Evaluation of Faecalibacterium 16S rDNA genetic markers for accurate identification of swine faecal waste by quantitative PCR

2016 ◽  
Vol 181 ◽  
pp. 193-200 ◽  
Author(s):  
Chuanren Duan ◽  
Yamin Cui ◽  
Yi Zhao ◽  
Jun Zhai ◽  
Baoyun Zhang ◽  
...  
Keyword(s):  
16S Rdna ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Accurate Identification

Corrigendum: Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil

2010 ◽  
Vol 56 (1) ◽  
pp. 87-87
Author(s):  
Miguel A. Providenti ◽  
Melissa Begin ◽  
Samielle Hynes ◽  
Christine Lamarche ◽  
David Chitty ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers

scholarly journals Identification, Detection, and Enumeration of Human Bifidobacterium Species by PCR Targeting the Transaldolase Gene

2002 ◽  
Vol 68 (5) ◽  
pp. 2420-2427 ◽  
Author(s):  
Teresa Requena ◽  
Jeremy Burton ◽  
Takahiro Matsuki ◽  
Karen Munro ◽  
Mary Alice Simon ◽  
...  
Keyword(s):  
Real Time ◽  
16S Rdna ◽  
Quantitative Pcr ◽  
Gene Sequence ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Bifidobacterium Longum ◽  
Bifidobacterium Adolescentis ◽  
Fecal Samples ◽  
Real Time Quantitative Pcr ◽  
Pcr Targeting

ABSTRACT Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

scholarly journals Quantitative PCR for Genetic Markers of Human Fecal Pollution

2009 ◽  
Vol 75 (17) ◽  
pp. 5507-5513 ◽  
Author(s):  
Orin C. Shanks ◽  
Catherine A. Kelty ◽  
Mano Sivaganesan ◽  
Manju Varma ◽  
Richard A. Haugland
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers ◽  
Quantitative Methods ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Target Dna ◽  
Pcr Assays ◽  
Wastewater Samples ◽  
Human Specific

ABSTRACT Assessment of health risk and fecal bacterial loads associated with human fecal pollution requires reliable host-specific analytical methods and a rapid quantification approach. We report the development of quantitative PCR assays for quantification of two recently described human-specific genetic markers targeting Bacteroidales-like cell surface-associated genes. Each assay exhibited a range of quantification from 10 to 1 � 106 copies of target DNA. For each assay, internal amplification controls were developed to detect the presence or absence of amplification inhibitors. The assays predominantly detected human fecal specimens and exhibited specificity levels greater than 97% when tested against 265 fecal DNA extracts from 22 different animal species. The abundance of each human-specific genetic marker in primary effluent wastewater samples collected from 20 geographically distinct locations was measured and compared to quantities estimated by real-time PCR assays specific for rRNA gene sequences from total Bacteroidales and enterococcal fecal microorganisms. Assay performances combined with the prevalence of DNA targets in sewage samples provide experimental evidence supporting the potential application of these quantitative methods for monitoring fecal pollution in ambient environmental waters.

scholarly journals Evaluation of Different PCR Assay Formats for Sensitive and Specific Detection of SARS-CoV-2 RNA

2020 ◽  
Author(s):  
Jeremy Ratcliff ◽  
Dung Nguyen ◽  
Monique Andersson ◽  
Peter Simmonds
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Nested Pcr ◽  
High Sensitivity ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Copy Detection ◽  
Accurate Identification ◽  
Laboratory Equipment ◽  
Rna Transcripts

ABSTRACTAccurate identification of individuals infected with SARS-CoV-2 is crucial for efforts to control the ongoing COVID-19 pandemic. Polymerase chain reaction (PCR)-based assays are the gold standard for detecting viral RNA in patient samples and are used extensively in clinical settings. Most currently used quantitative PCR (RT-qPCRs) rely upon real-time detection of PCR product using specialized laboratory equipment. To enable the application of PCR in resource-poor or non-specialist laboratories, we have developed and evaluated a nested PCR method for SARS-CoV-2 RNA using simple agarose gel electrophoresis for product detection. Using clinical samples tested by conventional qPCR methods and RNA transcripts of defined RNA copy number, the nested PCR based on the RdRP gene demonstrated high sensitivity and specificity for SARS-CoV-2 RNA detection in clinical samples, but showed variable and transcript length-dependent sensitivity for RNA transcripts. Samples and transcripts were further evaluated in an additional N protein real-time quantitative PCR assay. As determined by 50% endpoint detection, the sensitivities of three RT-qPCRs and nested PCR methods varied substantially depending on the transcript target with no method approaching single copy detection. Overall, these findings highlight the need for assay validation and optimization and demonstrate the inability to precisely compare viral quantification from different PCR methodologies without calibration.

Factors affecting the presence of human-associated and fecal indicator real-time quantitative PCR genetic markers in urban-impacted recreational beaches

2014 ◽  
Vol 64 ◽  
pp. 196-208 ◽  
Author(s):  
Marirosa Molina ◽  
Shayla Hunter ◽  
Mike Cyterski ◽  
Lindsay A. Peed ◽  
Catherine A. Kelty ◽  
...  
Keyword(s):  
Real Time ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Fecal Indicator ◽  
Factors Affecting ◽  
Real Time Quantitative Pcr ◽  
Recreational Beaches

scholarly journals Quantitative PCR for Detection and Enumeration of Genetic Markers of Bovine Fecal Pollution

2007 ◽  
Vol 74 (3) ◽  
pp. 745-752 ◽  
Author(s):  
Orin C. Shanks ◽  
Emina Atikovic ◽  
A. Denene Blackwood ◽  
Jingrang Lu ◽  
Rachel T. Noble ◽  
...  
Keyword(s):  
Genetic Marker ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Animal Species ◽  
Fecal Pollution ◽  
Rrna Gene ◽  
Broad Distribution ◽  
Assay Performance ◽  
Target Dna ◽  
Bovine Feces

ABSTRACTAccurate assessment of health risks associated with bovine (cattle) fecal pollution requires a reliable host-specific genetic marker and a rapid quantification method. We report the development of quantitative PCR assays for the detection of two recently described bovine feces-specific genetic markers and a method for the enumeration of these markers using a Markov chain Monte Carlo approach. Both assays exhibited a range of quantification from 25 to 2 × 106copies of target DNA, with a coefficient of variation of <2.1%. One of these assays can be multiplexed with an internal amplification control to simultaneously detect the bovine-specific genetic target and presence of amplification inhibitors. The assays detected only cattle fecal specimens when tested against 204 fecal DNA extracts from 16 different animal species and also demonstrated a broad distribution among individual bovine samples (98 to 100%) collected from five geographically distinct locations. The abundance of each bovine-specific genetic marker was measured in 48 individual samples and compared to quantitative PCR-enumerated quantities of rRNA gene sequences representing totalBacteroidetes,Bacteroides thetaiotaomicron, and enterococci in the same specimens. Acceptable assay performance combined with the prevalence of DNA targets across different cattle populations provides experimental evidence that these quantitative assays will be useful in monitoring bovine fecal pollution in ambient waters.

scholarly journals Development of DNA Melt Curve Analysis for the Identification of Lepidopteran Pests in Almonds and Pistachios

Insects ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 553
Author(s):  
Rohith Vulchi ◽  
Kent M. Daane ◽  
Jacob A. Wenger
Keyword(s):  
Quantitative Pcr ◽  
Curve Analysis ◽  
Oriental Fruit Moth ◽  
Choristoneura Rosaceana ◽  
Accurate Identification ◽  
Amyelois Transitella ◽  
Melt Temperature ◽  
Melt Curve Analysis ◽  
Lepidopteran Pests ◽  
Indian Meal

Almonds and pistachios are fed upon by a diverse assemblage of lepidopteran insects, several of which are economically important pests. Unfortunately, identification of these pests can be difficult, as specimens are frequently damaged during collection, occur in traps with non-target species, and are morphologically similar up to their third instar. Here, we present a quantitative PCR based melt curve analysis for simple, rapid, and accurate identification of six lepidopteran pests of almonds and pistachios: navel orangeworm (Amyelois transitella), peach twig borer (Anarsia lineatella), oriental fruit moth (Grapholita molesta), obliquebanded leafroller (Choristoneura rosaceana), raisin moth (Cadra figulilella), and Indian meal moth (Plodia interpunctella). In this approach, the dissociation (melt) temperature(s) of a 658 bp section of cytochrome c oxidase subunit 1 was determined using quantitative PCR (qPCR). Within these six species, the distribution and the number of melt peak temperatures provide an unambiguous species level identification that is reproducible when unsheared DNA can be extracted. The test is robust across a variety of sampling approaches including insects removed from sticky card traps, museum specimens, and samples that were left in the field for up to 7 days. The melt curve’s simplicity allows it to be performed in any basic molecular biology laboratory with a quantitative PCR.

Validation and application of quantitative PCR assays using host-specific Bacteroidales genetic markers for swine fecal pollution tracking

2017 ◽  
Vol 231 ◽  
pp. 1569-1577 ◽  
Author(s):  
Lihua Fan ◽  
Jiangbing Shuai ◽  
Ruoxue Zeng ◽  
Hongfei Mo ◽  
Suhua Wang ◽  
...  
Keyword(s):  
Quantitative Pcr ◽  
Genetic Markers ◽  
Fecal Pollution ◽  
Pcr Assays

Identification and application of AFLP-derived genetic markers for quantitative PCR-based tracking of Bacillus and Paenibacillus spp. released in soil

2009 ◽  
Vol 55 (10) ◽  
pp. 1166-1175 ◽  
Author(s):  
Miguel A. Providenti ◽  
Melissa Begin ◽  
Samielle Hynes ◽  
Christine Lamarche ◽  
David Chitty ◽  
...  
Keyword(s):  
Detection Limit ◽  
Quantitative Pcr ◽  
Genetic Markers ◽  
Paenibacillus Polymyxa ◽  
Soil Core ◽  
Environmental Persistence ◽  
Natural Systems ◽  
Length Polymorphisms ◽  
Amplified Fragment Length Polymorphisms ◽  
Different Strains

In this study, we show that noncoding sequences from amplified fragment length polymorphisms (AFLPs) can provide robust and sensitive genetic markers suitable for PCR-based discrimination of closely related strains of Bacillus and Paenibacillus , and quantitative PCR (qPCR)-based tracking of the strains in complex natural systems like soil. Quantitative PCR was accurate in the ~1 × 109 to ~1 × 104 colony forming units (CFU)/g soil range. The detection limit was improved to ~1 × 102 CFU/g when amplicons were analyzed by gel electrophoresis. Studies with laboratory-contained intact soil-core microcosms indicated that environmental persistence trends vary among different strains. For example, Bacillus circulans ATCC 9500, Bacillus amyloliquefaciens DSL 13563-0, Bacillus licheniformis ATCC 12713, Paenibacillus polymyxa NRRL B-4317, and 3 Bacillus subtilis strains (ATCC 6051A, ATCC 55405, and NRRL B-941) died down to below the 1 × 102 CFU/g detection limit by days 28–105. In contrast, over a 105-day period, B. licheniformis ATCC 55406, Bacillus megaterium NRRL B-14308, and P. polymyxa strains ATCC 55407 and DSL 13540-4 died down but persisted at levels just above the detection limit, whereas Bacillus thuringiensis ATCC 13367 experienced a less than 10-fold decrease in cell numbers.

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