Abstract：
:Site-directed mutagenesis is one of the most widely used techniques in life sciences. Here we describe an improved and simplified method for introducing mutations at desired sites. It consists of an inverse PCR using a plasmid template and two partially complementary primers. The synthesis step is followed by annealing of the PCR product's sticky ends, which are generated by exonuclease digestion. This method is fast, extremely efficient and cost-effective. It can be used to introduce large insertions and deletions, but also for multiple point mutations in a single step. To show the principle and to prove the efficiency of the method, we present a series of basic mutations (insertions, deletions, point mutations) on pUC19 plasmid DNA.
journal_name
J Microbiol Methodsjournal_title
Journal of microbiological methodsauthors
Füzik T,Ulbrich P,Ruml Tdoi
10.1016/j.mimet.2014.08.003subject
Has Abstractpub_date
2014-11-01 00:00:00pages
67-71eissn
0167-7012issn
1872-8359pii
S0167-7012(14)00237-1journal_volume
106pub_type
杂志文章abstract：:Monoclonal antibody and polyclonal antibody specific to Cronobacter spp. had been reported in previous studies. However, the preparation of single-chain variable fragment (scFv) was faster and convenient. Hence, the aim of this study was to construct a scFv using outer membrane protein A (OmpA) of C. sakazakii as anti...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.08.004
更新日期：2016-10-01 00:00:00
abstract：:Filamentous fungi have shown great potential in the pretreatment of lignocellulosic biomass and their use in bio-processes based on Solid State Fermentation (SSF) opens promising perspectives for plant biomass valorization. Obviously, quantification of the fungal biomass throughout the fermentation is a crucial parame...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.05.024
更新日期：2015-08-01 00:00:00
abstract：:The European Union Reference Laboratory (EURL) for Listeria monocytogenes (Lm) collaborates with a network of 35 National Reference Laboratories (NRLs) throughout Europe. Most of these NRLs are in charge of detecting and typing Lm strains from food, environment and animals, which are isolated nationally. The past few ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.06.001
更新日期：2014-09-01 00:00:00
abstract：:Recently, we demonstrated that a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS) can be used to determine the molecular weight of polymerase chain reaction (PCR) products of intact 16S rRNA regions and to profile their restriction digests. This is the first time that MALDI-T...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(01)00314-1
更新日期：2002-02-01 00:00:00
abstract：:Bacillus subtilis pheS was genetically modified to obtain a counter-selection marker with high selection efficiency in Bacillus amyloliquefaciens. The application of the new replication-thermosensitive integrative vector pNZTM1, containing this marker, pheSBsT255S/A309G, with a two-step replacement recombination proce...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.03.011
更新日期：2018-05-01 00:00:00
abstract：:Centrifugation is a necessary concentrating step for the detection of Mycobacterium tuberculosis in a liquid culture. However, centrifugation is biologically hazardous and presents an obstacle in the development of an automated culture system. A bead-based bacterial concentration method, TRICORE, was recently develope...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2012.04.018
更新日期：2012-09-01 00:00:00
abstract：:Plant-associated bacteria produce quorum sensing (QS) signals for community (biofilm) formation and functioning in the rhizosphere. The QS-positive biofilm-forming rhizobacteria that excel benefits to the plants are now gaining increased importance for agricultural use due to their high competitiveness. However, there...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2018.11.018
更新日期：2019-01-01 00:00:00
abstract：:A temperature-gradient incubator (TGI) is described, which produces a thermal gradient over 34 aluminium modules (15x30x5 cm) intersected by 2-mm layers of partly insulating graphite foil (SigraFlex Universal). The new, sandwich-designed TGI has 30 rows of six replicate sample wells for incubation of 28-ml test tubes....
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(01)00361-x
更新日期：2002-03-01 00:00:00
abstract：:The aim of the present investigation was to develop a novel approach for rapid identification and differentiation of Pseudomonas aeruginosa clones from Canadian cystic fibrosis patients by capillary electrophoresis-mass spectrometry. We screened P. aeruginosa isolates for lipopolysaccharide structure and presence/abse...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2008.10.008
更新日期：2009-02-01 00:00:00
abstract：:To facilitate routine laboratories in the effective diagnosis of brucellosis, we report a robust and rapid multiplex PCR assay, which allows for the differentiation of all nine currently recognised Brucella species. This includes the recently described species B. microti, B. inopinata, B. ceti and B. pinnipedialis. ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.10.015
更新日期：2010-01-01 00:00:00
abstract：:Sensitive real-time sequence detection methods based on two different chemistries were developed for Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of Johne's disease in cattle. One is based on the detection of SYBR Green bound to PCR products and the second method is more specific, detecting t...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2005.04.004
更新日期：2005-12-01 00:00:00
abstract：:The cDNA conversion of RNA molecules is a prerequisite for their analysis. In the case of prokaryotic RNAs, cDNA conversion is difficult due to a lack of the long poly(A) tails that are found in eukaryotic mRNAs. The full cDNAs for eukaryotic mRNAs can be amplified by the reverse transcription polymerase chain reactio...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2005.05.011
更新日期：2006-03-01 00:00:00
abstract：:An optimized Citrobacter braakii phytase gene, appA-c, was chemically synthesized by oligonucleotides synthesis and over-lap PCR method. The appA-c gene encoding 423 amino acids was cloned into expression vector pPIC9 and transformed into methylotropic yeast Pichia pastoris. From about 2000 transformants, 400 transfor...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2009.08.018
更新日期：2009-10-01 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：:There have been many recent reviews published on MALDI-TOF MS (matrix assisted laser desorption/ionization time-of-flight) MS (mass spectrometry) for identification of bacteria particularly with relevance to clinical microbiology. MALDI-TOF MS is now a mature technique for bacterial identification with great promise. ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章,评审
doi：10.1016/j.mimet.2013.01.004
更新日期：2013-03-01 00:00:00
abstract：:In order to reduce the risk of transmission of infectious diseases through transplantation of tissue allografts, one should examine tissues for the presence of microorganisms. However, there are no detailed tissue banking guidelines describing the culture method or incubation time to be used. Therefore, we compared tw...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2007.05.014
更新日期：2007-08-01 00:00:00
abstract：:Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis. Rapid and accurate diagnosis is crucial to tuberculosis control and prevention. A series of diagnostic methods has been available for MTB detection; however, new rapid, simple and affordable methods are needed. In this study, a multiple...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2017.11.003
更新日期：2018-02-01 00:00:00
abstract：:To establish a simple and rapid high-resolution melting curve (HRM) method, 5 different strains of Salmonella were identified by adding DNA denaturants at different concentrations into the HRM system to change the characteristics of DNA melting and to obtain different Tm (dissolving temperature) values of DNA from dif...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2019.105806
更新日期：2020-05-01 00:00:00
abstract：:Now-a-days increasing emergence of antibiotic-resistant pathogenic microorganisms is one of the biggest challenges for management of disease. In the present study comparative genomics, metabolic pathways analysis and additional parameters were defined for the identification of 94 non-homologous essential proteins in S...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2014.03.009
更新日期：2014-06-01 00:00:00
abstract：:Bacillus thuringiensis is a sporulating bacterium, which produces parasporal inclusions toxic to insects. Widely used methodologies to isolate Bt from soil consist of a thermal shock treatment followed by selective germination of spores. The results presented here suggest that a preliminary 5 h dry-heat treatment larg...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2008.06.008
更新日期：2008-10-01 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：:Haemophilus parasuis infection is of considerable economic importance in the swine industry due to high morbidity and mortality in naive swine populations. Accurate detection and identification of the causative agent are difficult, yet necessary, for disease control. In this study, a simple and rapid method of cross-p...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2017.11.005
更新日期：2018-01-01 00:00:00
abstract：:Oxalate catabolism, which can have both medical and environmental implications, is performed by phylogenetically diverse bacteria. The formyl-CoA-transferase gene was chosen as a molecular marker of the oxalotrophic function. Degenerated primers were deduced from an alignment of frc gene sequences available in databas...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2008.09.020
更新日期：2009-02-01 00:00:00
abstract：:Polyhydroxyalkanoates (PHAs) are important for biodegradable plastic production, and prokaryotes play a very important role in PHA production. PHA synthase is a key enzyme for the polymerization of PHAs. There are four classes of PHA synthase. The phaC gene is necessary for the production of all classes of PHA synthas...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2015.06.012
更新日期：2015-09-01 00:00:00
abstract：:5'-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx(1) and stx(2) genes when challenged with referenc...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2007.05.016
更新日期：2007-09-01 00:00:00
abstract：:A discontinuous lactate dehydrogenase coupled assay is described for the evaluation of the pyruvate carboxylase activity (Pc, EC 6.4.1.1) in a glutamate overproducing strain of Corynebacterium glutamicum. After an initial permeabilisation period of the cells, the method consisted of the fluorometric determination of t...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/s0167-7012(99)00104-9
更新日期：1999-12-01 00:00:00
abstract：:Listeria monocytogenes is an important foodborne pathogen responsible for non-invasive and invasive diseases in the elderly, pregnant women, neonates and immunocompromised populations. This bacterium has many similarities with other non-pathogenic Listeria species which makes its detection from food and environmental ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章,评审
doi：10.1016/j.mimet.2012.01.002
更新日期：2012-03-01 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：:The direct counts of bacteria inhabiting coral mucus were performed by flow cytometry testing four fluorescent dyes (SYBR®Green I, HCS, TOPRO®3, SYTO®62) with three different scleractinian species. Results obtained with SYTO62 were the most reliable based on the comparison with standardized epifluorescence counts and ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/j.mimet.2016.05.032
更新日期：2016-09-01 00:00:00
abstract：:Two rapid in situ enumeration methods using fluorescent probes were used to assess the physiological activities of Klebsiella pneumoniae biofilms on stainless steel. Fluorescent dyes, 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and rhodamine 123 (Rh 123), were chosen to perform this study. CTC is a soluble redox ...
journal_title：Journal of microbiological methods
pub_type： 杂志文章
doi：10.1016/0167-7012(94)90058-2
更新日期：1994-01-01 00:00:00
