Abstract：
:A rapid sonication method for lysis of Gram-positive bacteria was evaluated for use in combination with quantitative real-time polymerase chain reaction (PCR) analyses for detection. Other criteria used for evaluation of lysis were microscopic cell count, colony forming units (cfu), optical density at 600 nm and total yield of DNA measured by PicoGreen fluorescence. The aim of this study was complete disruption of cellular structures and release of DNA without the need for lysing reagents and time-consuming sample preparation. The Gram-positive bacterium Bacillus cereus was used as a model organism for Gram-positive bacteria. It was demonstrated by real-time PCR that maximum yield of DNA was obtained after 3 to 5 min of sonication. The yield of DNA was affected by culture age and the cells from a 4-h-old culture in the exponential phase of growth gave a higher yield of DNA after 5 min of sonication than a 24-h-old culture in the stationary phase of growth. The 4-h-old culture was also more sensitive for lysis caused by heating. The maximum yield of DNA, evaluated by real-time PCR, from a culture of the Gram-negative bacterium Escherichia coli, was obtained after 20 s of sonication. However, the yield of target DNA from E. coli rapidly decreased after 50 s of sonication due to degradation of DNA. Plate counting (cfu), microscopic counting and absorbance at 600 nm showed that the number of viable and structurally intact B. cereus cells decreased rapidly with sonication time, whereas the yield of DNA increased as shown by PicoGreen fluorescence and real-time PCR. The present results indicate that 3-5 min of sonication is sufficient for lysis and release of DNA from samples of Gram-positive bacteria.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Fykse EM,Olsen JS,Skogan Gdoi
10.1016/s0167-7012(03)00091-5subject
Has Abstractpub_date
2003-10-01 00:00:00pages
1-10issue
1eissn
0167-7012issn
1872-8359pii
S0167701203000915journal_volume
55pub_type
杂志文章abstract：:Quantitative PCR (qPCR) assays targeting cld (developed in this work) and pcrA (previously described) were used to quantify these perchlorate-related genes in a perchlorate-reducing enrichment culture. Transcript copies were quantified in perchlorate-reducing Rhodocyclaceae strain JDS4. Oxygen and nitrate inhibited ex...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.09.002
更新日期：2010-11-01 00:00:00
abstract：:The skin microbiota is characterized by high intra- and inter-variability among individuals, due to a multitude of intrinsic and extrinsic parameters such as genetics, lifestyles or pollution. This variability may be heightened due to sampling method as the skin is a multilayer organ and its outermost layer consists o...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2020.105880
更新日期：2020-04-01 00:00:00
abstract：BACKGROUND:The development of oral vaccines using yeast surface display technology is an area of intensive study in vaccine development, but the protein level displayed on yeast surfaces is not currently high enough to obtain a robust immune response. METHODS:To address this issue, we established an efficient and simp...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2017.02.002
更新日期：2017-04-01 00:00:00
abstract：:Confocal Raman Microspectroscopy (CRM) can be used as a tool for the in situ evaluation of the chemical composition of living, fully submerged, unstained biofilms. In this study the estimation of the local water content in Pseudomonas aeruginosa PA01 biofilms is given as an example. The ratio of the area of the O-H st...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2008.05.012
更新日期：2008-09-01 00:00:00
abstract：:Phenotypic differentiation between Campylobacter fetus (C. fetus) subspecies fetus and C. fetus subspecies venerealis is hampered by poor reliability and reproducibility of biochemical assays. AFLP (amplified fragment length polymorphism) and MLST (multilocus sequence typing) are the molecular standards for C. fetus s...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.06.005
更新日期：2013-10-01 00:00:00
abstract：:Quantitative real-time reverse transcriptase PCR (qRT-PCR) has become the method choice for quantification of gene expression changes, however, the accuracy of the method depends on the stability of reference genes. Ralstonia pseudosolanacearum (R. pseudosolanacearum) is an important plant pathogen, infecting >450 pla...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.04.004
更新日期：2018-05-01 00:00:00
abstract：:The performance of 3 blood culture bottles (BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Anaerobic Lytic/F) were analyzed with clinical specimens collected from 688 Emergency Department patients. A total of 270 strains belonging to 33 species were identified, with E. coli and S. aureus as the most frequently detected....
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.09.008
更新日期：2016-11-01 00:00:00
abstract：:Staphylococcus aureus is a versatile bacterial opportunist responsible for a wide spectrum of infections. Several genomes of this major human pathogen have been publicly available for almost 10 years, but comprehensive links between virulence or epidemicity and genome content of the bacterium are still missing. This p...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.12.025
更新日期：2011-02-01 00:00:00
abstract：:Bacterial identification methods used in routine identification of pathogens in medical microbiology include a combination approach of biochemical tests, mass spectrometry or molecular biology techniques. Extensive publicly-available databases of DNA sequence data from pathogenic bacteria have been amassed in recent y...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.03.016
更新日期：2019-05-01 00:00:00
abstract：:We developed and evaluated RNA aptamers to analyze their potential for use in detecting Salmonella Enteritidis. The selected aptamer was observed to specifically bind to Salmonella Enteritidis without any cross-reactivity to other Salmonella serovars. Thus, this study suggests that aptamers specific to Salmonella Ente...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.01.014
更新日期：2012-04-01 00:00:00
abstract：:Leishmaniasis is a disease, caused by Leishmania parasites, which infect humans and animals, posing a major social and economic burden worldwide. The need for accurate and sensitive disease diagnosis led to the widespread adoption of PCR amplification. Detection of the amplification products (i.e. gel electrophoresis)...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.05.027
更新日期：2016-08-01 00:00:00
abstract：:The antimicrobial activity of lime oil was significantly enhanced against Staphylococcus epidermidis in a pulsed electromagnetic field. These findings support other workers who found that electromagnetic fields augment antimicrobial activity. ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2010.09.015
更新日期：2010-11-01 00:00:00
abstract：:Bacterial metabolic activity is associated with the onset and progression mechanisms of oral biofilm-mediated disease; however, at present no method to monitor bacterial metabolism exists, especially for periodontopathic bacteria. Therefore, we aimed to establish a novel method for monitoring the metabolic activity of...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.05.015
更新日期：2015-08-01 00:00:00
abstract：:Culture-independent studies using next generation sequencing have revolutionized microbial ecology, however, oomycete ecology in soils is severely lagging behind. The aim of this study was to improve and validate standard techniques for using high throughput sequencing as a tool for studying oomycete communities. The ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.01.013
更新日期：2015-03-01 00:00:00
abstract：:Streptococcus agalactiae is the principal cause of neonatal infections; however, for its study, genetic tools are scarce. We have developed pG+off, an expression vector with a tetracycline inducible promoter that allows a tightly-controllable gene expression in S. agalactiae. Using a reporter gene, we have shown that ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.10.020
更新日期：2014-01-01 00:00:00
abstract：:Various types of microfluidic systems have been developed to detect bacterial pathogens. However, most of these require enrichment steps that take at least several hours when detecting bacteria that are present with a low number of cells and, in addition, fabrication requires complicated assembly steps. In this study,...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.12.001
更新日期：2017-01-01 00:00:00
abstract：:I formulated a systematic approach to screen for colicin resistance-associated genes by using an attenuated colicin mutant and demonstrated its utility in a screen of genes related to colicin E5 resistance. Screening of an Escherichia coli genome library revealed rstA as a partial resistance gene to colicin E5. Transc...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.03.003
更新日期：2014-05-01 00:00:00
abstract：:Systems that automate extraction of nucleic acid from cells or viruses in complex clinical matrices have tremendous value even in the absence of an integrated downstream detector. We describe our bench-top automated workstation that integrates our previously-reported extraction method - TruTip - with our newly-develop...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.03.021
更新日期：2018-05-01 00:00:00
abstract：:Global public health is threatened by the emergence of potentially dangerous antibiotic drug-resistant strains of Mycobacterium tuberculosis. Point mutations in certain M. tuberculosis genes are associated with the resistance of M. tuberculosis strains to antibiotic drugs. The purpose of this study was to develop a su...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2005.04.026
更新日期：2005-12-01 00:00:00
abstract：:Small-colony variants (SCVs) are slow-growing subpopulations of various auxotrophic bacterial strains. Thymidine-dependent SCVs (TD-SCVs) are unable to synthesize thymidine; hence, these variants fail to grow in a medium without thymidine. In this study, we used 10 TD-SCVs of Staphylococcus aureus, of which four strai...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.01.007
更新日期：2015-03-01 00:00:00
abstract：:Clostridium botulinum is the etiological agent of botulism. Due to food-borne poisoning and the potential use of the extremely toxic botulinum neurotoxin (BoNT) from C. botulinum in bioterror or biocrime related actions, reliable high resolution typing methods for discriminating C. botulinum strains are needed. Partia...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.11.003
更新日期：2014-01-01 00:00:00
abstract：:Activated charcoal is a carbonaceous adsorbent with a high internal porosity, and hence a large internal surface area. Cells of a strain of Escherichia coli O157:H7 seeded into oyster tissue homogenates were completely bound to untreated charcoal after an incubation period of 15 min at room temperature. In contrast, a...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2006.09.012
更新日期：2007-02-01 00:00:00
abstract：:Studying the mechanisms of Campylobacter pathogenesis is complicated by the lack of simple animal models that mimic the disease seen in humans. In vitro cell culture methods provide a useful alternative to investigate the interactions between Campylobacter and the host epithelium that occur during infection. In the ge...
journal_title：Journal of microbiological methods
pub_type： 杂志文章,评审
doi：10.1016/j.mimet.2004.12.003
更新日期：2005-05-01 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：:Here we demonstrate a cost effective and scalable microbial ecology sequencing platform using the Ion Torrent Personal Genome Machine (PGM). We assessed both PCR amplified 16S rRNA and shotgun metagenomic approaches and generated 100,000+ to 1,000,000+ reads using 'post-light' based sequencing technology within differ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.07.008
更新日期：2012-10-01 00:00:00
abstract：:Linear B-cell epitopes ((93)AKEFEAAAL(101) and (66)PQLTDVLN(73)) of CfaB were genetically fused to ltb-(gly)5-cfaB(1-25). Sera of rabbits immunized with fusion proteins reacted strongly with solid-phase bound ETEC bacteria bearing CFA/I fimbriae. Sera failed to agglutinate or inhibit hemagglutination promoted by CFA/I...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.07.035
更新日期：2014-11-01 00:00:00
abstract：:Neuraminidase was produced by 32.1% and 28.5% of Porphyromonas from dogs with and without periodontitis, respectively; and by 31.8% of bacteria from humans. The presence of neuraminidase in Porphyromonas spp. suggests that this enzyme can be involved with the pathogenesis of the periodontal disease, and the use of thi...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2013.06.017
更新日期：2013-09-01 00:00:00
abstract：:The fluorescent redox dye 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), combined with fluorescein-labeled antibodies, was tested for the simultaneous detection of the respiratory electron transport system (ETS) activity and enumeration of Giardia cysts and Cryptosporidium oocysts by spectral microfluorometry and epi...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(01)00249-4
更新日期：2001-07-30 00:00:00
abstract：:Despite its long-standing history as a model organism, Neurospora crassa has limited tools for repetitive gene deletions utilizing recyclable self-excising marker systems. Here we describe, for the first time, the functionality of a bacterial recombination system employing β-recombinase acting on six recognition seque...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.12.004
更新日期：2013-03-01 00:00:00
abstract：:In recent years the global rise in antibiotic resistance and environmental consciousness lead to a renewed fervour to find and develop novel antibiotics, including antifungals. However, the influence of the environment on antifungal activity is often disregarded and many in vitro assays may cause the activity of certa...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.09.025
更新日期：2012-12-01 00:00:00
