scholarly journals Misidentification of Bordetella bronchiseptica as Bordetella pertussis using a newly described real-time PCR targeting the pertactin gene

2007 ◽  
Vol 56 (12) ◽  
pp. 1608-1610 ◽  
Author(s):  
Karen B. Register ◽  
Tracy L. Nicholson
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
Gene Sequence ◽  
Significant Proportion ◽  
Bordetella Bronchiseptica ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Avian Origin ◽  
Pcr Targeting

Recently, a real-time PCR (RT-PCR) assay based on sequence from the gene for pertactin was proposed for identification of Bordetella pertussis. Here, it is reported that the B. pertussis pertactin gene sequence for the region that encompasses the RT-PCR probe and primers is nearly identical to that of many Bordetella bronchiseptica strains of human and avian origin. Additionally, it is demonstrated that such strains are erroneously identified as B. pertussis using the RT-PCR assay. These data suggest that the use of the assay without confirmatory testing may result in erroneous identification of a significant proportion of human isolates of B. bronchiseptica as B. pertussis.

scholarly journals Ultrasensitive detection of Bacillus anthracis by real time PCR targeting a polymorphism in multi-copy 16S rRNA genes and their transcripts

2021 ◽  
Author(s):  
Peter Braun ◽  
Martin Duy-Thanh Nguyen ◽  
Mathias C Walter ◽  
Gregor Grass
Keyword(s):  
16S Rrna ◽  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Single Copy ◽  
Rrna Genes ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Species Specific ◽  
Pcr Targeting

The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific SNP present in 2-5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene allele afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with a LoD of 6.3 copies/reaction. In a dilution-series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This will at least provide results equaling the DNA-based implementation if no RNA is present but will be superior even at the lowest residual rRNA concentrations.

scholarly journals Ultrasensitive Detection of Bacillus anthracis by Real-Time PCR Targeting a Polymorphism in Multi-Copy 16S rRNA Genes and Their Transcripts

2021 ◽  
Vol 22 (22) ◽  
pp. 12224
Author(s):  
Peter Braun ◽  
Martin Duy-Thanh Nguyen ◽  
Mathias C. Walter ◽  
Gregor Grass
Keyword(s):  
16S Rrna ◽  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Single Copy ◽  
Rrna Genes ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Species Specific ◽  
Pcr Targeting

The anthrax pathogen Bacillus anthracis poses a significant threat to human health. Identification of B. anthracis is challenging because of the bacterium’s close genetic relationship to other Bacillus cereus group species. Thus, molecular detection is founded on species-specific PCR targeting single-copy genes. Here, we validated a previously recognized multi-copy target, a species-specific single nucleotide polymorphism (SNP) present in 2–5 copies in every B. anthracis genome analyzed. For this, a hydrolysis probe-based real-time PCR assay was developed and rigorously tested. The assay was specific as only B. anthracis DNA yielded positive results, was linear over 9 log10 units, and was sensitive with a limit of detection (LoD) of 2.9 copies/reaction. Though not exhibiting a lower LoD than established single-copy PCR targets (dhp61 or PL3), the higher copy number of the B. anthracis–specific 16S rRNA gene alleles afforded ≤2 unit lower threshold (Ct) values. To push the detection limit even further, the assay was adapted for reverse transcription PCR on 16S rRNA transcripts. This RT-PCR assay was also linear over 9 log10 units and was sensitive with an LoD of 6.3 copies/reaction. In a dilution series of experiments, the 16S RT-PCR assay achieved a thousand-fold higher sensitivity than the DNA-targeting assays. For molecular diagnostics, we recommend a real-time RT-PCR assay variant in which both DNA and RNA serve as templates (thus, no requirement for DNase treatment). This can at least provide results equaling the DNA-based implementation if no RNA is present but is superior even at the lowest residual rRNA concentrations.

scholarly journals A specific real-time PCR assay for the detection of Bordetella pertussis

2007 ◽  
Vol 56 (7) ◽  
pp. 918-920 ◽  
Author(s):  
Benoit Vincart ◽  
Ricardo De Mendonça ◽  
Sylvianne Rottiers ◽  
Françoise Vermeulen ◽  
Marc J. Struelens ◽  
...  
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
Analytical Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Pcr Assays ◽  
Reference Strains ◽  
Respiratory Specimens

A novel real-time PCR (RT-PCR) assay was developed for detection of Bordetella pertussis in respiratory specimens by targeting the pertactin gene. In vitro evaluation with reference strains and quality control samples showed analytical sensitivity equivalent to and specificity superior to those of PCR assays which target the IS481 element. The pertactin-based RT-PCR assay offers better discrimination between B. pertussis and other Bordetella species than previously described assays.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

Detection of dengue virus serotypes by single-tube multiplex RT-PCR and multiplex real-time PCR assay

2021 ◽  
Author(s):  
Kundan Tandel ◽  
Mahadevan Kumar ◽  
G.S. Bhalla ◽  
S.P.S. Shergill ◽  
Vijaya Swarnim ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Real Time ◽  
Real Time Pcr ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Single Tube ◽  
Dengue Virus Serotypes

scholarly journals Addition of a Homologous Internal Control to a Real-Time PCR Assay for Detection of Bordetella pertussis

2005 ◽  
Vol 51 (12) ◽  
pp. 2404-2406 ◽  
Author(s):  
Christoph Koidl ◽  
Michael Bozic ◽  
Jörg Berg ◽  
Markus Stöcher ◽  
Gerhard Mühlbauer ◽  
...  
Keyword(s):  
Real Time ◽  
Bordetella Pertussis ◽  
Internal Control ◽  
Real Time Pcr ◽  
Pcr Assay

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.

Isolation and detection of Shiga toxin-producing Escherichia coli in clinical stool samples using conventional and molecular methods

2009 ◽  
Vol 58 (7) ◽  
pp. 905-911 ◽  
Author(s):  
Matthew W. Gilmour ◽  
Linda Chui ◽  
Theodore Chiu ◽  
Dobryan M. Tracz ◽  
Kathryn Hagedorn ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Molecular Methods ◽  
Genetic Profile ◽  
Pcr Assay ◽  
Suspension Array ◽  
Stool Samples ◽  
Pcr Targeting

The isolation of Shiga toxin-producing Escherichia coli (STEC) other than serogroup O157 from clinical stool samples is problematic due to the lack of differential phenotypic characteristics from non-pathogenic E. coli. The development of molecular reagents capable of identifying both toxin and serogroup-specific genetic determinants holds promise for a more comprehensive characterization of stool samples and isolation of STEC strains. In this study, 876 stool samples from paediatric patients with gastroenteritis were screened for STEC using a cytotoxicity assay, commercial immunoassay and a conventional PCR targeting Shiga-toxin determinants. In addition, routine culture methods for isolating O157 STEC were also performed. The screening assays identified 45 stools presumptively containing STEC, and using non-differential culture techniques a total of 20 O157 and 22 non-O157 strains were isolated. These included STEC serotypes O157 : H7, O26 : H11, O121 : H19, O26 : NM, O103 : H2, O111 : NM, O115 : H18, O121 : NM, O145 : NM, O177 : NM and O5 : NM. Notably, multiple STEC serotypes were isolated from two clinical stool samples (yielding O157 : H7 and O26 : H11, or O157 : H7 and O103 : H2 isolates). These data were compared to molecular serogroup profiles determined directly from the stool enrichment cultures using a LUX real-time PCR assay targeting the O157 fimbrial gene lpfA, a microsphere suspension array targeting allelic variants of espZ and a gnd-based molecular O-antigen serogrouping method. The genetic profile of individual stool cultures indicated that the espZ microsphere array and lpfA real-time PCR assay could accurately predict the presence and provide preliminary typing for the STEC strains present in clinical samples. The gnd-based molecular serogrouping method provided additional corroborative evidence of serogroup identities. This toolbox of molecular methods provided robust detection capabilities for STEC in clinical stool samples, including co-infection of multiple serogroups.

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