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 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 MOLECULAR INVESTIGATION OF HEMOTROPIC MYCOPLASMAS IN HUMAN BEINGS, DOGS AND HORSES IN A RURAL SETTLEMENT IN SOUTHERN BRAZIL

2015 ◽  
Vol 57 (4) ◽  
pp. 353-357 ◽  
Author(s):  
Rafael Felipe da Costa VIEIRA ◽  
Odilon VIDOTTO ◽  
Thállitha Samih Wischral Jayme VIEIRA ◽  
Ana Márcia Sá GUIMARAES ◽  
Andrea Pires dos SANTOS ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Sybr Green ◽  
Rrna Genes ◽  
Human Beings ◽  
Pcr Assay ◽  
Factors Associated ◽  
Human Sample ◽  
Successful Amplification ◽  
Species Specific

SUMMARY The aims of this study were to determine the prevalence of hemoplasmas in a rural Brazilian settlement's population of human beings, their dogs and horses, highly exposed to tick bites; to identify the tick species parasitizing dogs and horses, and analyze factors associated with their infection. Blood samples from 132 dogs, 16 horses and 100 humans were screened using a pan-hemoplasma SYBR green real-time PCR assay followed by a species-specific TaqMan real-time PCR. A total of 59/132 (44.7%) dog samples were positive for hemoplasmas (21 Mycoplasma haemocanisalone, 12 ' Candidatus Mycoplasma haematoparvum' alone and 21 both). Only 1/100 (1.0%) human sample was positive by qPCR SYBR green, with no successful amplification of 16S rRNA or 23 rRNA genes despite multiple attempts. All horse samples were negative. Dogs >1 year of age were more likely to be positive for hemoplasmas ( p= 0.0014). In conclusion, although canine hemoplasma infection was highly prevalent, cross-species hemoplasma transmission was not observed, and therefore may not frequently occur despite overexposure of agents and vectors.

scholarly journals Development and Assessment of a Real-Time PCR Assay for Rapid and Sensitive Detection of a Novel Thermotolerant Bacterium, Lactobacillus thermotolerans, in Chicken Feces

2005 ◽  
Vol 71 (8) ◽  
pp. 4214-4219 ◽  
Author(s):  
Abu Sadeque Md Selim ◽  
Piyanuch Boonkumklao ◽  
Teruo Sone ◽  
Apinya Assavanig ◽  
Masaru Wada ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Real Time ◽  
Real Time Pcr ◽  
Pure Culture ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Chicken Feces

ABSTRACT A new real-time PCR assay was successfully developed using a TaqMan fluorescence probe for specific detection and enumeration of a novel bacterium, Lactobacillus thermotolerans, in chicken feces. The specific primers and probe were designed based on the L. thermotolerans 16S rRNA gene sequences, and these sequences were compared to those of all available 16S rRNA genes in the GenBank database. The assay, targeting 16S rRNA gene, was evaluated using DNA from a pure culture of L. thermotolerans, DNA from the closely related bacteria Lactobacillus mucosae DSM 13345T and Lactobacillus fermentum JCM 1173T, and DNA from other lactic acid bacteria in quantitative experiments. Serial dilutions of L. thermotolerans DNA were used as external standards for calibration. The minimum detection limit of this technique was 1.84 × 103 cells/ml of an L. thermotolerans pure culture. The assay was then applied to chicken feces in two different trials. In the first trial, the cell population was 104 cells/g feces on day 4 and 105 cells/g feces on days 11 to 18. However, cell populations of 106 to 107 cells/g feces were detected in the second trial. The total bacterial count, measured by 4′,6-diamidino-2-phenylindole (DAPI) staining, was approximately 1011 cells/g feces. These results suggest that in general, L. thermotolerans is a normal member of the chicken gut microbiota, although it is present at relatively low levels in the feces.

scholarly journals Power Analysis for Real-Time PCR Quantification of Genes in Activated Sludge and Analysis of the Variability Introduced by DNA Extraction

2003 ◽  
Vol 69 (11) ◽  
pp. 6597-6604 ◽  
Author(s):  
Hebe M. Dionisi ◽  
Gerda Harms ◽  
Alice C. Layton ◽  
Igrid R. Gregory ◽  
Jack Parker ◽  
...  
Keyword(s):  
16S Rrna ◽  
Activated Sludge ◽  
Real Time ◽  
Real Time Pcr ◽  
Power Analysis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Pcr Assay ◽  
Pcr Assays

ABSTRACT The aims of this study were to determine the power of discrimination of the real-time PCR assay for monitoring fluctuations in microbial populations within activated sludge and to identify sample processing points where methodological changes are needed to minimize the variability in target quantification. DNA was extracted using a commercially available kit from mixed liquor samples taken from the aeration tank of four bench-scale activated-sludge reactors operating at 2-, 5-, 10-, and 20-day solid retention times, with mixed-liquor volatile suspended solid (MLVSS) values ranging from 260 to 2,610 mg/liter. Real-time PCR assays for bacterial and Nitrospira 16S rRNA genes were chosen because they represent, respectively, a highly abundant and a less-abundant bacterial target subject to clustering within the activated sludge matrix. The mean coefficient of variation in DNA yields (measured as microgram of DNA per milligram of MLVSS) in triplicate extractions of 12 different samples was 12.2%. Based on power analyses, the variability associated with DNA extraction had a small impact on the overall variability of the real-time PCR assay. Instead, a larger variability was associated with the PCR assay. The less-abundant target (Nitrospira 16S rRNA gene) had more variability than the highly abundant target (bacterial 16S rRNA gene), and samples from the lower-biomass reactors had more variability than samples from the higher-biomass reactors. Power analysis of real-time PCR assays indicated that three to five samples were necessary to detect a twofold increase in bacterial 16S rRNA genes, whereas three to five samples were required to detect a fivefold increase in Nitrospira 16S rRNA genes.

scholarly journals Real-Time PCR Assay for a Unique Chromosomal Sequence of Bacillus anthracis

2004 ◽  
Vol 42 (12) ◽  
pp. 5825-5831 ◽  
Author(s):  
E. Bode ◽  
W. Hurtle ◽  
D. Norwood
Keyword(s):  
Bacillus Anthracis ◽  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Chromosomal Sequence

scholarly journals Characterization of Plasmodium ovale curtisi and P. ovale wallikeri in Western Kenya Utilizing a Novel Species-specific Real-time PCR Assay

2015 ◽  
Vol 9 (1) ◽  
pp. e0003469 ◽  
Author(s):  
Robin H. Miller ◽  
Clifford O. Obuya ◽  
Elizabeth W. Wanja ◽  
Bernhards Ogutu ◽  
John Waitumbi ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Novel Species ◽  
Plasmodium Ovale ◽  
Pcr Assay ◽  
Western Kenya ◽  
Plasmodium Ovale Curtisi ◽  
Species Specific

Species-specific identification of variola, monkeypox, cowpox, and vaccinia viruses by multiplex real-time PCR assay

2011 ◽  
Vol 175 (2) ◽  
pp. 163-169 ◽  
Author(s):  
Sergei N. Shchelkunov ◽  
Dmitrii N. Shcherbakov ◽  
Rinat A. Maksyutov ◽  
Elena V. Gavrilova
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Specific Identification ◽  
Vaccinia Viruses ◽  
Species Specific

scholarly journals Detection of Dientamoeba fragilis in animal faeces using species specific real time PCR assay

2016 ◽  
Vol 227 ◽  
pp. 42-47 ◽  
Author(s):  
Douglas Chan ◽  
Joel Barratt ◽  
Tamalee Roberts ◽  
Owen Phillips ◽  
Jan Šlapeta ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Pcr Assay ◽  
Dientamoeba Fragilis ◽  
Species Specific

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.

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