Corrigendum to “A handheld real time thermal cycler for bacterial pathogen detection” [Biosens. Bioelectron. 18 (2003) 1115–1123]

Fecal coliforms have been used as indicators to evaluate health risks associated with the microbiological quality of water for many years. Recent studies have challenged their ability to accurately predict bacterial numbers in the natural environment. DNA-based assays are proposed candidates to replace existing methods, but protocols suited for standardized direct-use have not yet been sufficiently developed. The objective of this study was to examine the feasibility of using real-time quantitative PCR (qPCR) to detect contamination from five waterborne bacterial pathogens in surface and treated drinking waters. Robust oligonucleotide primers were assembled to target virulence-associated genes. Primers were found to have high specificity and increased sensitivity for low pathogen loads of 10 cells/mL, as determined experimentally via qPCR. Detection of pathogenic cells directly from an environmental matrix has also been demonstrated using a filtration-extraction procedure. The developed protocols have shown their potential for use in conjunction with traditional indicator techniques.

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Development and evaluation of rapid novel isothermal amplification assays for important veterinary pathogens:Chlamydia psittaciandChlamydia pecorum

PeerJ ◽

10.7717/peerj.3799 ◽

2017 ◽

Vol 5 ◽

pp. e3799 ◽

Cited By ~ 26

Author(s):

Martina Jelocnik ◽

Md. Mominul Islam ◽

Danielle Madden ◽

Cheryl Jenkins ◽

James Branley ◽

...

Keyword(s):

Real Time ◽

Pathogen Detection ◽

Point Of Care ◽

Chlamydia Psittaci ◽

Isothermal Amplification ◽

Diagnostic Tools ◽

Clinical Samples ◽

Conserved Gene ◽

Thermal Cycler ◽

Species Specific

BackgroundChlamydia psittaciandChlamydia pecorumare important veterinary pathogens, with the former also being responsible for zoonoses, and the latter adversely affecting koala populations in Australia and livestock globally. The rapid detection of these organisms is still challenging, particularly at the point-of-care (POC). In the present study, we developed and evaluated rapid, sensitive and robustC. psittaci-specific andC. pecorum-specific Loop Mediated Isothermal Amplification (LAMP) assays for detection of these pathogens.Methods and MaterialsThe LAMP assays, performed in a Genie III real-time fluorometer, targeted a 263 bp region of theC. psittaci-specific Cps_0607 gene or a 209 bp region of aC. pecorum-specific conserved gene CpecG_0573, and were evaluated using a range of samples previously screened using species-specific quantitative PCRs (qPCRs). Species-specificity forC. psittaciandC. pecorumLAMP targets was tested against DNA samples from related chlamydial species and a range of other bacteria. In order to evaluate pathogen detection in clinical samples,C. psittaciLAMP was evaluated using a total of 26 DNA extracts from clinical samples from equine and avian hosts, while forC. pecorumLAMP, we tested a total of 63 DNA extracts from clinical samples from koala, sheep and cattle hosts. A subset of 36C. pecorumsamples was also tested in a thermal cycler (instead of a real-time fluorometer) using newly developed LAMP and results were determined as an end point detection. We also evaluated rapid swab processing (without DNA extraction) to assess the robustness of these assays.ResultsBoth LAMP assays were demonstrated to species-specific, highly reproducible and to be able to detect as little as 10 genome copy number/reaction, with a mean amplification time of 14 and 24 min forC. psittaciandC. pecorum, respectively. When testing clinical samples, the overall congruence between the newly developed LAMP assays and qPCR was 92.3% forC. psittaci(91.7% sensitivity and 92.9% specificity); and 84.1% forC. pecorum(90.6% sensitivity and 77.4% specificity). For a subset of 36C. pecorumsamples tested in a thermal cycler using newly developed LAMP, we observed 34/36 (94.4%) samples result being congruent between LAMP performed in fluorometer and in thermal cycler. Rapid swab processing method evaluated in this study also allows for chlamydial DNA detection using LAMP.DiscussionIn this study, we describe the development of novel, rapid and robustC. psittaci-specific andC. pecorum-specific LAMP assays that are able to detect these bacteria in clinical samples in either the laboratory or POC settings. With further development and a focus on the preparation of these assays at the POC, it is anticipated that both tests may fill an important niche in the repertoire of ancillary diagnostic tools available to clinicians.

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Design and validation of oligonucleotide primers suitable for waterborne bacterial pathogen detection via real-time qPCR

10.32920/ryerson.14661288.v1 ◽

2021 ◽

Author(s):

Shawn Thomas Clark

Keyword(s):

Real Time ◽

Pathogen Detection ◽

Extraction Procedure ◽

Microbiological Quality ◽

Bacterial Pathogen ◽

High Specificity ◽

Fecal Coliforms ◽

Oligonucleotide Primers ◽

Real Time Quantitative Pcr ◽

Quality Of Water

Fecal coliforms have been used as indicators to evaluate health risks associated with the microbiological quality of water for many years. Recent studies have challenged their ability to accurately predict bacterial numbers in the natural environment. DNA-based assays are proposed candidates to replace existing methods, but protocols suited for standardized direct-use have not yet been sufficiently developed. The objective of this study was to examine the feasibility of using real-time quantitative PCR (qPCR) to detect contamination from five waterborne bacterial pathogens in surface and treated drinking waters. Robust oligonucleotide primers were assembled to target virulence-associated genes. Primers were found to have high specificity and increased sensitivity for low pathogen loads of 10 cells/mL, as determined experimentally via qPCR. Detection of pathogenic cells directly from an environmental matrix has also been demonstrated using a filtration-extraction procedure. The developed protocols have shown their potential for use in conjunction with traditional indicator techniques.

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Engineered Combined-Positive-Control Template for Real-Time Reverse Transcription-PCR in Multiple-Pathogen-Detection Assays

Journal of Clinical Microbiology ◽

10.1128/jcm.05987-11 ◽

2011 ◽

Vol 50 (3) ◽

pp. 1057-1060 ◽

Cited By ~ 22

Author(s):

M. Kodani ◽

J. M. Winchell

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Pathogen Detection ◽

Positive Control ◽

Reverse Transcription Pcr ◽

Multiple Pathogen

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Abstract 11119: Real-Time Nanopore Sequencing for Bacterial Pneumonia After Out-of-Hospital Cardiac Arrest, a Pilot Proof-of-Concept Study

Circulation ◽

10.1161/circ.144.suppl_2.11119 ◽

2021 ◽

Vol 144 (Suppl_2) ◽

Author(s):

Alexandra Weissman ◽

Mariam Bramah Lawani ◽

Thomas Rohan ◽

Clifton W CALLAWAY

Keyword(s):

Patient Care ◽

Pathogen Detection ◽

Bacterial Pathogen ◽

Improve Patient Care ◽

Antibiotic Administration ◽

Nanopore Sequencing ◽

Proof Of Concept ◽

Chi Square ◽

Significant Difference ◽

Improve Patient

Introduction: Pneumonia is common after OHCA but is difficult to diagnose in the first 72 hours following ROSC, this results in early untargeted antibiotic administration based on non-specific imaging and laboratory findings. Antibiotic resistance is rising, is influenced by untargeted antibiotic administration, and can increase patient morbidity and mortality as well as healthcare costs. Precision methods of bacterial pathogen detection in OHCA patients are needed to improve patient care. This proof-of-concept pilot study aimed to assess feasibility of bacterial pathogen sequencing and comparability of sequencing results to clinical culture after OHCA. Methods: Blood and bronchoalveolar lavage (BAL) were obtained from residual clinical specimens collected within 12 hours of ROSC. Bacterial DNA was extracted using the Qiagen PowerLyzer PowerSoil DNA kit, sequenced using the MinION nanopore sequencer, and analyzed with Oxford Nanopore Technologies’ EPI2ME bioinformatics software. Sequencing results were compared to culture results using McNemar’s chi-square statistic. Study-defined pneumonia was based on presence of at least two characteristics within 72 hours of ROSC: fever (temperature ≥38°C); persistent leukocytosis >15,000 or leukopenia <3,500 for 48 hours; persistent chest radiography infiltrates for 48 hours per clinical radiology read; bacterial pathogen cultured. Results: We enrolled 38 consecutive OHCA subjects: mean age 61.8 years (18.0); 16 (42%) female; 25 (66%) White, 7 (18%) Black, 6 (16%) “Other” race; 7 subjects (18%) survived and 31 (82%) died; 16 (42%) subjects had pneumonia. Sequencing results were available in 12 hours while culture results were available in 48-72 hours after collection. There was a non-significant difference in the proportion of the same pathogens identified for each method per McNemar’s chi-square: p = 0.38, difference of 0.095 (-0.095, 0.286). Conclusions: Nanopore sequencing detects pathogenic bacteria comparable to clinical microbiologic culture and in less time. This technology can produce a paradigm shift in early bacterial pathogen detection in OHCA survivors, which can improve patient care. The technology is applicable to other patient populations and for viral and fungal pathogens.

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Utilizing the Public GenomeTrakr Database for Foodborne Pathogen Traceback v1

10.17504/protocols.io.bxw3ppgn ◽

2021 ◽

Author(s):

Ruth E Timme ◽

Maria Sanchez ◽

Marc Allard

Keyword(s):

Real Time ◽

Foodborne Pathogens ◽

Pathogen Detection ◽

Foodborne Pathogen ◽

Reference Database ◽

Surveillance Program ◽

Environmental Isolates ◽

The Public ◽

The World ◽

The Usa

This protocol outlines the all the steps necessary to become a GenomeTrakr data contributor. GenomeTrakr is an international genomic reference database of mostly food and environmental isolates from foodborne pathogens. The data and analyses are housed at the National Center for Biotechnology Information (NCBI), which is a database freely available to anyone in the world. The Pathogen Detection browser at NCBI computes daily cluster results adding the newly submitted data to the existing phylogenetic clusters of closely related genomes. Contributors to this database can see how their new isolates are related to the real-time foodborne pathogen surveillance program established in the USA and a few other countries, and at the same time adding valuable new data to the reference database. ------ Although originally published as a Chapter in Methods and Protocols, Foodborne Bacterial Pathogens, the protocol has since been adapted and split into four separate protocols all of which are contained in this collection.

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Identification of Field Tolerance and Resistance to Mal Secco Disease in a Citrus Germplasm Collection in Sicily

Agronomy ◽

10.3390/agronomy10111806 ◽

2020 ◽

Vol 10 (11) ◽

pp. 1806

Author(s):

Riccardo Russo ◽

Marco Caruso ◽

Carmen Arlotta ◽

Angela Roberta Lo Piero ◽

Elisabetta Nicolosi ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Pathogen Detection ◽

Germplasm Collection ◽

Research Centre ◽

Molecular Screening ◽

Sources Of Resistance ◽

Strong Negative Correlation ◽

Canopy Volume ◽

Mal Secco

Mal secco is a tracheomycotic disease caused by the fungus Plenodomus tracheiphilus (Petri) Gruyter, Aveskamp, and Verkley that has caused severe damage and loss of yield in the citrus industry in the Mediterranean area, for 100 years. While the disease can affect different cultivated citrus species, lemon (C. × limon var. limon (L.) Burm. f.) and citron are the most susceptible. The identification of resistant or field-tolerant clones and hybrids is a major goal for lemon growers and breeders. To identify sources of resistance or tolerance to the disease, we performed a phenotypic survey on a lemon and lemon-like open-field germplasm planted at CREA (Research Centre for Olive, Fruit and Citrus Crops), Italy, in an area with high pathogen pressure. Phenotyping was performed visually, four times, for three consecutive years, on a total of 50 accessions, with two or three replicate trees per accession. Moreover, molecular screening based on real-time PCR was performed, for two consecutive years, on twigs, young leaves, and mature leaves of all plants, to detect the pathogen in the absence of clear symptoms. The accessions were categorized into seven groups based on the presence of visual symptoms, real-time PCR pathogen detection, and canopy volume. The results revealed sources of tolerance in lemon and citron hybrids. The molecular screening identified P. tracheiphilus in all lemon clones, with mean Ct values ranging from 17 to 39. The screening also identified P. tracheiphilus in clones without clear symptoms, indicating their ability to tolerate the disease. Moreover, a strong negative correlation was found between the Ct values in twigs and symptom severity (r = −0.72). This indicates that the DNA from twigs is the most appropriate for use in performing reliable phenotyping of mal secco susceptibility in adult plants. An autotetraploid lemon (Doppio Lentini) seems to be immune to the disease, under natural pressure, since P. tracheiphilus was not detected by real-time PCR and visual screening. Overall, the data obtained are a valuable resource for identifying both the most tolerant lemon varieties suitable for areas with high pathogen pressure and the best breeding parents for the introgression of resistance genes into lemon genotypes.

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