scholarly journals Nanopore sequencing for the detection and the identification of Xylella fastidiosa subspecies and sequence types from naturally infected plant material

2019 ◽  
Author(s):  
Luigi Faino ◽  
Valeria Scala ◽  
Alessio Albanese ◽  
Vanessa Modesti ◽  
Alessandro Grottoli ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Material ◽  
Xylella Fastidiosa ◽  
Infected Plant ◽  
Diagnostic Tools ◽  
Nanopore Sequencing ◽  
Direct Dna Sequencing ◽  
Detection And Identification ◽  
Infected Plant Material

SummaryXylella fastidiosa (Xf) is a polyphagous gram-negative bacterial plant pathogen that can infect more than 300 plant species. It is endemic in America while, in 2013, Xf subsp. pauca was for the first time reported in Europe on olive tree in the Southern Italy. The availability of fast and reliable diagnostic tools is indispensable for managing current and future outbreaks of Xf.In this work, we used the Oxford Nanopore Technologies (ONT) device MinION platform for detecting and identifying Xf at species, subspecies and Sequence Type (ST) level straight from infected plant material. The study showed the possibility to detect Xf by direct DNA sequencing and identify the subspecies in highly infected samples. In order to improve sensitivity, Nanopore amplicon sequencing was assessed. Using primers within the set of the seven MLST officially adopted for identifying Xf at type strain level, we developed a workflow consisting in a multiple PCR and an ad hoc pipeline to generate MLST consensus after Nanopore-sequencing of the amplicons. The here-developed combined approach achieved a sensitivity higher than real-time PCR allowing within few hours, the detection and identification of Xf at ST level in infected plant material, also at low level of contamination.Originality Significance StatementIn this work we developed a methodology that allows the detection and identification of Xylella fastidiosa in plant using the Nanopore technology portable device MinION. The approach that we develop resulted more sensitive than methods currently used for detecting X. fastidiosa, like real-time PCR. This approach can be extensively used for X. fastidiosa detection and it may pave the road for the detection of other tedious vascular pathogens.

scholarly journals Fast Identification of Yersinia pestis, Bacillus anthracis and Francisella tularensis Based on Conventional PCR

2013 ◽  
Vol 62 (4) ◽  
pp. 453-455 ◽  
Author(s):  
ALEKSANDRA A. ZASADA ◽  
KAMILA FORMIŃSKA ◽  
KATARZYNA ZACHARCZUK
Keyword(s):  
Public Health ◽  
Real Time ◽  
Francisella Tularensis ◽  
Real Time Pcr ◽  
Health Impact ◽  
Diagnostic Tools ◽  
Conventional Pcr ◽  
The Public ◽  
Detection And Identification ◽  
Appropriate Treatment

Rapid and accurate diagnostic tools for detection and identification of Y pestis, B. anthracis and F. tularensis are essential for timely initial appropriate treatment of exposed individuals, which will be critical to their survival, as well as for reduction of the public health impact and the spread of the disease. The paper presents application of fast polymerases and fast dry electrophoresis in conventional PCR as an alternative for real-time PCR application for detection and identification of the above pathogens. The proposed method takes less than 50 min. to obtain final results of the tests and is cheaper than real-time PCR.

scholarly journals Conventional and Real-Time PCRs for Detection of Erwinia piriflorinigrans Allow Its Distinction from the Fire Blight Pathogen, Erwinia amylovora

2014 ◽  
Vol 80 (8) ◽  
pp. 2390-2398 ◽  
Author(s):  
Silvia Barbé ◽  
Edson Bertolini ◽  
Montserrat Roselló ◽  
Pablo Llop ◽  
María M. López
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Plant Material ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Epidemiological Studies ◽  
Molecular Characteristics ◽  
Content Type ◽  
Detection And Identification ◽  
Erwinia Piriflorinigrans

ABSTRACTErwinia piriflorinigransis a new pathogenic species of the bacterial genusErwiniathat has been described recently in Spain. Accurate detection and identification ofE. piriflorinigransare challenging because its symptoms on pear blossoms are similar to those caused byErwinia amylovora, the causal agent of fire blight. Moreover, these two species share phenotypic and molecular characteristics. Two specific and sensitive conventional and real-time PCR protocols were developed to identify and detectE. piriflorinigransand to differentiate it fromE. amylovoraand other species of this genus. These protocols were based on sequences from plasmid pEPIR37, which is present in all strains ofE. piriflorinigransanalyzed. After the stability of the plasmid was demonstrated, the specificities of the protocols were confirmed by the amplification of allE. piriflorinigransstrains tested, whereas 304 closely related pathogenic and nonpathogenicErwiniastrains and microbiota from pear trees were not amplified. In sensitivity assays, 103cells/ml extract were detected in spiked plant material by conventional or real-time PCR, and 102cells/ml were detected in DNA extracted from spiked plant material by real-time PCR. The protocols developed here succeeded in detectingE. piriflorinigransin 102 out of 564 symptomatic and asymptomatic naturally infected pear samples (flowers, cortex stem tissue, leaves, shoots, and fruitlets), in necroticPyracanthasp. blossoms, and in necrotic pear and apple tissues infected with bothE. amylovoraandE. piriflorinigrans. Therefore, these new tools can be used in epidemiological studies that will enhance our understanding of the life cycle ofE. piriflorinigransin different hosts and plant tissues and its interaction withE. amylovora.

scholarly journals A single-run, real-time PCR for detection and identification ofBorrelia burgdorferi sensu latospecies, based on thehbbgene sequence

2006 ◽  
Vol 259 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Denis Portnoï ◽  
Natacha Sertour ◽  
Elisabeth Ferquel ◽  
Martine Garnier ◽  
Guy Baranton ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Detection And Identification

Development of a single-tube duplex EvaGreen real-time PCR for the detection and identification of EHV-1 and EHV-4

2014 ◽  
Vol 98 (9) ◽  
pp. 4179-4186 ◽  
Author(s):  
Zhe Hu ◽  
Chao Zhu ◽  
Hao Chang ◽  
Wei Guo ◽  
Diqiu Liu ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Single Tube ◽  
Detection And Identification

scholarly journals Primers designed for the detection of grapevine pathogens spreading with propagating material by quantitative real-time PCR

2015 ◽  
Vol 21 (1-2) ◽  
Author(s):  
N. Czotter ◽  
E. Manduláné Farkas ◽  
R. Lózsa ◽  
I. Ember ◽  
G. Szûcsné Varga ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Dna Amplification ◽  
Molecular Techniques ◽  
Latent Infections ◽  
Quantitative Real Time Pcr ◽  
Detection And Identification

Several grapevine pathogens are disseminated by propagating material as systemic, but latent infections. Their detection and identification have a basic importance in the production and handling of propagating stocks. Thus several sensitive and reliable diagnostic protocols mostly based on molecular techniques have been developed. Of these methods quantitative real-time PCR (q-PCR) has recently got an emerging importance. Here we collected primer data for the detection and identification of grapevine pathogens which are important in the production of propagating stocks by q-PCR. Additional novel techniques that use DNA amplification, hybridization and  sequencing are also briefly reviewed.

scholarly journals Rapid Detection and Identification of Uveitis Pathogens by Qualitative Multiplex Real-Time PCR

2018 ◽  
Vol 59 (1) ◽  
pp. 582 ◽  
Author(s):  
Paulo J. M. Bispo ◽  
Samaneh Davoudi ◽  
Matthew L. Sahm ◽  
Ai Ren ◽  
John Miller ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Rapid Detection ◽  
Detection And Identification

scholarly journals Rapid and Sensitive Direct Detection and Identification of Poliovirus from Stool and Environmental Surveillance Samples by Use of Nanopore Sequencing

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Alexander G. Shaw ◽  
Manasi Majumdar ◽  
Catherine Troman ◽  
Áine O’Toole ◽  
Blossom Benny ◽  
...  
Keyword(s):  
Cell Culture ◽  
Real Time ◽  
Environmental Samples ◽  
Direct Detection ◽  
Nanopore Sequencing ◽  
Wild Type ◽  
Consensus Sequences ◽  
Real Time Analysis ◽  
Detection And Identification ◽  
Culture Algorithm

ABSTRACT Global poliovirus surveillance involves virus isolation from stool and environmental samples, intratypic differential (ITD) by PCR, and sequencing of the VP1 region to distinguish vaccine (Sabin), vaccine-derived, and wild-type polioviruses and to ensure an appropriate response. This cell culture algorithm takes 2 to 3 weeks on average between sample receipt and sequencing. Direct detection of viral RNA using PCR allows faster detection but has traditionally faced challenges related to poor sensitivity and difficulties in sequencing common samples containing poliovirus and enterovirus mixtures. We present a nested PCR and nanopore sequencing protocol that allows rapid (<3 days) and sensitive direct detection and sequencing of polioviruses in stool and environmental samples. We developed barcoded primers and a real-time analysis platform that generate accurate VP1 consensus sequences from multiplexed samples. The sensitivity and specificity of our protocol compared with those of cell culture were 90.9% (95% confidence interval, 75.7% to 98.1%) and 99.2% (95.5% to 100.0%) for wild-type 1 poliovirus, 92.5% (79.6% to 98.4%) and 98.7% (95.4% to 99.8%) for vaccine and vaccine-derived serotype 2 poliovirus, and 88.3% (81.2% to 93.5%) and 93.2% (88.6% to 96.3%) for Sabin 1 and 3 poliovirus alone or in mixtures when tested on 155 stool samples in Pakistan. Variant analysis of sequencing reads also allowed the identification of polioviruses and enteroviruses in artificial mixtures and was able to distinguish complex mixtures of polioviruses in environmental samples. The median identity of consensus nanopore sequences with Sanger or Illumina sequences from the same samples was >99.9%. This novel method shows promise as a faster and safer alternative to cell culture for the detection and real-time sequencing of polioviruses in stool and environmental samples.

scholarly journals Development of Conventional Multiplex PCR: A Rapid Technique for Simultaneous Detection of Soil-Transmitted Helminths

Pathogens ◽  
2019 ◽  
Vol 8 (3) ◽  
pp. 152 ◽  
Author(s):  
Vivornpun Sanprasert ◽  
Ruthairat Kerdkaew ◽  
Siriporn Srirungruang ◽  
Sarit Charuchaibovorn ◽  
Kobpat Phadungsaksawasdi ◽  
...  
Keyword(s):  
Multiplex Pcr ◽  
Real Time ◽  
Real Time Pcr ◽  
Intestinal Parasites ◽  
Simultaneous Detection ◽  
Diagnostic Tools ◽  
Multiple Infections ◽  
Parasitic Infections ◽  
Soil Transmitted Helminths ◽  
Stool Samples

Soil-transmitted helminths (STHs) are the most common intestinal parasites infecting humans worldwide. STH infections are a major cause of morbidity and disability. Accurate diagnostic tools are pivotal for assessing the exact prevalence of parasitic infections. Microscopic examination and culture techniques have been used to observe the presence of eggs or larvae of parasites in stool samples, but they are time-consuming and have low sensitivity. Therefore, accurate, simple, and inexpensive diagnostic techniques are still required for simultaneous detection of STH infections. Although molecular-based techniques, such as real-time PCR and multiplex real-time PCR, have been developed, they are not suitable for routine diagnosis due to the requirement for expensive reagents and instruments. In this study, we established a conventional multiplex PCR for simultaneous rapid detection of Ascaris lumbricoides, Necator americanus, and Strongyloides stercoralis in stool samples. Our results show that the multiplex PCR could detect the DNA of STHs at a very low target gene concentrations (lower than 1 pg) with no cross-amplification. Multiplex PCR had five times higher sensitivity than the formalin–ethyl acetate concentration technique (FECT) in the detection of multiple infections, and two times higher for detection of S. stercoralis. However, multiplex PCR was comparable to FECT in the detection of A. lumbricoides and N. americanus. In conclusion, this method could be used as an alternative method for the detection of STHs, especially for S. stercoralis.

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