Adaptation of a Phytophthora ramorum Real-Time Polymerase Chain Reaction Assay Based on a Mitochondrial Gene Region for Use on the Cepheid SmartCycler

2010 ◽  
Vol 11 (1) ◽  
pp. 28
Author(s):  
K. E. Sechler ◽  
M. M. Carras ◽  
N. Shishkoff ◽  
P. W. Tooley
Keyword(s):  
Real Time ◽  
Mitochondrial Gene ◽  
Cross Reactivity ◽  
Phytophthora Species ◽  
Phytophthora Ramorum ◽  
Rt Pcr ◽  
Field Samples ◽  
Symptomatic Tissue ◽  
Polymerase Chain ◽  
High Degree

Detection of Phytophthora ramorum in US commercial nurseries has led to a number of quarantine regulations. Methods such as real-time PCR (RT-PCR) provide rapid and reliable detection that can supplement attempts to culture P. ramorum from symptomatic tissue. We adapted and optimized a previously described mitochondrial gene-based RT-PCR assay for use with a Cepheid SmartCycler v.1 and ready-to-use lyophilized PCR beads. The detection limit was 10 fg of P. ramorum genomic DNA. No cross-reactivity was observed on the SmartCycler for seven additional Phytophthora species tested, which included species known to cross-react in other assays as well as recently described species Phytophthora foliorum and P. kernoviae. The SmartCycler assay described here was used to detect P. ramorum in a set of 2008 California field samples with a high degree of accuracy. Accepted for publication 13 October 2009. Published 13 February 2010.

scholarly journals Real-time fluorogenic reverse transcription polymerase chain reaction assay for the specific detection of Bagaza virus

2012 ◽  
Vol 24 (5) ◽  
pp. 959-963 ◽  
Author(s):  
Dolores Buitrago ◽  
Ana Rocha ◽  
Cristina Tena-Tomás ◽  
Marta Vigo ◽  
Montserrat Agüero ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Reverse Transcription ◽  
Diagnostic Methods ◽  
Nucleotide Sequencing ◽  
Alectoris Rufa ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Field Samples ◽  
Polymerase Chain

In September 2010, an outbreak of disease in 2 wild bird species (red-legged partridge, Alectoris rufa; ring-necked pheasant, Phasianus colchicus) occurred in southern Spain. Bagaza virus (BAGV) was identified as the etiological agent of the outbreak. BAGV had only been reported before in Western Africa (Central African Republic, Senegal) and in India. The first occurrence of BAGV in Spain stimulated a demand for rapid, reliable, and efficacious diagnostic methods to facilitate the surveillance of this disease in the field. This report describes a real-time reverse transcription polymerase chain reaction (RT-PCR) method based on a commercial 5’-Taq nuclease-3’ minor groove binder DNA probe and primers targeting the Bagaza NS5 gene. The method allowed the detection of BAGV with a high sensitivity, whereas other closely related flaviviruses ( Usutu virus, West Nile virus, and Japanese encephalitis virus) were not detected. The assay was evaluated using field samples of red-legged partridges dead during the outbreak ( n = 11), as well as samples collected from partridges during surveillance programs ( n = 81). The results were compared to those obtained with a pan-flaviviral hemi-nested RT-PCR followed by nucleotide sequencing, which was employed originally to identify the virus involved in the outbreak. The results obtained with both techniques were 100% matching, indicating that the newly developed real-time RT-PCR is a valid technique for BAGV genome detection, useful in both diagnosis and surveillance studies.

scholarly journals Detection and Quantification of Phytophthora ramorum from California Forests Using a Real-Time Polymerase Chain Reaction Assay

2004 ◽  
Vol 94 (10) ◽  
pp. 1075-1083 ◽  
Author(s):  
Katherine J. Hayden ◽  
David Rizzo ◽  
Justin Tse ◽  
Matteo Garbelotto
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Pathogen Detection ◽  
Phytophthora Species ◽  
Phytophthora Ramorum ◽  
Plant Diseases ◽  
Chain Reaction ◽  
Nested Polymerase Chain Reaction ◽  
New Host ◽  
Polymerase Chain

The timely and accurate detection of pathogens is a critical aid in the study of the epidemiology and biology of plant diseases. In the case of regulated organisms, the availability of a sensitive and reliable assay is essential when trying to achieve early detection of the pathogen. We developed and tested a real-time, nested polymerase chain reaction (PCR) assay for the detection of Phytophthora ramorum, causal agent of sudden oak death. This technique then was implemented as part of a widespread environmental screen throughout California. The method here described is sensitive, detecting less than 12 fg of pathogen DNA, and is specific for P. ramorum when tested across 21 Phytophthora spp. Hundreds of symptomatic samples from 33 sites in 14 California counties were assayed, resulting in the discovery of 10 new host species and 23 infested areas, including 4 new counties. With the exception of a single host, PCR-based discovery of new hosts and infested areas always was confirmed by traditional pathogen isolations and inoculation studies. Nonetheless, molecular diagnostics were key in early pathogen detection, and steered the direction of further research on this newly discovered and generalist Phytophthora species.

scholarly journals Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

2006 ◽  
Vol 96 (4) ◽  
pp. 336-345 ◽  
Author(s):  
Paul W. Tooley ◽  
Frank N. Martin ◽  
Marie M. Carras ◽  
Reid D. Frederick
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Limit Of Detection ◽  
Multiplex Assay ◽  
Phytophthora Ramorum ◽  
Chain Reaction ◽  
Plant Dna ◽  
Field Samples ◽  
Polymerase Chain ◽  
Fluorescent Polymerase Chain Reaction

A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10-5 dilution of extracted tissue of artificially infected rhododendron ‘Cunningham's White’, and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.

1504: Molecular Diagnosis and Staging of Prostate Cancer Using Clusterin in Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR)

2006 ◽  
Vol 175 (4S) ◽  
pp. 485-486
Author(s):  
Sabarinath B. Nair ◽  
Christodoulos Pipinikas ◽  
Roger Kirby ◽  
Nick Carter ◽  
Christiane Fenske
Keyword(s):  
Prostate Cancer ◽  
Polymerase Chain Reaction ◽  
Real Time ◽  
Molecular Diagnosis ◽  
Reverse Transcription ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Polymerase Chain

scholarly journals Investigations, actions and learning from an outbreak of SARS-CoV-2 infection among healthcare workers in the United Kingdom

2020 ◽  
pp. 175717742097679
Author(s):  
Kordo Saeed ◽  
Emanuela Pelosi ◽  
Nitin Mahobia ◽  
Nicola White ◽  
Christopher Labdon ◽  
...  
Keyword(s):  
Real Time ◽  
Healthcare Workers ◽  
Healthcare Facility ◽  
Rt Pcr ◽  
Serum Samples ◽  
The United Kingdom ◽  
Key Issues ◽  
Current Infection ◽  
Polymerase Chain ◽  
A Current

Background: We report an outbreak of SARS coronavirus-2 (SARS-CoV-2) infection among healthcare workers (HCW) in an NHS elective healthcare facility. Methodology: A narrative chronological account of events after declaring an outbreak of SARS-CoV-2 among HCWs. As part of the investigations, HCWs were offered testing during the outbreak. These were: (1) screening by real-time reverse transcriptase polymerase chain reaction (RT- PCR) to detect a current infection; and (2) serum samples to determine seroprevalence. Results: Over 180 HCWs were tested by real-time RT-PCR for SARS-CoV-2 infection. The rate of infection was 15.2% (23.7% for clinical or directly patient-facing HCWs vs. 4.8% in non-clinical non-patient-facing HCWs). Of the infected HCWs, 57% were asymptomatic. Seroprevalence (SARS-CoV-2 IgG) among HCWs was 13%. It was challenging to establish an exact source for the outbreak. The importance of education, training, social distancing and infection prevention practices were emphasised. Additionally, avoidance of unnecessary transfer of patients and minimising cross-site working for staff and early escalation were highlighted. Establishing mass and regular screening for HCWs are also crucial to enabling the best care for patients while maintaining the wellbeing of staff. Conclusion: To our knowledge, this is the first UK outbreak report among HCWs and we hope to have highlighted some key issues and learnings that can be considered by other NHS staff and HCWs globally when dealing with such a task in future.

scholarly journals Anomaly Identification during Polymerase Chain Reaction for Detecting SARS-CoV-2 Using Artificial Intelligence Trained from Simulated Data

Molecules ◽  
2020 ◽  
Vol 26 (1) ◽  
pp. 20
Author(s):  
Reynaldo Villarreal-González ◽  
Antonio J. Acosta-Hoyos ◽  
Jaime A. Garzon-Ochoa ◽  
Nataly J. Galán-Freyle ◽  
Paola Amar-Sepúlveda ◽  
...  
Keyword(s):  
Artificial Intelligence ◽  
Big Data ◽  
Real Time ◽  
Binary Classification ◽  
Simulated Data ◽  
Classification Model ◽  
Rt Pcr ◽  
Simon Bolivar ◽  
Polymerase Chain ◽  
Available Information

Real-time reverse transcription (RT) PCR is the gold standard for detecting Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), owing to its sensitivity and specificity, thereby meeting the demand for the rising number of cases. The scarcity of trained molecular biologists for analyzing PCR results makes data verification a challenge. Artificial intelligence (AI) was designed to ease verification, by detecting atypical profiles in PCR curves caused by contamination or artifacts. Four classes of simulated real-time RT-PCR curves were generated, namely, positive, early, no, and abnormal amplifications. Machine learning (ML) models were generated and tested using small amounts of data from each class. The best model was used for classifying the big data obtained by the Virology Laboratory of Simon Bolivar University from real-time RT-PCR curves for SARS-CoV-2, and the model was retrained and implemented in a software that correlated patient data with test and AI diagnoses. The best strategy for AI included a binary classification model, which was generated from simulated data, where data analyzed by the first model were classified as either positive or negative and abnormal. To differentiate between negative and abnormal, the data were reevaluated using the second model. In the first model, the data required preanalysis through a combination of prepossessing. The early amplification class was eliminated from the models because the numbers of cases in big data was negligible. ML models can be created from simulated data using minimum available information. During analysis, changes or variations can be incorporated by generating simulated data, avoiding the incorporation of large amounts of experimental data encompassing all possible changes. For diagnosing SARS-CoV-2, this type of AI is critical for optimizing PCR tests because it enables rapid diagnosis and reduces false positives. Our method can also be used for other types of molecular analyses.

scholarly journals A New Multiplex Real-Time RT-PCR for Simultaneous Detection and Differentiation of Avian Bornaviruses

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1358
Author(s):  
Brigitte Sigrist ◽  
Jessica Geers ◽  
Sarah Albini ◽  
Dennis Rubbenstroth ◽  
Nina Wolfrum
Keyword(s):  
Real Time ◽  
Simultaneous Detection ◽  
Epidemiological Studies ◽  
Virus Species ◽  
Rt Pcr ◽  
P Gene ◽  
Field Samples ◽  
Causative Agents ◽  
Species Specific ◽  
Proventricular Dilatation Disease

Avian bornaviruses were first described in 2008 as the causative agents of proventricular dilatation disease (PDD) in parrots and their relatives (Psittaciformes). To date, 15 genetically highly diverse avian bornaviruses covering at least five viral species have been discovered in different bird orders. Currently, the primary diagnostic tool is the detection of viral RNA by conventional or real-time RT-PCR (rRT-PCR). One of the drawbacks of this is the usage of either specific assays, allowing the detection of one particular virus, or of assays with a broad detection spectrum, which, however, do not allow for the simultaneous specification of the detected virus. To facilitate the simultaneous detection and specification of avian bornaviruses, a multiplex real-time RT-PCR assay was developed. Whole-genome sequences of various bornaviruses were aligned. Primers were designed to recognize conserved regions within the overlapping X/P gene and probes were selected to detect virus species-specific regions within the target region. The optimization of the assay resulted in the sensitive and specific detection of bornaviruses of Psittaciformes, Passeriformes, and aquatic birds. Finally, the new rRT-PCR was successfully employed to detect avian bornaviruses in field samples from various avian species. This assay will serve as powerful tool in epidemiological studies and will improve avian bornavirus detection.

scholarly journals Monitoring Current-Season Spread of Potato virus Y in Potato Fields Using ELISA and Real-Time RT-PCR

Plant Disease ◽  
2013 ◽  
Vol 97 (5) ◽  
pp. 641-644 ◽  
Author(s):  
Manphool S. Fageria ◽  
Mathuresh Singh ◽  
Upeksha Nanayakkara ◽  
Yvan Pelletier ◽  
Xianzhou Nie ◽  
...  
Keyword(s):  
Real Time ◽  
Potato Virus ◽  
Potato Virus Y ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Current Season ◽  
Seed Market ◽  
Polymerase Chain ◽  
Time Of Harvest

The current-season spread of Potato virus Y (PVY) was investigated in New Brunswick, Canada, in 11 potato fields planted with six different cultivars in 2009 and 2010. In all, 100 plants selected from each field were monitored for current-season PVY infections using enzyme-linked immunosorbent assay (ELISA) and real-time reverse-transcription polymerase chain reaction (RT-PCR) assay. Average PVY incidence in fields increased from 0.6% in 2009 and 2% in 2010 in the leaves to 20.3% in 2009 and 21.9% in 2010 in the tubers at the time of harvest. In individual fields, PVY incidence in tubers reached as high as 37% in 2009 and 39% in 2010 at the time of harvest. Real-time RT-PCR assay detected more samples with PVY from leaves than did ELISA. A higher number of positive samples was also detected with real-time RT-PCR from growing tubers compared with the leaves collected from the same plant at the same sampling time. PVY incidence determined from the growing tubers showed a significant positive correlation with the PVY incidence of tubers after harvest. Preharvest testing provides another option to growers to either top-kill the crop immediately to secure the seed market when the PVY incidence is low or leave the tubers to develop further for table or processing purposes when incidence of PVY is high.

scholarly journals Polycistronic transcription of fused cassettes and identification of translation initiation signals in an unusual gene cassette array from Pseudomonas aeruginosa

F1000Research ◽  
2015 ◽  
Vol 2 ◽  
pp. 99
Author(s):  
Érica L. Fonseca ◽  
Ana Carolina Paulo Vicente
Keyword(s):  
Real Time ◽  
Northern Blot ◽  
Gene Cassette ◽  
Rt Pcr ◽  
Recombination Site ◽  
Gene Cassettes ◽  
Polycistronic Transcription ◽  
Polymerase Chain ◽  
Fused Gene ◽  
Gene Cassette Array

The gene cassettes found in class 1 integrons are generally promoterless units composed by an open reading frame (ORF), a short 5’ untranslated region (UTR) and a 3’ recombination site (attC). Fused gene cassettes are generated by partial or total loss of the attC from the first cassette in an array, creating, in some cases, a fusion with the ORF from the next cassette. These structures are rare and little is known about their mechanisms of mobilization and expression. The aim of this study was to evaluate the dynamic of mobilization and transcription of the gcu14-blaGES-1/aacA4 gene cassette array, which harbours a fused gene cassette represented by blaGES-1/aacA4. The cassette array was analyzed by Northern blot and real-time reverse transcription-polymerase chain reaction (RT-PCR) in order to assess the transcription mechanism of blaGES-1/aacA4 fused cassette. Also, inverse polymerase chain reactions (PCR) were performed to detect the free circular forms of gcu14, blaGES-1 and aacA4. The Northern blot and real time RT-PCR revealed a polycistronic transcription, in which the fused cassette blaGES-1/aacA4 is transcribed as a unique gene, while gcu14 (with a canonical attC recombination site) has a monocistronic transcription. The gcu14 cassette, closer to the weak configuration of cassette promoter (PcW), had a higher transcription level than blaGES-1/aacA4, indicating that the cassette position affects the transcript amounts. The presence of ORF-11 at attI1, immediately preceding gcu14, and of a Shine-Dalgarno sequence upstream blaGES-1/aacA4 composes a scenario for the occurrence of array translation. Inverse PCR generated amplicons corresponding to gcu14, gcu14-aacA4 and gcu14-blaGES-1/aacA4 free circular forms, but not to blaGES-1 and aacA4 alone, indicating that the GES-1 truncated attC is not substrate of integrase activity and that these genes are mobilized together as a unique cassette. This study was original in showing the transcription of fused cassettes and in correlating cassette position with transcription.

scholarly journals Investigation of Different Commercially Available Real Time-Polymerase Chain Reaction Kits for SARS-CoV-2 Diagnosis

2021 ◽  
Author(s):  
Rajeev Kumar Jain ◽  
Nagaraj Perumal ◽  
Rakesh Shrivastava ◽  
Kamlesh Kumar Ahirwar ◽  
Jaya Lalwani ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Real Time ◽  
Internal Control ◽  
Housekeeping Genes ◽  
Housekeeping Gene ◽  
Specific Gene ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Gene Target ◽  
Polymerase Chain

Introduction: The whole world is facing an ongoing global health emergency of COVID-19 disease caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is a gold standard in the detection of SARS-CoV-2 infection. Presently, many single tube multiple gene target RT-PCR kits have been developed and are commercially available for Coronavirus Disease 2019 (COVID-19) diagnosis. Aim: To evaluate the performance of seven COVID-19 RT-PCR kits (DiagSure, Meril, VIRALDTECT II, TruPCR, Q-line, Allplex and TaqPath) which are commercially available for COVID-19 RT-PCR diagnosis. Materials and Methods: This observational study was conductedat the State Virology Laboratory (SVL), Gandhi Medical College, Bhopal, Madhya Pradesh, India. Seven commercially available kits have been evaluated on the basis of: (i) number of SARS-CoV-2 specific gene target; (ii) human housekeeping genes as internal control; (iii) RT-PCR run time; and (iv) kit performances to correctly detect SARS-CoV-2 positive and negative RNA samples. A total of 50 RNA samples (left over RNA) were included, master mix preparation, template addition and RT-PCR test has been performed according to kits literature. At the end of PCR run, mean and standard deviation of obtained cut-off of all kits were calculated using Microsoft Excel. Results: All seven RT-PCR kits performed satisfactory regarding the reproducibility and they could correctly identify 30 positive and 20 negative RNA samples. RNA samples (group C) having low viral loads with a high Cycle threshold (Ct) value (>30) were also detected by all these seven kits. Obtained Ct values of each group was in parallel range in comparison with the initial testing Ct values. Kits were found to be superior which contains primers and probes for three SARS-CoV-2 specific gene targets, have human housekeeping gene as internal control and taking less time to complete RT-PCR. Conclusion: All seven COVID-19 RT-PCR kits included in this study demonstrated satisfactory performance and can be used for the routine molecular diagnosis of COVID-19 disease.

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