants from GM plants in a single multiplexed quantitative PCR (qPCR) reaction. Following initial testing and optimization via PCR and singleplex-to-multiplex qPCR on both plasmid and plant DNA, TaqMan qPCR probes with different fluorescence wavelengths were designed to target actin (a positive-control plant gene), P35S, P3 (a CaMV-specific gene), and TNOS. We tested the specificity of our quadruplex qPCR assay using different DNA extracts from organic watercress and both organic and GM canola, all with and without CaMV infection, and by using commercial and industrial samples. The limit of detection (LOD) of each target was determined to be 1% for actin, 0.001% for P35S, and 0.01% for both P3 and TNOS. CONCLUSIONS:This assay was able to distinguish CaMV-infected plants from GM plants in a single multiplexed qPCR reaction for all samples tested in this study, suggesting that this protocol is broadly applicable and readily transferrable to any interested parties with a qPCR platform

Development of novel multiplex microsatellite polymerase chain reactions to enable high-throughput population genetic studies of Schistosoma haematobium

© 2015 Webster et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. The attached file is the published version of the article