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.

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 Rapid identification of Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii with a multiplex PCR assay

2014 ◽  
Vol 63 (9) ◽  
pp. 1154-1159 ◽  
Author(s):  
Te-Li Chen ◽  
Yi-Tzu Lee ◽  
Shu-Chen Kuo ◽  
Su-Pen Yang ◽  
Chang-Phone Fung ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Multiplex Pcr ◽  
Acinetobacter Calcoaceticus ◽  
Rapid Identification ◽  
23S Rrna ◽  
Pcr Assay ◽  
Complex Species ◽  
Multiplex Pcr Assay ◽  
Acinetobacter Pittii ◽  
Reference Strains

Acinetobacter baumannii, Acinetobacter nosocomialis and Acinetobacter pittii are clinically relevant members of the Acinetobacter calcoaceticus–A. baumannii (Acb) complex and important nosocomial pathogens. These three species are genetically closely related and phenotypically similar; however, they differ in their epidemiology, antibiotic resistance and pathogenicity. In this study, we investigated the use of a multiplex PCR-based assay designed to detect internal fragments of the 16S–23S rRNA intergenic region and the gyrB and recA genes. The assay was capable of differentiating A. baumannii, A. nosocomialis and A. pittii in a reliable manner. In 23 different reference strains and 89 clinical isolates of Acinetobacter species, the assay accurately identified clinically relevant Acb complex species except those ‘between 1 and 3’ or ‘close to 13TU’. None of the non-Acb complex species was misidentified. In an analysis of 1034 positive blood cultures, the assay had a sensitivity of 92.4 % and specificity of 98.2 % for Acb complex identification. Our results show that a single multiplex PCR assay can reliably differentiate clinically relevant Acb complex species. Thus, this method may be used to better understand the clinical differences between infections caused by these species.

scholarly journals Accurate and Rapid Identification of Longan Arillus and Litchi Semen by a Multiplex PCR Assay

Plants ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 948
Author(s):  
Wook Jin Kim ◽  
Sungyu Yang ◽  
Goya Choi ◽  
Inkyu Park ◽  
Pureum Noh ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Dna Markers ◽  
Herbal Medicines ◽  
High Specificity ◽  
Single Species ◽  
Rapid Identification ◽  
Genetic Identification ◽  
Pcr Assay ◽  
Multiplex Pcr Assay ◽  
Specificity And Sensitivity

Dimocarpus longan, Litchi chinensis, and Nephelium lappaceum are commercially valuable subtropical and tropical fruits of the Sapindaceae family. Arillus and seeds of the three species have very similar morphologies; however, the arillus of D. longan is used as the herbal medicine Longan Arillus and seeds of L. chinensis are used as Litchi Semen in Korean and Chinese pharmacopoeias. The adulteration of herbal medicines with inauthentic species, including the use of Aril and seed fractions acquired from a single species for two herbal medicines (e.g., Longan Arillus and Litchi Semen), is often driven by economic motives. DNA markers are a tool for the detection of adulterants in commercial products. To establish rapid and reliable assays for the genetic identification of authentic Longan Arillus and Litchi Semen, we developed DNA markers with high specificity and sensitivity based on internal transcribed spacer (ITS) sequences. The newly developed DNA markers and multiplex PCR assay may contribute to efforts to protect against adulteration, quality control, and the standardization of herbal medicines.

Development of a multiplex PCR assay for the detection of key genes associated with Pasteurella multocida subspecies

2021 ◽  
pp. 104063872110634
Author(s):  
Barbara Ujvári ◽  
Hubert Gantelet ◽  
Tibor Magyar
Keyword(s):  
Multiplex Pcr ◽  
Pasteurella Multocida ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Biochemical Tests ◽  
Specific Primers ◽  
Multiplex Pcr Assay ◽  
Subspecies Level ◽  
Species Specific ◽  
Reference Strains

The ability to distinguish among the subspecies of Pasteurella multocida isolates is important epidemiologically; however, classification at the subspecies level based on the results of conventional biochemical tests (fermentation of sorbitol and dulcitol) is reportedly not accurate in all cases. Therefore, we developed a rapid, multiplex PCR assay to differentiate among the 3 subspecies of P. multocida. The PCR assay includes the P. multocida species–specific primers KMT1SP6 and KMT1T7 as an internal amplification control, with a newly designed gatD (galactitol-1-phosphate-5-dehydrogenase)-specific primer pair (unique for subsp. gallicida), and primers targeting a 16S rRNA gene region specific for subsp. septica. The subspecies specificity of the PCR was demonstrated by applying the test to a collection of 70 P. multocida isolates, including the Heddleston serovar reference strains; all isolates and strains were assigned correctly. The PCR assay is a sensitive, specific, and highly effective method for the identification of P. multocida subspecies, and an alternative to biochemical test–based differentiation. A possible relationship was noticed between P. multocida subspecies and lipopolysaccharide (LPS) genotype; all but one of the subsp. gallicida strains were isolated only from avian hosts and represented L1 LPS genotype. Subsp. multocida and subsp. septica isolates were classified into 5 and 4 different LPS genotypes, respectively, of which L3 was the only LPS genotype shared between these 2 subspecies.

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 Species Identification and Strain Typing of Staphylococcus agnetis and Staphylococcus hyicus Isolates from Bovine Milk by Use of a Novel Multiplex PCR Assay and Pulsed-Field Gel Electrophoresis

2017 ◽  
Vol 55 (6) ◽  
pp. 1778-1788 ◽  
Author(s):  
P. R. F. Adkins ◽  
J. R. Middleton ◽  
M. J. Calcutt ◽  
G. C. Stewart ◽  
L. K. Fox
Keyword(s):  
Multiplex Pcr ◽  
Species Identification ◽  
Gel Electrophoresis ◽  
Bovine Milk ◽  
Gene Sequencing ◽  
Pulsed Field Gel Electrophoresis ◽  
Strain Typing ◽  
Content Type ◽  
Pulsed Field ◽  
Multiplex Pcr Assay

ABSTRACTStaphylococcus hyicusandStaphylococcus agnetisare two coagulase-variable staphylococcal species that can be isolated from bovine milk and are difficult to differentiate. The objectives of this study were to characterize isolates of bovine milk origin from a collection that had previously been characterized as coagulase-positiveS. hyicusbased on phenotypic species identification methods and to develop a PCR-based method for differentiatingS. hyicus,S. agnetis, andStaphylococcus aureus. Isolates (n= 62) were selected from a previous study in which milk samples were collected from cows on 15 dairy herds. Isolates were coagulase tested and identified to the species level using housekeeping gene sequencing. A multiplex PCR to differentiateS. hyicus,S. agnetis, andS. aureuswas developed. Pulsed-field gel electrophoresis was conducted to strain type the isolates. Based on gene sequencing, 44/62 of the isolates were determined to be eitherS. agnetis(n= 43) orS. hyicus(n= 1). Overall, 88% (37/42) of coagulase-positiveS. agnetisisolates were found to be coagulase positive at 4 h. The herd-level prevalence of coagulase-positiveS. agnetisranged from 0 to 2.17%. Strain typing identified 23 different strains. Six strains were identified more than once and from multiple cows within the herd. Three strains were isolated from cows at more than one time point, with 41 to 264 days between samplings. These data suggest thatS. agnetisis likely more prevalent on dairy farms thanS. hyicus. Also, someS. agnetisisolates in this study appeared to be contagious and associated with persistent infections.

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