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 Genetic Analysis of the Cronobacter sakazakii O4 to O7 O-Antigen Gene Clusters and Development of a PCR Assay for Identification of All C. sakazakii O Serotypes

2012 ◽  
Vol 78 (11) ◽  
pp. 3966-3974 ◽  
Author(s):  
Yamin Sun ◽  
Min Wang ◽  
Quan Wang ◽  
Boyang Cao ◽  
Xin He ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Cronobacter Sakazakii ◽  
Pcr Assay ◽  
Gram Negative ◽  
Content Type ◽  
Antigen Gene ◽  
O Antigen ◽  
Multiplex Pcr Assay ◽  
Food Borne ◽  
Invasive Infections

ABSTRACTThe Gram-negative bacteriumCronobacter sakazakiiis an emerging food-borne pathogen that causes severe invasive infections in neonates. Variation in the O-antigen lipopolysaccharide in the outer membrane provides the basis for Gram-negative bacteria serotyping. The O-antigen serotyping scheme forC. sakazakii, which includes seven serotypes (O1 to O7), has been recently established, and the O-antigen gene clusters and specific primers for threeC. sakazakiiserotypes (O1, O2, and O3) have been characterized. In this study, theC. sakazakiiO4, O5, O6, and O7 O-antigen gene clusters were sequenced, and gene functions were predicted on the basis of homology.C. sakazakiiO4 shared a similar O-antigen gene cluster withEscherichia coliO103. The general features and anomalies of all sevenC. sakazakiiO-antigen gene clusters were evaluated and the relationship between O-antigen structures and their gene clusters were investigated. Serotype-specific genes for O4 to O7 were identified, and a molecular serotyping method for allC. sakazakiiO serotypes, a multiplex PCR assay, was developed by screening against 136 strains ofC. sakazakiiand closely related species. The sensitivity of PCR-based serotyping method was determined to be 0.01 ng of genomic DNA and 103CFU of each strain/ml. This study completes the elucidation ofC. sakazakiiO-antigen genetics and provides a molecular method suitable for the identification ofC. sakazakiiO1 to O7 strains.

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 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 “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 A Multiplex PCR Assay for the Detection of Food-borne Pathogens in Meat Products

2010 ◽  
Vol 30 (4) ◽  
pp. 590-596 ◽  
Author(s):  
Hyoun-Wook Kim ◽  
Ji-Hyun Kim ◽  
Seong-Ryul Rhim ◽  
Kyung-A Lee ◽  
Cheon-Jei Kim ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Meat Products ◽  
Pcr Assay ◽  
Food Borne Pathogens ◽  
Multiplex Pcr Assay ◽  
Food Borne

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.

scholarly journals Advanced Multiplex PCR Assay for Differentiation of Brucella Species

2011 ◽  
Vol 77 (18) ◽  
pp. 6726-6728 ◽  
Author(s):  
Sung-Il Kang ◽  
Moon Her ◽  
Jong Wan Kim ◽  
Ji-Yeon Kim ◽  
Kyung Yuk Ko ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Brucella Species ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
Brucella Canis ◽  
Brucella Suis ◽  
Primer Sets

ABSTRACTTwo new primer sets of a 766- and a 344-bp fragment were introduced into the conventional Bruce-ladder PCR assay. This novel multiplex PCR assay rapidly and concisely discriminatesBrucella canisandBrucella microtifromBrucella suisstrains and also may differentiate all of the 10Brucellaspecies.

scholarly journals Multiplex PCR for Simultaneous Detection of Bacteria of the Genus Listeria, Listeria monocytogenes, and Major Serotypes and Epidemic Clones of L. monocytogenes

2007 ◽  
Vol 73 (19) ◽  
pp. 6299-6304 ◽  
Author(s):  
Yi Chen ◽  
Stephen J. Knabel
Keyword(s):  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Simultaneous Detection ◽  
Important Food ◽  
Inexpensive Method ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Food Borne

ABSTRACT A multiplex PCR assay which combines detection of bacteria of the genus Listeria, Listeria monocytogenes serotypes 1/2a and 4b, and epidemic clones I, II, and III of L. monocytogenes was developed. The assay provides a rapid, reliable, and inexpensive method for screening and subgrouping this important food-borne pathogen.

scholarly journals One-Step Identification of Five Prominent Chicken Salmonella Serovars and Biotypes

2015 ◽  
Vol 53 (12) ◽  
pp. 3881-3883 ◽  
Author(s):  
Chunhong Zhu ◽  
Min Yue ◽  
Shelley Rankin ◽  
François-Xavier Weill ◽  
Joachim Frey ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Genomic Data ◽  
Pcr Assay ◽  
Content Type ◽  
Multiplex Pcr Assay ◽  
One Step ◽  
Salmonella Serovars

Based on bacterial genomic data, we developed a one-step multiplex PCR assay to identifySalmonellaand simultaneously differentiate the two invasive avian-adaptedS. entericaserovar Gallinarum biotypes Gallinarum and Pullorum, and the most frequent, specific, and asymptomatic colonizers of chickens, serovars Enteritidis, Heidelberg, and Kentucky.

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