scholarly journals “Pathotyping” Multiplex PCR Assay for Haemophilus parasuis: a Tool for Prediction of Virulence

2017 ◽  
Vol 55 (9) ◽  
pp. 2617-2628 ◽  
Author(s):  
Kate J. Howell ◽  
Lucy A. Weinert ◽  
Sarah E. Peters ◽  
Jinhong Wang ◽  
Juan Hernandez-Garcia ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Generalized Linear Model ◽  
Bacterial Species ◽  
Haemophilus Parasuis ◽  
Pcr Assay ◽  
Current Standard ◽  
Content Type ◽  
The United Kingdom ◽  
Multiplex Pcr Assay ◽  
Upper Respiratory

ABSTRACTHaemophilus parasuisis a diverse bacterial species that is found in the upper respiratory tracts of pigs and can also cause Glässer's disease and pneumonia. A previous pangenome study ofH. parasuisidentified 48 genes that were associated with clinical disease. Here, we describe the development of a generalized linear model (termed a pathotyping model) to predict the potential virulence of isolates ofH. parasuisbased on a subset of 10 genes from the pangenome. A multiplex PCR (mPCR) was constructed based on these genes, the results of which were entered into the pathotyping model to yield a prediction of virulence. This new diagnostic mPCR was tested on 143 field isolates ofH. parasuisthat had previously been whole-genome sequenced and a further 84 isolates from the United Kingdom from cases ofH. parasuis-related disease in pigs collected between 2013 and 2014. The combination of the mPCR and the pathotyping model predicted the virulence of an isolate with 78% accuracy for the original isolate collection and 90% for the additional isolate collection, providing an overall accuracy of 83% (81% sensitivity and 93% specificity) compared with that of the “current standard” of detailed clinical metadata. This new pathotyping assay has the potential to aid surveillance and disease control in addition to serotyping data.

scholarly journals Multiplex PCR Assay for Detection and Identification of Clostridium botulinum Types A, B, E, and F in Food and Fecal Material

2001 ◽  
Vol 67 (12) ◽  
pp. 5694-5699 ◽  
Author(s):  
Miia Lindström ◽  
Riikka Keto ◽  
Annukka Markkula ◽  
Mari Nevas ◽  
Sebastian Hielm ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Bacterial Species ◽  
Type A ◽  
Food Samples ◽  
Type B ◽  
Fecal Material ◽  
Pcr Assay ◽  
Multiplex Pcr Assay

ABSTRACT Botulism is diagnosed by detecting botulinum neurotoxin andClostridium botulinum cells in the patient and in suspected food samples. In this study, a multiplex PCR assay for the detection of Clostridium botulinum types A, B, E, and F in food and fecal material was developed. The method employs four new primer pairs with equal melting temperatures, each being specific to botulinum neurotoxin gene type A, B, E, or F, and enables a simultaneous detection of the four serotypes. A total of 43 C. botulinum strains and 18 strains of other bacterial species were tested. DNA amplification fragments of 782 bp for C. botulinum type A alone, 205 bp for type B alone, 389 bp for type E alone, and 543 bp for type F alone were obtained. Other bacterial species, including C. sporogenes and the nontoxigenic nonproteolytic C. botulinum-like organisms, did not yield a PCR product. Sensitivity of the PCR for types A, E, and F was 102 cells and for type B was 10 cells per reaction mixture. With a two-step enrichment, the detection limit in food and fecal samples varied from 10−2 spore/g for types A, B, and F to 10−1 spore/g of sample material for type E. Of 72 natural food samples investigated, two were shown to contain C. botulinum type A, two contained type B, and one contained type E. The assay is sensitive and specific and provides a marked improvement in the PCR diagnostics of C. botulinum.

scholarly journals Differentiating Botulinum Neurotoxin-Producing Clostridia with a Simple, Multiplex PCR Assay

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Charles H. D. Williamson ◽  
Adam J. Vazquez ◽  
Karen Hill ◽  
Theresa J. Smith ◽  
Roxanne Nottingham ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Gene Cluster ◽  
Botulinum Neurotoxin ◽  
Comparative Genomic ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Group I ◽  
Pcr Assays ◽  
Group Ii

ABSTRACT Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present (ha positive [ha +] or orfX +). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene (bont) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes, and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type (ha + or orfX +) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest.

scholarly journals Multiplex PCR Assay Targeting a Diguanylate Cyclase-Encoding Gene,cgcA, To Differentiate Species within the Genus Cronobacter

2012 ◽  
Vol 79 (2) ◽  
pp. 734-737 ◽  
Author(s):  
L. Carter ◽  
L. A. Lindsey ◽  
C. J. Grim ◽  
V. Sathyamoorthy ◽  
K. G. Jarvis ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Diguanylate Cyclase ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplexed Pcr ◽  
Multiplex Pcr Assay ◽  
Food Borne ◽  
Encoding Gene

ABSTRACTIn a comparison to the widely usedCronobacter rpoBPCR assay, a highly specific multiplexed PCR assay based oncgcA, a diguanylate cyclase gene, that identified all of the targeted six species among 305Cronobacterisolates was designed. This assay will be a valuable tool for identifying suspectedCronobacterisolates from food-borne investigations.

scholarly journals Multiplex PCR for Identification of Two Capsular Types in Epidemic KPC-Producing Klebsiella pneumoniae Sequence Type 258 Strains

2014 ◽  
Vol 58 (7) ◽  
pp. 4196-4199 ◽  
Author(s):  
Liang Chen ◽  
Kalyan D. Chavda ◽  
Jacqueline Findlay ◽  
Gisele Peirano ◽  
Katie Hopkins ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Multiplex Pcr ◽  
Capsular Polysaccharide ◽  
Sequence Type ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Polysaccharide Synthesis ◽  
Sequence Types

ABSTRACTWe developed a multiplex PCR assay capable of identifying two capsular polysaccharide synthesis sequence types (sequence type 258 [ST258]cps-1andcps-2) in epidemicKlebsiella pneumoniaeST258 strains. The assay performed with excellent sensitivity (100%) and specificity (100%) for identifyingcpstypes in 60 ST258K. pneumoniaesequenced isolates. The screening of 419 ST258 clonal isolates revealed a significant association betweencpstype andK. pneumoniaecarbapenemase (KPC) variant:cps-1is largely associated with KPC-2, whilecps-2is primarily associated with KPC-3.

scholarly journals Development of a One-Step Multiplex PCR Assay for Differential Detection of Major Mycobacterium Species

2017 ◽  
Vol 55 (9) ◽  
pp. 2736-2751 ◽  
Author(s):  
Hansong Chae ◽  
Seung Jung Han ◽  
Su-Young Kim ◽  
Chang-Seok Ki ◽  
Hee Jae Huh ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Beijing Genotype ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Accurate Identification ◽  
Reliable Technique ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
One Step ◽  
Reference Strains

ABSTRACTThe prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between theMycobacterium tuberculosiscomplex (MTBC) and NTM usingrv0577or RD750, (ii) differentiateM. tuberculosis(M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespreadM. tuberculosisBeijing genotype by targetingmtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium,M. intracellulare,M. abscessus,M. massiliense, andM. kansasii) by targeting IS1311, DT1,mass_3210, andmkan_rs12360. An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targetedMycobacteriumspecies. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103and 104CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinicalM. tuberculosisand NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC,M. tuberculosis,M. tuberculosisBeijing genotype, and major NTM species.

scholarly journals Development of a Multiplex PCR Assay for Rapid Molecular Serotyping of Haemophilus parasuis

2015 ◽  
Vol 53 (12) ◽  
pp. 3812-3821 ◽  
Author(s):  
Kate J. Howell ◽  
Sarah E. Peters ◽  
Jinhong Wang ◽  
Juan Hernandez-Garcia ◽  
Lucy A. Weinert ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
In Silico ◽  
High Specificity ◽  
Haemophilus Parasuis ◽  
Content Type ◽  
Molecular Serotyping ◽  
Multiplex Pcr Assay ◽  
Indirect Hemagglutination ◽  
Capsule Locus ◽  
Reference Strains

Haemophilus parasuiscauses Glässer's disease and pneumonia in pigs. Indirect hemagglutination (IHA) is typically used to serotype this bacterium, distinguishing 15 serovars with some nontypeable isolates. The capsule loci of the 15 reference strains have been annotated, and significant genetic variation was identified between serovars, with the exception of serovars 5 and 12. A capsule locus andin silicoserovar were identified for all but two nontypeable isolates in our collection of >200 isolates. Here, we describe the development of a multiplex PCR, based on variation within the capsule loci of the 15 serovars ofH. parasuis, for rapid molecular serotyping. The multiplex PCR (mPCR) distinguished between all previously described serovars except 5 and 12, which were detected by the same pair of primers. The detection limit of the mPCR was 4.29 × 105ng/μl bacterial genomic DNA, and high specificity was indicated by the absence of reactivity against closely related commensalPasteurellaceaeand other bacterial pathogens of pigs. A subset of 150 isolates from a previously sequencedH. parasuiscollection was used to validate the mPCR with 100% accuracy compared to thein silicoresults. In addition, the twoin silico-nontypeable isolates were typeable using the mPCR. A further 84 isolates were analyzed by mPCR and compared to the IHA serotyping results with 90% concordance (excluding those that were nontypeable by IHA). The mPCR was faster, more sensitive, and more specific than IHA, enabling the differentiation of 14 of the 15 serovars ofH. parasuis.

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples

2017 ◽  
Vol 80 (2) ◽  
pp. 295-301 ◽  
Author(s):  
Dele Ogunremi ◽  
Susan Nadin-Davis ◽  
Andrée Ann Dupras ◽  
Imelda Gálvan Márquez ◽  
Katayoun Omidi ◽  
...  
Keyword(s):  
Salmonella Typhimurium ◽  
Multiplex Pcr ◽  
Internal Control ◽  
Broiler Chickens ◽  
Salmonella Enteritidis ◽  
Bacterial Species ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Retail Outlets ◽  
Dna Fragment

ABSTRACT A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella-specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non-Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

scholarly journals A Multiplex PCR Assay to Detect and Differentiate Select Agent Strains of Ralstonia solanacearum

Plant Disease ◽  
2015 ◽  
Vol 99 (3) ◽  
pp. 333-341 ◽  
Author(s):  
Michael J. Stulberg ◽  
Jonathan Shao ◽  
Qi Huang
Keyword(s):  
United States ◽  
Multiplex Pcr ◽  
Ralstonia Solanacearum ◽  
Species Complex ◽  
Bacterial Species ◽  
The United States ◽  
Pcr Assay ◽  
Plant Dna ◽  
Multiplex Pcr Assay ◽  
Verification Methods

Ralstonia solanacearum race 3 biovar 2 strains are considered select agents by the U.S. government because they are not endemic to the United States and have the potential to cause brown rot in our potato production fields. Simple and accurate methods are needed for quick identification prior to more discriminating but time-consuming verification methods. We developed a multiplex PCR assay that identifies R. solanacearum species complex strains, signals whether the strain detected is a select agent, and controls for false negatives associated with PCR inhibition or unsuccessful DNA extractions in one reaction. We identified unique sequences of non-phage-related DNA for the R. solanacearum species complex strains, and for select agent strains, using in silico genome subtraction. We also designed and included an internal plant DNA control assay. Our multiplex PCR assay correctly identified 90 R. solanacearum species complex strains and 34 select agent strains, while not recognizing five out-group bacterial species. Additionally, the multiplex PCR assay facilitated the detection of plant DNA and R. solanacearum from infected tomato, potato, geranium, and tobacco plants. Our rapid, accurate, and reliable detection assay can help government officials make timely and appropriate recommendations to exclude this bacterium from the United States.

scholarly journals Novel Multiplex PCR Assay for Detection of Chlorhexidine-Quaternary Ammonium, Mupirocin, and Methicillin Resistance Genes, with Simultaneous Discrimination of Staphylococcus aureus from Coagulase-Negative Staphylococci

2017 ◽  
Vol 55 (6) ◽  
pp. 1857-1864 ◽  
Author(s):  
Jo-Ann McClure ◽  
Johanna Zaal DeLongchamp ◽  
John M. Conly ◽  
Kunyan Zhang
Keyword(s):  
Staphylococcus Aureus ◽  
Multiplex Pcr ◽  
Resistance Genes ◽  
Quaternary Ammonium ◽  
Methicillin Resistance ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Coagulase Negative Staphylococci ◽  
Content Type ◽  
Multiplex Pcr Assay

ABSTRACTMethicillin-resistantStaphylococcus aureus(MRSA) is a clinically significant pathogen that is resistant to a wide variety of antibiotics and responsible for a large number of nosocomial infections worldwide. The Agency for Healthcare Research and Quality and the Centers for Disease Control and Prevention recently recommended the adoption of universal mupirocin-chlorhexidine decolonization of all admitted intensive care unit patients rather than MRSA screening with targeted treatments, which raises a serious concern about the selection of resistance to mupirocin and chlorhexidine in strains of staphylococci. Thus, a simple, rapid, and reliable approach is paramount in monitoring the prevalence of resistance to these agents. We developed a simple multiplex PCR assay capable of screeningStaphylococcusisolates for the presence of antiseptic resistance genes for chlorhexidine and quaternary ammonium compounds, as well as mupirocin and methicillin resistance genes, while simultaneously discriminatingS. aureusfrom coagulase-negative staphylococci (CoNS). The assay incorporates 7 PCR targets, including theStaphylococcus16S rRNA gene (specifically detectingStaphylococcusspp.),nuc(distinguishingS. aureusfrom CoNS),mecA(distinguishing MRSA from methicillin-susceptibleS. aureus),mupAandmupB(identifying high-level mupirocin resistance), andqacandsmr(identifying chlorhexidine and quaternary ammonium resistance). Our assay demonstrated 100% sensitivity, specificity, and accuracy in a total of 23 variant antiseptic- and/or antibiotic-resistant control strains. Further validation of our assay using 378 randomly selected and previously well-characterized local clinical isolates confirmed its feasibility and practicality. This may prove to be a useful tool for multidrug-resistantStaphylococcusmonitoring in clinical laboratories, particularly in the wake of increased chlorhexidine and mupirocin treatments.

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