Abstract:
:Long-read sequencing technologies are having a major impact on our approaches to studying non-model organisms and microbial communities. By significantly reducing the cost and facilitating the genome assembly pipelines, any laboratory can now develop its own genomics program regardless of the complexity of the genome studied. The most crucial current challenge is to develop efficient protocols for extracting genomic DNA (gDNA) with high quality and integrity adapted to the organism of interest. This can be particularly complex for obligate pathogens that must maintain intimate interactions inside infected host tissues. Here we propose a simple and cost-effective method for high molecular weight gDNA extraction from spores of Plasmopara halstedii, an obligate biotroph oomycete pathogen responsible for downy mildew in sunflower. We optimized the yield, the quality and the integrity of the extracted gDNA by fine-tuning three critical parameters, the grinding, the lysis temperature and the lysis duration. We obtained gDNA with a fragment size distribution reaching a peak ranging from 79 to 145 kb. More than half of the extracted gDNA consisted of DNA fragments larger than 42 kb, with 23% of fragments larger than 100 kb. We then demonstrated the relevance of this protocol for long-read sequencing using PacBio RSII technology. With this protocol, we were able to obtain a mean read length of 9.3 kb, a max read length of 71 kb and an N50 of 13.3 kb. The development of such DNA extraction protocols is an essential prerequisite for fully exploiting technologies requiring high molecular weight gDNA (e.g. long-read sequencing or optical mapping). These technological advances will help generate data to answer questions such as the role of newly duplicated gene clusters, repeated regions, genomic structural variations or to define number of chromosomes that still remains undefined in many species of pathogenic fungi and oomycetes.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Penouilh-Suzette C,Fourré S,Besnard G,Godiard L,Pecrix Ydoi
10.1016/j.mimet.2020.106054subject
Has Abstractpub_date
2020-09-12 00:00:00pages
106054eissn
0167-7012issn
1872-8359pii
S0167-7012(20)30770-3journal_volume
178pub_type
杂志文章abstract::Nosocomial infections caused by Stenotrophomonas maltophilia resistant to SXT are increasingly reported worldwide. In this study, a novel melting-curve based multiplex real-time PCR assay for the simultaneous detection of the ssrA and sul1, sul2 and sul3 genes was first established. The assays were performed on a Roch...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2018.12.002
更新日期:2019-01-01 00:00:00
abstract::Cryptosporidium is a zoonotic coccidian parasite associated with diarrhea, and the disinfectant-resistant oocysts are threats to public health even in industrialized countries. In order to make an accurate assessment of the risk to public health, a detection method that has a high recovery rate of oocysts in water is ...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/s0167-7012(03)00209-4
更新日期:2003-12-01 00:00:00
abstract::An improved approach for simultaneous detection of Cryptosporidium parvum and Giardia lamblia (oo)cysts in soil is described. Recoveries>70% were obtained for concentrations>55 and 21 (oo)cysts g(-1) for C. parvum and G. lamblia, respectively. The limits of detection were determined to be<5 (oo)cysts g(-1) soil. ...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2013.07.019
更新日期:2013-09-01 00:00:00
abstract::A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microp...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2010.11.013
更新日期:2011-02-01 00:00:00
abstract::Loop-mediated isothermal amplification (LAMP) is considered as one of the alternatives to the conventional PCR and it is an inexpensive portable diagnostic system with minimal power consumption. The present work describes the application of LAMP in real-time photon detection and quantitative analysis of nucleic acids ...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2016.12.002
更新日期:2017-02-01 00:00:00
abstract::Two novel modifications of continuous culture with gradual change of dilution rate (A-stat): D-stat and auxo-accelerostat were evaluated in the studies of the effect of changing individual environmental parameters (T, pH, pO(2), substrate concentration, etc.) on growth characteristics of different microorganisms. Comm...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/s0167-7012(03)00143-x
更新日期:2003-10-01 00:00:00
abstract::To prevent postoperative infectious complications, a urinary tract infection should be either diagnostically excluded or treated prior to ureterorenoscopy (URS). URS is a frequently performed endoscopic surgery for urological stone removal. Although the urinary dipstick test represents a simple and cost-effective meth...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2020.105933
更新日期:2020-06-01 00:00:00
abstract::Fourier-transform infrared (FT-IR) spectroscopy is known as a high-resolution method for the rapid identification of pure cultures of microorganisms. Here, we evaluated FT-IR as a method for the quantification of bacterial populations in binary mixed cultures consisting of Pseudomonas putida and Rhodococcus ruber. A c...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2011.05.001
更新日期:2011-08-01 00:00:00
abstract::Serological tests, such as agglutination and indirect hemagglutination assay (IHA), have been used to identify antibodies against Pasteurella multocida in poultry sera, but none are highly sensitive. An enzyme-linked immunosorbent assays (ELISA) has been used with varying degrees of success in attempts to monitor sero...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2017.08.018
更新日期:2017-11-01 00:00:00
abstract::Race 3, biovar 2 strains of Ralstonia solanacearum are quarantined pathogens in Europe and Canada and Select Agent pathogens in the United States. The biovar classification of R. solanacearum strains is based on their biochemical abilities to utilize a carbohydrate panel. The standard biovar test uses bromothymol blue...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2011.12.007
更新日期:2012-02-01 00:00:00
abstract::In Europe, alternative methods for the detection of food-borne pathogens can be used instead of the standard ISO/CEN reference protocol, if validated according to the protocol outlined in ISO 16140, 2003. In this study, the performance of two novel methods for the detection of Salmonella sp. using real-time PCR techno...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2010.10.004
更新日期:2011-01-01 00:00:00
abstract::This contribution honoring David C. White (DC) summarizes the five years I interacted with him on a daily basis in his laboratory. Over this time we worked on many different projects all tied together by the unifying principle now recognized as central to bacterial life in nature: biofilms. My goal is to convey some o...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2007.07.003
更新日期:2008-07-01 00:00:00
abstract::The spores and conidia of most fungi have very thick and resistant cell walls that severely impede the staining with fluorescent dyes to allow epifluorescence microscopy to be employed for their direct detection and quantification in natural habitats. In this study, oxidation by sodium hypochlorite and microwave irrad...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2004.07.016
更新日期:2004-12-01 00:00:00
abstract::We evaluated the performance of two MALDI-TOF MS systems for the identification of clinically important Gram-positive cocci. Vitek MS and Microflex LT correctly identified 97.2% and 94.7%, respectively. Both systems offer reliable and rapid identification of clinically important Gram-positive cocci isolated in clinica...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2015.03.020
更新日期:2015-06-01 00:00:00
abstract::AFLP is a selective restriction fragment amplification method generating DNA fingerprints for microbial isolates. We present high-throughput AFLP (htAFLP) to characterize molecular markers associated with bacterial phenotypes. Methicillin-resistant and -susceptible isolates of Staphylococcus aureus have been used for ...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2007.07.013
更新日期:2007-10-01 00:00:00
abstract::In the current study, a duplex PCR-ELISA method was developed targeting the specific genes, invA of Salmonella spp. and rfbE of Escherichia coli O157: H7, to detect one or both bacteria in food. In brief, PCR product amplified by PCR primer labeled with digoxin at the 5'-end and a probe labeled with biotin at the 3'-e...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2018.10.017
更新日期:2018-11-01 00:00:00
abstract::MTS or {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl}-2H-tetrazolium, inner salt) is converted into soluble formazan by mitochondrial dehydrogenase of viable cells, thus serving as an indicator of cell viability. Accordingly, a MTS-based assay was developed to evaluate anti-leishmanial acti...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2005.10.011
更新日期:2006-07-01 00:00:00
abstract::Contamination of cereals with Fusarium species is one of the major sources of mycotoxins in food and feed. Quantification of biomass of Fusarium species is essential to understand the interactions of individual species in disease development. In this study quantitative real-time PCR assays based on the elongation fact...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2008.10.016
更新日期:2009-03-01 00:00:00
abstract::Microscopic discrimination between extracellular and invasive, intracellular bacteria is a valuable technique in microbiology and immunology. We describe a novel fluorescence staining protocol, called FITC-biotin-avidin (FBA) staining, which allows the differentiation between extracellular and intracellular bacteria a...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2004.05.008
更新日期:2004-10-01 00:00:00
abstract::Methods to assess the diversity of the diazotroph assemblage in the rhizosphere of the salt marsh cordgrass, Spartina alterniflora were examined. The effectiveness of nifH PCR-denaturing gradient gel electrophoresis (DGGE) was compared to that of nifH clone library analysis. Seventeen DGGE gel bands were sequenced and...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2008.02.005
更新日期:2008-05-01 00:00:00
abstract::Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and chromogenic media are widely used in clinical microbiology laboratories to facilitate the rapid selection and identification of pathogens. The aim of this study was to evaluate whether usage of chromogenic media limits the ...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2017.03.001
更新日期:2017-05-01 00:00:00
abstract::O157, O26, and O111 are the most important O serogroups of enterohemorrhagic Escherichia coli worldwide. Recently we reported a strategy for discriminating these serotypes from the others using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) based on the S10-spc-alpha operon...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2015.11.005
更新日期:2015-12-01 00:00:00
abstract::Limited biological samples of microbial origin often yield insufficient amounts of genomic DNA, making application of standard techniques of genetic analysis, like amplified fragment length polymorphism (AFLP), virtually impossible. The Phi29 DNA polymerase based whole genome amplification (WGA) method has the potenti...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2005.03.009
更新日期:2005-11-01 00:00:00
abstract::Escherichia coli O157:H7 has frequently been associated with foodborne infections and is considered an adulterant in raw non-intact beef in the U.S. Shiga toxin-producing E. coli (STEC) belonging to serogroups O26, O45, O103, O111, O121, and O145 (known as the "big six" non-O157) were estimated to cause >70% of foodbo...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2016.07.005
更新日期:2016-10-01 00:00:00
abstract::Molecular diagnostic devices are increasingly finding utility in clinical laboratories. Demonstration of the effectiveness of these devices is dependent upon comparing results from clinical samples tested with the new device to an alternative testing method. The preparation of mock clinical specimens will be necessary...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2016.11.005
更新日期:2017-01-01 00:00:00
abstract::This paper reports a novel electrochemical immunosensor for the sensitive detection of staphylococcal enterotoxin A (SEA) based on self-assembly monolayer (SAM) and protein A immobilization on gold electrode. Three different methods of protein A immobilization were tested: physical adsorption, cross-linking using glut...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2012.05.016
更新日期:2012-10-01 00:00:00
abstract::Microarrays have allowed gene expression profiling to progress from the gene level to the genome level, and oligonucleotide microarrays have become the platform of choice for large-scale, targeted gene expression studies. cDNA arrays and spotted oligonucleotide arrays have gradually given way to in situ synthesized ol...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2013.12.009
更新日期:2014-02-01 00:00:00
abstract::We performed a comparison of four selected protocols from the literature using microtitre-plate and colorimetric biomass assay for evaluation of enterococci biofilm formation ability and optimized a protocol after the identification and stringent performance of biofilm formation steps. The optimized protocol uses a dy...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2010.11.007
更新日期:2011-02-01 00:00:00
abstract::Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated ...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2016.08.022
更新日期:2016-11-01 00:00:00
abstract::Gene expression analysis provides significant insight to understand regulatory mechanisms of biology, yet acquisition and reproduction of quality data, as well as data confirmation and verification remain challenging due to a lack of proper quality controls across different assay platforms. We present a set of six uni...
journal_title:Journal of microbiological methods
pub_type: 杂志文章
doi:10.1016/j.mimet.2006.10.014
更新日期:2007-03-01 00:00:00