scholarly journals Emergence of Salmonella enterica serovar 4,[5],12:i:- as the primary serovar identified from swine clinical samples and development of a multiplex real-time PCR for improved Salmonella serovar-level identification

2019 ◽  
Vol 31 (6) ◽  
pp. 818-827 ◽  
Author(s):  
Samantha A. Naberhaus ◽  
Adam C. Krull ◽  
Laura K. Bradner ◽  
Karen M. Harmon ◽  
Paulo Arruda ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Enrichment Culture ◽  
The United States ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Diagnostic Laboratory ◽  
Diagnostic Samples ◽  
Group B

Rapid identification of the infecting Salmonella serovar from porcine diagnostic samples is vital to allow implementation of appropriate on-farm treatment and management decisions. Although identification at the serogroup level can be rapidly achieved at most veterinary diagnostic laboratories, final Salmonella serovar identification often takes several weeks because of the limited number of reference laboratories performing the complex task of serotyping. Salmonella serogroup B, currently the dominant serogroup identified from swine clinical samples in the United States, contains serovars that vary from highly pathogenic to minimally pathogenic in swine. We determined the frequency of detection of individual group B serovars at the Iowa State Veterinary Diagnostic Laboratory from 2008 to 2017, and validated a multiplex real-time PCR (rtPCR) to distinguish pathogenic serogroup B serovars from those of lesser pathogenicity. Our results indicate that, since 2014, Salmonella enterica ssp. enterica serovar 4,[5],12:i:- has been the dominant serovar identified from swine clinical samples at the ISU-VDL, with S. Typhimurium now the second most common serovar identified. We developed a rtPCR to allow rapid differentiation of samples containing S. 4,[5],12:i:- and S. Typhimurium from samples containing serovars believed to be of less pathogenicity, such as S. Agona and S. Derby. When combined with enrichment culture, this rtPCR has the ability to significantly improve the time to final serovar identification of the 2 most commonly identified pathogenic Salmonella serovars in swine, and allows rapid implementation of serovar-specific intervention strategies.

scholarly journals Sequential Quadriplex Real-Time PCR for Identifying 20 Common emm Types of Group A Streptococcus

2020 ◽  
Vol 59 (1) ◽  
pp. e01764-20
Author(s):  
Srinivasan Velusamy ◽  
Katherine Jordak ◽  
Madeline Kupor ◽  
Sopio Chochua ◽  
Lesley McGee ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Group A Streptococcus ◽  
High Specificity ◽  
The United States ◽  
Rapid Identification ◽  
Invasive Group ◽  
Outbreak Investigations ◽  
Group A ◽  
Culture Negative

ABSTRACTWe developed a sequential quadriplex real-time PCR-based method for rapid identification of 20 emm types commonly found in invasive group A Streptococcus (iGAS) strains recovered through the Centers for Disease Control and Prevention’s Active Bacterial Core surveillance. Each emm real-time PCR assay showed high specificity and accurately identified the respective target emm type, including emm subtypes in the United States. Furthermore, this method is useful for rapid typing of GAS isolates and culture-negative specimens during outbreak investigations.

scholarly journals Harmonization of Bordetella pertussis Real-Time PCR Diagnostics in the United States in 2012

2014 ◽  
Vol 53 (1) ◽  
pp. 118-123 ◽  
Author(s):  
Margaret M. Williams ◽  
Thomas H. Taylor ◽  
David M. Warshauer ◽  
Monte D. Martin ◽  
Ann M. Valley ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
The United States ◽  
Extraction Methods ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Content Type

Real-time PCR (rt-PCR) is an important diagnostic tool for the identification ofBordetella pertussis,Bordetella holmesii, andBordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in theB. pertussisgenome and 32 to 65 copies inB. holmesii. The CDC developed a multitarget PCR assay to differentiateB. pertussis,B. holmesii, andB. parapertussisand provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these threeBordetellaspecies in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viableBordetellaand non-Bordetellaspecies in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiatedB. pertussisandB. holmesiiand 68% and 72% identifiedB. parapertussis. IS481cycle threshold (CT) values forB. pertussissamples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiatedB. pertussisandB. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCRBordetelladiagnostic protocols in USPHLs compared to that of the previous survey.

RAPID IDENTIFICATION AND TYPING OF FUNGAL DNA FROM CLINICAL SAMPLES BY REAL TIME PCR

Mycoses ◽  
2002 ◽  
Vol 45 (S2) ◽  
pp. 26-26
Author(s):  
S. Jalal ◽  
E. Norberg ◽  
J. Tollemar ◽  
J. Loeffler ◽  
L. Klingspor
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Fungal Dna

scholarly journals Real-Time PCR Assays for Rapid Identification of Common Aphanomyces astaci Genotypes

2021 ◽  
Vol 9 ◽  
Author(s):  
Marco Di Domenico ◽  
Valentina Curini ◽  
Riccardo Caprioli ◽  
Carla Giansante ◽  
Agata Mrugała ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Central Italy ◽  
The United States ◽  
Wide Distribution ◽  
Cross Reactivity ◽  
Etiologic Agent ◽  
Clinical Samples ◽  
Introduction Pathways ◽  
Aphanomyces Astaci

The oomycete Aphanomyces astaci is the etiologic agent of crayfish plague, a disease that has seriously impacted the populations of European native crayfish species. The introduction of non-indigenous crayfish of North American origin and their wide distribution across Europe have largely contributed to spread of crayfish plague in areas populated by indigenous crayfish. Tracking A. astaci genotypes may thus be a useful tool for investigating the natural history of crayfish plague in its European range, as well as the sources and introduction pathways of the pathogen. In this study, we describe the development of real-time PCR TaqMan assays aiming to distinguish the five genotype groups of A. astaci (A–E) previously defined by their distinct RAPD patterns. The method was evaluated using DNA extracts from pure A. astaci cultures representing the known genotype groups, and from A. astaci-positive crayfish clinical samples collected mostly during crayfish plague outbreaks that recently occurred in Central Italy and Czechia. The assays do not cross-react with each other, and those targeting genotype groups A, B, D, and E seem sufficiently specific to genotype the pathogen from infected crayfish in the areas invaded by A. astaci (particularly Europe). The unusual A. astaci genotype “SSR-Up” documented from crayfish plague outbreaks in Czechia and chronically infected Pontastacus leptodactylus in the Danube is detected by the group B real-time PCR. The assay originally developed to detect group C (one not yet documented from crayfish plague outbreaks) showed cross-reactivity with Aphanomyces fennicus; the A. astaci genotype “rust1” described in the United States from Faxonius rusticus is detected by that assay as well. Analyses of additional markers (such as sequencing of the nuclear internal transcribed spacer or mitochondrial ribosomal subunits) may complement such cases when the real-time PCR-based genotyping is not conclusive. Despite some limitations, the method is a robust tool for fast genotyping of A. astaci genotype groups common in Europe, both during crayfish plague outbreaks and in latent infections.

Prevalence of Salmonella enterica, Listeria monocytogenes, and Escherichia coli Virulence Factors in Bulk Tank Milk and In-Line Filters from U.S. Dairies†

2011 ◽  
Vol 74 (5) ◽  
pp. 759-768 ◽  
Author(s):  
JO ANN S. VAN KESSEL ◽  
JEFFREY S. KARNS ◽  
JASON E. LOMBARD ◽  
CHRISTINE A. KOPRAL
Keyword(s):  
Escherichia Coli ◽  
Listeria Monocytogenes ◽  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Raw Milk ◽  
The United States ◽  
Bulk Tank Milk ◽  
Milk Products ◽  
Bulk Tank

The zoonotic bacteria Salmonella enterica, Listeria monocytogenes, and Escherichia coli are known to infect dairy cows while not always causing clinical signs of disease. These pathogens are sometimes found in raw milk, and human disease outbreaks due to these organisms have been associated with the consumption of raw milk or raw milk products. Bulk tank milk (BTM) samples (536) and in-line milk filters (519) collected from dairy farms across the United States during the National Animal Health Monitoring System's Dairy 2007 study were analyzed by real-time PCR for the presence of S. enterica and pathogenic forms of E. coli and by culture techniques for the presence of L. monocytogenes. S. enterica was detected in samples from 28.1% of the dairy operations, primarily in milk filters. Salmonella was isolated from 36 of 75 PCR-positive BTM samples and 105 of 174 PCR-positive filter samples, and the isolates were serotyped. Cerro, Kentucky, Muenster, Anatum, and Newport were the most common serotypes. L. monocytogenes was isolated from 7.1% of the dairy operations, and the 1/2a complex was the most common serotype, followed by 1/2b and 4b (lineage 1). Shiga toxin genes were detected in enrichments from 15.2% of the BTM samples and from 51.0% of the filters by real-time PCR. In most cases, the cycle threshold values for the PCR indicated that toxigenic strains were not a major part of the enrichment populations. These data confirm those from earlier studies showing significant contamination of BTM by zoonotic bacterial pathogens and that the consumption of raw milk and raw milk products presents a health risk.

Non-culture detection of Streptococcus agalactiae (Lancefield group B Streptococcus) in clinical samples by real-time PCR

2012 ◽  
Vol 61 (8) ◽  
pp. 1086-1090 ◽  
Author(s):  
Aruni de Zoysa ◽  
Kirstin Edwards ◽  
Saheer Gharbia ◽  
Anthony Underwood ◽  
André Charlett ◽  
...  
Keyword(s):  
Real Time ◽  
Streptococcus Agalactiae ◽  
Real Time Pcr ◽  
Group B Streptococcus ◽  
Clinical Samples ◽  
Group B ◽  
Lancefield Group

scholarly journals Development of a 5′-Nuclease Real-Time PCR Assay Targeting fliP for the Rapid Identification of Burkholderia mallei in Clinical Samples

2006 ◽  
Vol 52 (2) ◽  
pp. 307-310 ◽  
Author(s):  
Herbert Tomaso ◽  
Holger C Scholz ◽  
Sascha Al Dahouk ◽  
Meike Eickhoff ◽  
Thomas M Treu ◽  
...  
Keyword(s):  
Real Time ◽  
United Arab Emirates ◽  
Real Time Pcr ◽  
Bacterial Load ◽  
Turnaround Time ◽  
Rapid Identification ◽  
Clinical Samples ◽  
Burkholderia Mallei ◽  
Pcr Assay ◽  
Bacterial Dna

Abstract Background: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. Methods: We developed a B. mallei-specific 5′-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. Results: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. Conclusions: Compared with conventional PCR, our rapid 5′-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

scholarly journals Development and Evaluation of Alternative Methods to Identify the Three Most Common Serotypes of Salmonella enterica Causing Clinical Infections in Kazakhstan

2021 ◽  
Vol 9 (11) ◽  
pp. 2319
Author(s):  
Sabyrkhan M. Barmak ◽  
Yuriy A. Sinyavskiy ◽  
Aidar B. Berdygaliev ◽  
Turegeldy Sh. Sharmanov ◽  
Irina S. Savitskaya ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enterica ◽  
Alternative Methods ◽  
Clinical Samples ◽  
Pcr Analysis ◽  
Screening Methods ◽  
Conventional Pcr ◽  
Diagnostic Efficacy ◽  
Microbial Cells

In this study, we aimed to compare the performance of conventional PCR and real-time PCR assays as screening methods for identification of three frequent, clinically significant Salmonella serovars in Kazakhstan. We determined the diagnostic efficacy of three molecular methods for detection of S. enterica subsp. enterica and typing S. Typhimurium, S. Enteritidis, and S. Virchow. A total of 137 clinical samples and 883 food samples were obtained in Almaty in 2018–2019. All tests showed high analytical specificity for detecting S. enterica and its corresponding serovariants (100%). The sensitivity of real-time PCR for each of the tested targets was 1–10 microbial cells and in conventional PCR 10–100 microbial cells. The trials with conventional PCR and real-time PCR had a diagnostic efficacy (DE) of 100% and 99.71%, respectively. The DE of real-time PCR and conventional PCR for detecting S. Enteritidis and S. Typhimurium was 99.90%, while the DE of conventional PCR and real-time PCR for detecting S. Virchow was 99.31% and 99.80%, respectively. The RAPD-PCR analysis of the genomic DNA of Salmonella enterica showed the genetic kinship of S. Enteritidis isolates, and the genetic heterogeneity of S. Typhimurium and S. Virchow isolates. Thus, the developed methods can be considered as alternatives to classical serotyping using antisera.

Sign in / Sign up

Export Citation Format

Share Document