Dynamic multiplex PCR: A pioneering approach to rapid, cost-effective, and high-quality microsatellite fragment analysis

Event Abstract Back to Event Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish Ana R. Ferreira1, Teresa M. Baptista2 and Clélia N. Afonso2* 1 Escola Superior de Turismo e Tecnologia do Mar, Politécnico de Leiria, Portugal 2 Escola Superior de Turismo e Tecnologia do Mar, Politécnico de Leiria, MARE – Marine and Environmental Sciences Centre, Portugal The aquaculture sector has registered a significant growth in the last years; however, the presence of infectious diseases is a serious threat, leading to huge losses in this sector. Bacteria belonging to the genus Vibrio (Toranzo et al., 2005) cause some of these diseases. It is of uttermost importance to create methods to detect precociously the presence of these bacteria in aquaculture, in order to prevent potential outbreaks that may arise and minimize the consequences (Martins et al., 2015; Shi et al., 2012; Sorroza et al., 2012). A critical step in the study of bacterial fish diseases is the correct identification of the infectious agent (Avendaño-Herrera et al., 2004), but classical microbiological methods does not allow distinguishing the different species of Vibrio and are not expedite enough (Kim & Lee, 2014). Furthermore, it is known that the precise identification of different species of the genus Vibrio is problematic, especially when dealing with related species (Kwok et al., 2002), namely due to the variability of the biochemical characteristics, which complicates the phenotypical identification of these bacteria (Nhung et al., 2007). The conventional polymerase chain reaction (PCR) tool provides rapid, specific and sensitive analysis of Vibrio species, but it time consuming and expensive, when dealing with a large number of targets. Multiplex polymerase chain reaction, commonly referred to as multiplex-PCR (m-PCR), is a type of PCR in which two or more target sequences can be amplified simultaneously, by including more than one pair of oligonucleotides of amplification (primers) in the reaction. This type of m-PCR is advantageous when looking at time and resources in the laboratory (Markoulatos et al., 2002), as well as to reduce expenses in reagents, without compromising the results. The aim of this study is to develop and validate a molecular tool that detects the presence of Vibrio anguillarum, Vibrio harveyi and Vibrio alginolyticus, three of the pathogens that cause more losses in aquaculture, using a multiplex-PCR tool. The standard bacterial strains were grown in Tryptone Soya Broth (TSB) and the growth curves were established. The bacterial DNA was extracted with NZY Tissue gDNA isolation kit (Nzytech) and compared to the boiling method. Different conditions for the multiplex-PCR were tested in order to establish the optimum conditions to be used. The multiplex PCR reactions were tested using a Taq polymerase and varying concentrations of MgCl2, being the ideal concentration of MgCl2 of 7.5 mM. Better results were obtained with 35 cycles of amplification and optimum annealing temperature at 58°C. The specificity of the primers was tested using purified DNA from the respective bacterial strains, yielding clear bands at the expected sizes. With respect to the sensitivity of the tool, there is a gradient of band intensity, which can be correlated with the concentration of DNA present. These results are in agreement with that described by Pinto et al. (2017), indicating a band intensity gradient, with m-PCR efficiently amplifying up to the concentration of 1 ng.μl-1. Results indicate that the multiplex PCR tool is suitable for the detection of the target pathogens and that this molecular tool is important especially when it is suspected that there are multiple infections derived from different pathogens in commercial aquaculture. The results indicate that this method allows a diagnosis of vibriosis in one working day and thus, it appears more convenient than the classical microbiological methods, which are time consuming and not always accurate. Acknowledgements This study had the support of Fundação para a Ciência e Tecnologia (FCT), through the strategic project UID/MAR/04292/2013 granted to MARE. References Avendaño-Herrera, R., Magariños, B., Toranzo, A.E., Beaz, R., Romalde, J.L. (2004). Species-specific polymerase chain reaction primers sets for the diagnosis of Tenacibaculum maritimum infection. Diseases of Aquatic Organisms, 62: 75-83. Kim J.Y. & Lee J-L (2014) Multipurpose assessment for the quantification of Vibrio spp. and total bacteria in fish and seawater using multiplex real-time polymerase chain reaction. J Sci Food Agric 94: 2807–2817. Kwok A. Y.C., Wilson J.T, Coulthart M., Ng L-K, Mutharia L., Chow A.C. (2002) Phylogenetic study and identification of human pathogenic Vibrio species based on partial hsp60 gene sequences. Can. J. Microbiol. 48: 903–910 Markoulatos P., Siafakas N., Moncany M. (2002) Multiplex Polymerase Chain Reaction: A Practical Approach. Journal of Clinical Laboratory Analysis 16: 47–51 Martins, P., R.V.V. Navarro, F.J.R.C. Coelho e N.C.M. Gomes. 2015. Development of a molecular methodology for fast detection of Photobacterium damselae subspecies in water samples. Aquaculture. 435: 137-142. Nhung P.H., Ohkusu K., Miyasaka J., Sun X.S., Ezaki T. (2007) Rapid and specific identification of 5 human pathogenic Vibrio species by multiplex polymerase chain reaction targeted to dnaJ gene. Diagnostic Microbiology and Infectious Disease 59: 271–275 Pinto M.P., Baptista T., Afonso C.C.A. (2017) Development of a new multiplex-PCR for the simultaneous detection of the fish pathogens Vibrio alginolyticus, Vibrio anguillarum, Vibrio harveyi and Edwardsiella tarda. Aquatic Living Resources 30: 1-6 Shi, Y.-H., Chen, J., Li, C.H., Lu, X.-J., Zhang, D.-M. et al. (2012). Detection of bacterial pathogens in aquaculture samples by DNA microarray analysis. Aquaculture, 338-341: 29-35. Sorroza L., Padilla D., Acosta F., Roman L., Grasso V., Veja J., Real F. (2012) Characterization of the probiotic strain Vagococcus fluvialis in the protection of European sea bass (Dicentrarchus labrax) against vibriosis by Vibrio anguillarum. Veterinary Microbiology 155: 369–373 Toranzo A.E., Magarinos B., Romalde J.L. (2005) A review of the main bacterial fish diseases in mariculture systems. Aquaculture 246: 37– 61 Keywords: Vibriosis, Fish pathology, Aquaculture, Disease Diagnostic, Multiplex-PCR Conference: IMMR'18 | International Meeting on Marine Research 2018, Peniche, Portugal, 5 Jul - 6 Jul, 2018. Presentation Type: Poster Presentation Topic: Blue Biotech Citation: Ferreira AR, Baptista TM and Afonso CN (2019). Development and validation of a Multiplex-PCR tool for the detection of Vibrio alginolyticus, Vibrio anguillarum and Vibrio harveyi in fish. Front. Mar. Sci. Conference Abstract: IMMR'18 | International Meeting on Marine Research 2018. doi: 10.3389/conf.FMARS.2018.06.00001 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Apr 2018; Published Online: 07 Jan 2019. * Correspondence: Prof. Clélia N Afonso, Escola Superior de Turismo e Tecnologia do Mar, Politécnico de Leiria, MARE – Marine and Environmental Sciences Centre, Peniche, 2520-641, Portugal, clelia@ipleiria.pt Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Ana R Ferreira Teresa M Baptista Clélia N Afonso Google Ana R Ferreira Teresa M Baptista Clélia N Afonso Google Scholar Ana R Ferreira Teresa M Baptista Clélia N Afonso PubMed Ana R Ferreira Teresa M Baptista Clélia N Afonso Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A hallmark property of non-functional clonotypes was used to develop a universal and fully computational method for detection and correction of multiplex PCR-specific quantitative bias in adaptive immune receptor repertoire.

A hallmark property of non-functional clonotypes was used to develop a universal and fully computational method for detection and correction of multiplex PCR-specific quantitative bias in adaptive immune receptor repertoire.

Comparison of Culture, Multiplex, and 5′ Nuclease Polymerase Chain Reaction Assays for the Rapid Detection of Yersinia enterocolitica in Swine and Pork Products

The human adaptive immune system is a primary defense against the vast world of potential pathogens. To counter this, B and T cells rearrange a diverse repertoire of surface receptors. This system functions in part by clonal expansion, so measuring changes in the abundance of both unique and total clones is necessary to understand the immune response. We developed immune profiling assays to amplify and sequence rearranged receptors. To efficiently capture receptor diversity we use a multiplex PCR approach. However, small variations in annealing kinetics can have large impacts on amplification efficiency causing PCR amplification bias (PAB). To address this issue, we developed a method to quantify and correct PAB introduced by multiplex PCR. We demonstrate this method using our T cell receptor γ (TCRG) assay. Our method uses a quantitated, synthetic immune repertoire standard consisting of every possible Variable(V) and Joining(J) gene combination. Following multiplex TCRG PCR amplification, we assessed the difference between the synthetic VJ composition before and after amplification and quantified PAB. We adjusted primer concentrations and redesigned primers to generate an unbiased multiplex PCR assay. Using this method we reduced PAB for the TCRG assay from a 340-fold to a 4.5-fold dynamic range. Now the abundance of receptor sequences is representative of the frequency of each receptor chain in the input sample, allowing for accurate quantification of the TCRG repertoire.

Introduction: The identification of the pathogens involved in pleural empyema has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercial multiplex pneumonia PCR assay to identify the pathogens involved in pleural empyema. Methods: Prospective, monocentric, observational study. Pleural fluid samples collected from 194 consecutive patients were analysed at the time of hospitalisation using conventional microbiology. Commercial multiplex pneumonia bacterial PCR was performed retrospectively. Results: There was a suspicion of empyema based on ultrasound findings in 18.5% of the cases, of these, 85.7% were diagnosed with empyema. When only taking into account the pathogens included in the multiplex PCR assay more pathogens were identified using the multiplex PCR assay than with conventional methods (23.3% vs. 6.7%, p=0.008). In those cases without suspicion of empyema, 9.7% had a final diagnosis of empyema. Using the same criteria, conventional methods identified pathogens in 0% of the cases and the multiplex PCR-based assay identified pathogens in 6.7% of the cases. The most common microorganisms identified in the cases diagnosed with empyema, were anaerobes (31) followed by gram-positive cocci (10) and gram-negative rods (4). Conclusion: The multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.

BackgroundThe identification of the pathogens in pleural effusion has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercially available multiplex bacterial PCR assay developed for pneumonia to identify the pathogens involved in pleural infection, particularly empyema.MethodsA prospective, monocentric, observational study including 194 patients with pleural effusion. Patients were evaluated based on imaging, laboratory values, pleura ultrasound and results of thoracentesis including conventional microbiology studies during hospitalisation. Multiplex bacterial PCR (Curetis Unyvero p55) was performed in batch and had no influence on therapeutic decisions.ResultsOverall, there were 51/197 cases with transudate and 146/197 with exudate. In 42% (n = 90/214) there was a clinical suspicion of parapneumonic effusion and the final clinical diagnosis of empyema was made in 29% (n = 61/214) of all cases. The most common microorganisms identified in the cases diagnosed with empyema were anaerobes [31] followed by gram-positive cocci [10] and gram-negative rods [4]. The multiplex PCR assay identified more of the pathogens on the panel than the conventional methods (23.3% (7/30) vs. 6.7% (2/30), p = 0.008).ConclusionThe multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the pneumonia panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.

Conventional octaplex PCR for the simultaneous identification of eight mainstream closely related Dendrobium species

To develop a multiplex PCR approach for simultaneous detection of Leuconostoc and Carnobacterium and its validation in meat products. Two multiplex PCR assays were developed using newly designed 16S rDNA-directed primers adapted to the current taxonomic situation of genera Leuconostoc and Carnobacterium that allow: (i) simultaneous detection of both genera, and members of the nonmotile species of genus Carnobacterium and (ii) identification in a single assay of the nonmotile species C. divergens, C. maltaromicum and C. gallinarum. Sensitivity values of 10(3) and 10(4) CFU g(-1) were determined for multiplex PCR detection of Carnobacterium and Leuconostoc, respectively, following artificially inoculated meat trials. In addition, both multiplex PCR assays were validated in 14 naturally contaminated samples covering nine types of meat products. Results obtained by colony identification were confirmed by PCR detection. The methods described in this study provide a rapid and reliable tool for PCR detection of Carnobacterium and Leuconostoc, in meat products, and for colony identification. This multiplex PCR approach will help in the analysis of the spoilage microbiota of refrigerated vacuum-packaged meat product in order to determine the appropriate preservation method.

Detection of total and hemolysin-producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tl, tdh and trh

Monitoring Minimal Residual Disease in AML By Patient Specific Mutational Fingerprint Using Multiplex PCR and Deep Sequencing

Background: The identification of the pathogens in pleural effusion has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercially available multiplex bacterial PCR assay developed for pneumonia to identify the pathogens involved in pleural infection, particularly empyema. Methods: A prospective, monocentric, observational study including 194 patients with pleural effusion. Patients were evaluated based on imaging, laboratory values, pleura ultrasound and results of thoracentesis including conventional microbiology studies during hospitalisation. Multiplex bacterial PCR (Curetis Unyvero p55) was performed in batch and had no influence on therapeutic decisions. Results: Overall, there were 51/197 cases with transudate and 146/197 with exudate. In 42% (n = 90/214) there was a clinical suspicion of parapneumonic effusion and the final clinical diagnosis of empyema was made in 29% (n = 61/214) of all cases. The most common microorganisms identified in the cases diagnosed with empyema were anaerobes [<xref ref-type="bibr" rid="ref31">31</xref>] followed by gram-positive cocci [<xref ref-type="bibr" rid="ref10">10</xref>] and gram-negative rods [<xref ref-type="bibr" rid="ref4">4</xref>]. The multiplex PCR assay identified more of the pathogens on the panel than the conventional methods (23.3% (7/30) vs. 6.7% (2/30), p = 0.008). Conclusion: The multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the pneumonia panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.

Y-chromosomal testing of brown bears (Ursus arctos): Validation of a multiplex PCR-approach for nine STRs suitable for fecal and hair samples

Development and Clinical Validation of a Multiplex Real-time PCR Assay for Herpes Simplex and Varicella Zoster Virus

BackgroundSchistosomiasis a neglected tropical disease endemic in Brazil. It is caused by the trematode Schistosoma mansoni, which is transmitted by snails of the genus Biomphalaria. Among measures used to control and eliminate schistosomiasis, accurate mapping and monitoring of snail breeding sites are recommended. Despite the limitations of parasitological methods, they are still used to identify infected snails. Loop-mediated isothermal amplification (LAMP) is a sensitive, rapid, and cost-effective diagnostic method for the identification of infected snails. In the work reported here, we aimed to validate the use of LAMP for the detection of S. mansoni in snails of the genus Biomphalaria.MethodsSnails were collected in five municipalities of the Mucuri Valley and Jequitinhonha Valley regions in the state of Minas Gerais, Brazil. Snails were pooled according to collection site and then squeezed for the detection of S. mansoni and other trematode larvae. Pooled snails were subjected to pepsin digestion and DNA extraction. Molecular assays were performed for species-specific identification and characterization of the samples. A previously described LAMP assay was adapted, evaluated, and validated using laboratory and field samples.ResultsUsing the parasitological method described here, S. mansoni cercariae were detected in snails from two collection sites, and cercariae of the family Spirorchiidae were found in snails from one site. The snails were identified by polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP). Biomphalaria glabrata, the main snail host of S. mansoni in Brazil, was detected in 72.2% of the collection sites. Biomphalaria kuhniana, which is resistant to S. mansoni infection, was found in the remaining sites. Multiplex, low stringency (LS), and conventional PCR allowed the detection of positive snails in four additional sites. Trematodes belonging to the families Strigeidae and Echinostomatidae were detected by multiplex PCR in two sites. The LAMP assay was effective in detecting the presence of S. mansoni infection in laboratory (7 days post-infection) and field samples with no cross-reactivity for other trematodes. When compared to LS and conventional PCR, LAMP showed 100% specificity, 85.7% sensitivity, and a κ index of 0.88.ConclusionsOur findings suggest that LAMP is a good alternative method for the detection and monitoring of transmission foci of S. mansoni, as it was three times as effective as the parasitological examination used here for the detection of infection, and is more directly applicable in the field than other molecular techniques.Graphical abstract