Detection and quantification of Histomonas meleagridis by real-time PCR targeting single copy genes

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.

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Detection and Quantification of Hepatitis C Virus (HCV) by MultiCode-RTx Real-Time PCR Targeting the HCV 3' Untranslated Region

Journal of Clinical Microbiology ◽

10.1128/jcm.02170-08 ◽

2009 ◽

Vol 47 (8) ◽

pp. 2635-2638 ◽

Cited By ~ 7

Author(s):

E. K. Mulligan ◽

J. J. Germer ◽

M. Q. Arens ◽

K. L. D'Amore ◽

A. Di Bisceglie ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Real Time ◽

Real Time Pcr ◽

Untranslated Region ◽

Pcr Targeting ◽

Detection And Quantification

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

International Journal of Molecular Sciences ◽

10.3390/ijms222212224 ◽

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.

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Real-Time PCR for Molecular Detection of Zoonotic and Non-Zoonotic Giardia spp. in Wild Rodents

Microorganisms ◽

10.3390/microorganisms9081610 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1610

Author(s):

Christian Klotz ◽

Elke Radam ◽

Sebastian Rausch ◽

Petra Gosten-Heinrich ◽

Toni Aebischer

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Gastrointestinal Disease ◽

Single Copy ◽

Marker Genes ◽

Small Rodent ◽

Analytical Sensitivity ◽

Single Copy Gene ◽

Wild Rodents ◽

Copy Gene

Giardiasis in humans is a gastrointestinal disease transmitted by the potentially zoonotic Giardia duodenalis genotypes (assemblages) A and B. Small wild rodents such as mice and voles are discussed as potential reservoirs for G. duodenalis but are predominantly populated by the two rodent species Giardia microti and Giardia muris. Currently, the detection of zoonotic and non-zoonotic Giardia species and genotypes in these animals relies on cumbersome PCR and sequencing approaches of genetic marker genes. This hampers the risk assessment of potential zoonotic Giardia transmissions by these animals. Here, we provide a workflow based on newly developed real-time PCR schemes targeting the small ribosomal RNA multi-copy gene locus to distinguish G. muris, G. microti and G. duodenalis infections. For the identification of potentially zoonotic G. duodenalis assemblage types A and B, an established protocol targeting the single-copy gene 4E1-HP was used. The assays were specific for the distinct Giardia species or genotypes and revealed an analytical sensitivity of approximately one or below genome equivalent for the multi-copy gene and of about 10 genome equivalents for the single-copy gene. Retesting a biobank of small rodent samples confirmed the specificity. It further identified the underlying Giardia species in four out of 11 samples that could not be typed before by PCR and sequencing. The newly developed workflow has the potential to facilitate the detection of potentially zoonotic and non-zoonotic Giardia species in wild rodents.

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Molecular Characterization of Giardia duodenalis in Children and Adults Sampled in Algeria

Microorganisms ◽

10.3390/microorganisms9010054 ◽

2020 ◽

Vol 9 (1) ◽

pp. 54

Author(s):

Salem Belkessa ◽

Daniel Thomas-Lopez ◽

Karim Houali ◽

Farida Ghalmi ◽

Christen Rune Stensvold

Keyword(s):

Real Time ◽

Molecular Characterization ◽

Real Time Pcr ◽

Intestinal Parasites ◽

Giardia Duodenalis ◽

Specific Pcr ◽

Stool Samples ◽

Pcr Targeting ◽

Assemblage B

The molecular epidemiology of giardiasis in Africa remains unclear. A study was carried out across four hospitals in Algeria. A total of 119 fecal samples from 55 children, 37 adults, and 27 individuals of undetermined age, all scored positive for intestinal parasites by microscopy, and were screened by real-time PCR for Giardia. Molecular characterization of Giardia was performed by assemblage-specific PCR and PCR targeting the triose phosphate isomerase gene (tpi). Of the 119 samples, 80 (67%) were Giardia-positive by real-time PCR. For 48 moderately-highly real-time PCR-positive samples, tpi genotyping assigned 22 samples to Assemblage A and 26 to Assemblage B. Contrary to Assemblage A, Assemblage B exhibited substantial genetic diversity and allelic heterozygosity. Assemblage-specific PCR proved to be specific for discriminating Assemblage A or B but not as sensitive as tpi genotyping. We confirmed that real-time PCR is more sensitive than microscopy for detecting Giardia in stool samples and that robust amplification and sequencing of the tpi gene is feasible when moderate-to-strongly real-time PCR-positive samples are used. This study is one of the few performed in Africa providing genotyping data on Giardia infections in humans. Both assemblages A and B were commonly seen and not associated with specific sociodemographic data.

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Single-copy detection of somatic variants from solid and liquid biopsy

Scientific Reports ◽

10.1038/s41598-021-85545-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Ana-Luisa Silva ◽

Paulina Klaudyna Powalowska ◽

Magdalena Stolarek ◽

Eleanor Ruth Gray ◽

Rebecca Natalie Palmer ◽

...

Keyword(s):

Real Time ◽

Single Molecule ◽

Real Time Pcr ◽

Clinical Decision Making ◽

High Sensitivity ◽

Clinical Decision ◽

Single Copy ◽

Turnaround Time ◽

Copy Detection ◽

Allele Specific

AbstractAccurate detection of somatic variants, against a background of wild-type molecules, is essential for clinical decision making in oncology. Existing approaches, such as allele-specific real-time PCR, are typically limited to a single target gene and lack sensitivity. Alternatively, next-generation sequencing methods suffer from slow turnaround time, high costs, and are complex to implement, typically limiting them to single-site use. Here, we report a method, which we term Allele-Specific PYrophosphorolysis Reaction (ASPYRE), for high sensitivity detection of panels of somatic variants. ASPYRE has a simple workflow and is compatible with standard molecular biology reagents and real-time PCR instruments. We show that ASPYRE has single molecule sensitivity and is tolerant of DNA extracted from plasma and formalin fixed paraffin embedded (FFPE) samples. We also demonstrate two multiplex panels, including one for detection of 47 EGFR variants. ASPYRE presents an effective and accessible method that simplifies highly sensitive and multiplexed detection of somatic variants.

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