Method for Rapid Detection and Identification of Chaetomium and Evaluation of Resistance to Peracetic Acid

2013 ◽  
Vol 76 (6) ◽  
pp. 999-1005 ◽  
Author(s):  
MOTOKAZU NAKAYAMA ◽  
KOUICHI HOSOYA ◽  
DAISUKE TOMIYAMA ◽  
TAKASHI TSUGUKUNI ◽  
TETSUHIRO MATSUZAWA ◽  
...  
Keyword(s):  
Peracetic Acid ◽  
Cell Walls ◽  
Acid Resistance ◽  
High Specificity ◽  
Specific Primers ◽  
Beverage Industry ◽  
Detection And Identification ◽  
Transmission Electron ◽  
Pcr Products ◽  
Primer Sets

In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the β-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

scholarly journals Detection and identification of phytoplasmas in peach based on woody indexing and molecular methods

2001 ◽  
Vol 7 (1) ◽  
Author(s):  
M. Németh ◽  
I. Ember ◽  
L. Krizbai ◽  
M. Kölber ◽  
R. Hangyál ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Rflp Analysis ◽  
Specific Primers ◽  
Universal Primer ◽  
Detection And Identification ◽  
Molecular Studies ◽  
Pcr Products ◽  
Phytoplasma Infection ◽  
Peach Trees ◽  
Different Parts

Symptoms resembling phytoplasma disease have been observed on peach trees in a seed-source plantation of stone fruits in south Hungary quite recently. In this publication we report on the results of woody indexing of symptomatic peach trees on GF 305 indicator in the field and under greenhouse conditions as well as on molecular studies. Phytoplasma infection detected on GF 305 indicators in greenhouse and field indexing was confirmed by PCR. Nested PCR was conducted using universal primer pairs followed by group and subgroup specific primers for the second amplification. RFLP analysis of nested PCR products was performed using Rsal restriction enzyme. Based on the results of molecular studies it can be concluded that phytoplasmas, belonging to the European stone fruit yellows subgroup (16SrX-B) were identified in peach trees. Further studies on symptomatic peach trees originating from different parts of Hungary are in progress.

A Rapid Method for Identifying Byssochlamys and Hamigera

2010 ◽  
Vol 73 (8) ◽  
pp. 1486-1492 ◽  
Author(s):  
MOTOKAZU NAKAYAMA ◽  
KOUICHI HOSOYA ◽  
TETSUHIRO MATSUZAWA ◽  
YUSUKE HIRO ◽  
AYUMI SAKO ◽  
...  
Keyword(s):  
Food Spoilage ◽  
Pcr Assay ◽  
Specific Primers ◽  
Closely Related Species ◽  
Heat Resistant ◽  
Specific Pcr ◽  
Pcr Products ◽  
Fungal Dna ◽  
Primer Sets ◽  
High Degree

Heat-resistant fungi, genera Byssochlamys, Talaromyces, Neosartorya, and Hamigera, contribute significantly to the spoilage of heat-processed acidic foods, due to the formation of heat-resistant ascospores. Here, we first evaluated the differences in the β-tubulin gene between Byssochlamys and Hamigera and developed specific primers to identify the Byssochlamys species fulva, nivea, and spectabilis, and Hamigera. Using primers designed for B. fulva and B. nivea (B1F/1R), specific PCR products were detected for B. fulva and B. nivea, as well as B. langunculariae and B. zollerniae, two closely related species. Similarly, the Pae4F/4R-1 and H2F/2R primers produced specific PCR products for B. spectabilis and Hamigera, respectively. Using these three primer sets, strains involved in acidic food spoilage and environmental contamination were not detected. The detection limits of all primer sets were 1 ng of DNA by PCR and 10 pg of DNA by nested PCR. Each PCR assay was specific, even if the sample was contaminated 1,000-fold by other fungal DNA. Thus, this method has proved to possess an extremely high degree of specificity.

Ultrastructure of commercial recycled pulp fibers for the production of packaging paper

Holzforschung ◽  
2005 ◽  
Vol 59 (6) ◽  
pp. 675-680 ◽  
Author(s):  
Jonas Brändström ◽  
Jean-Paul Joseleau ◽  
Alain Cochaux ◽  
Nathalie Giraud-Telme ◽  
Katia Ruel
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Significant Proportion ◽  
Pulp Fibers ◽  
Chemical Pulp ◽  
Recycled Pulp ◽  
Transmission Electron ◽  
Large Heterogeneity ◽  
Recycled Fibers ◽  
Packaging Paper

Abstract Transmission electron microscopy was used to investigate the ultrastructure of recycled pulp fibers originating from a household collection plant and intended for the production of packaging paper. Three recovered paper grades and recycling processes, including pulping, screening, cleaning and refining, were assessed with emphasis on surface and internal fibrillation as well as xylan localization. Results showed a large heterogeneity with respect to fiber ultrastructure within and between the grades. Screening and cleaning steps had no detectable effects, but refining clearly increased cell-wall delamination and surface fibrillation. Immunolabeling of xylans showed that they were distributed rather evenly across the cell walls. They were also present on fines. Two different mechanisms for fiber delamination and surface fibrillation were found, one which implies that internal and external fibrillation take place simultaneously across the cell wall, and another which implies successive peeling of layers or sub-layers from the outside towards the inside. It is suggested that recycled fibers of chemical pulp origin undergo the former mechanism and recycled fibers that contain lignin binding the cell wall matrix give rise to the latter peeling mechanism. Because several recycled fibers were severely delaminated and almost fractured, we suggest that to produce a good packaging paper, it is important that recycled pulp should contain a significant proportion of fibers with high intrinsic strength.

scholarly journals Induction of Multiple Prophages from a Marine Bacterium: a Genomic Approach

2006 ◽  
Vol 72 (7) ◽  
pp. 4995-5001 ◽  
Author(s):  
Feng Chen ◽  
Kui Wang ◽  
Jeneen Stewart ◽  
Robert Belas
Keyword(s):  
Mitomycin C ◽  
Marine Bacterium ◽  
Pcr Amplification ◽  
Pulsed Field Gel Electrophoresis ◽  
Bacterial Genomes ◽  
Dominant Type ◽  
Transmission Electron ◽  
Viral Particles ◽  
Genomic Approach ◽  
Primer Sets

ABSTRACT Approximately 70% of sequenced bacterial genomes contain prophage-like structures, yet little effort has been made to use this information to determine the functions of these elements. The recent genomic sequencing of the marine bacterium Silicibacter sp. strain TM1040 revealed five prophage-like elements in its genome. The genomes of these prophages (named prophages 1 to 5) are approximately 74, 30, 39, 36, and 15 kb long, respectively. To understand the function of these prophages, cultures of TM1040 were treated with mitomycin C to induce the production of viral particles. A significant increase in viral counts and a decrease in bacterial counts when treated with mitomycin C suggested that prophages were induced from TM1040. Transmission electron microscopy revealed one dominant type of siphovirus, while pulsed-field gel electrophoresis demonstrated two major DNA bands, equivalent to 35 and 75 kb, in the lysate. PCR amplification with primer sets specific to each prophage detected the presence of prophages 1, 3, and 4 in the viral lysate, suggesting that these prophages are inducible, but not necessarily to the same level, while prophages 2 and 5 are likely defective or non-mitomycin C-inducible phages. The combination of traditional phage assays and modern microbial genomics provides a quick and efficient way to investigate the functions and inducibility of prophages, particularly for a host harboring multiple prophages with similar sizes and morphological features.

scholarly journals Characterization of Passage-Selected Vancomycin-Resistant Staphylococcus aureus Strains of Diverse Parental Backgrounds

2000 ◽  
Vol 44 (2) ◽  
pp. 294-303 ◽  
Author(s):  
Richard F. Pfeltz ◽  
Vineet K. Singh ◽  
Jennifer L. Schmidt ◽  
Michael A. Batten ◽  
Christopher S. Baranyk ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Walls ◽  
Methicillin Resistance ◽  
Surface Hydrophobicity ◽  
Whole Cell ◽  
Transmission Electron ◽  
Vancomycin Mics ◽  
Vancomycin Resistant ◽  
Doubling Times

ABSTRACT A series of 12 Staphylococcus aureus strains of various genetic backgrounds, methicillin resistance levels, and autolytic activities were subjected to selection for the glycopeptide-intermediate S. aureus (GISA) susceptibility phenotype on increasing concentrations of vancomycin. Six strains acquired the phenotype rapidly, two did so slowly, and four failed to do so. The vancomycin MICs for the GISA strains ranged from 4 to 16 μg/ml, were stable to 20 nonselective passages, and expressed resistance homogeneously. Neither ease of acquisition of the GISA phenotype nor the MIC attained correlated with methicillin resistance hetero- versus homogeneity or autolytic deficiency or sufficiency. Oxacillin MICs were generally unchanged between parent and GISA strains, although the mec members of both isogenic methicillin-susceptible and methicillin-resistant pairs acquired the GISA phenotype more rapidly and to higher MICs than did their susceptible counterparts. Transmission electron microscopy revealed that the GISA strains appeared normal in the absence of vancomycin but had thickened and diffuse cell walls when grown with vancomycin at one-half the MIC. Common features among GISAs were reduced doubling times, decreased lysostaphin susceptibilities, and reduced whole-cell and zymographic autolytic activities in the absence of vancomycin. This, with surface hydrophobicity differences, indicated that even in the absence of vancomycin the GISA cell walls differed from those of the parents. Autolytic activities were further reduced by the inclusion of vancomycin in whole-cell and zymographic studies. The six least vancomycin-susceptible GISA strains exhibited an increased capacity to remove vancomycin from the medium versus their parent lines. This study suggests that while some elements of the GISA phenotype are strain specific, many are common to the phenotype although their expression is influenced by genetic background. GISA strains with similar glycopeptide MICs may express individual components of the phenotype to different extents.

Species-specific duplex PCR for the detection of Pratylenchus penetrans

Nematology ◽  
2009 ◽  
Vol 11 (6) ◽  
pp. 847-857 ◽  
Author(s):  
Lieven Waeyenberge ◽  
Nicole Viaene ◽  
Maurice Moens
Keyword(s):  
Internal Control ◽  
Dna Sequences ◽  
Rrna Gene ◽  
Duplex Pcr ◽  
Specific Primers ◽  
5.8S Rrna Gene ◽  
5.8S Rrna ◽  
Wide Range ◽  
Pcr Products ◽  
Species Specific

Abstract ITS1, the 5.8S rRNA gene and ITS2 of the rDNA region were sequenced from 20 different Pratylenchus species. Additionally, the same region was sequenced from seven populations of P. penetrans. After purifying, cloning and sequencing the PCR products, all sequences were aligned in order to find unique sites suitable for the design of species-specific primers for P. penetrans. Since ITS regions showed variability between and even within populations of P. penetrans, only three small DNA sequences were suitable for the construction of three potentially useful species-specific primers. New species-specific primers were paired with existing universal ITS primers and tested in all possible primer combinations. The best performing primer set, supplemented with a universal 28S rDNA primer set that served as an internal control, was tested in duplex PCR. The ideal annealing temperature, Mg2+ concentration and primer ratios were then determined for the most promising primer set. The optimised duplex PCR was subsequently tested on a wide range of different Pratylenchus spp. and 25 P. penetrans populations originating from all over the world. To test the sensitivity, the duplex PCR was conducted on DNA extracted from a single P. penetrans nematode mixed with varying amounts of nematodes belonging to another Pratylenchus species. Results showed that a reliable and sensitive P. penetrans species-specific duplex PCR was constructed.

Variation in bacterial endosymbionts associated with the date palm hopper,Ommatissus lybicuspopulations

2017 ◽  
Vol 108 (2) ◽  
pp. 271-281 ◽  
Author(s):  
S. Karimi ◽  
H. Izadi ◽  
M. Askari Seyahooei ◽  
A. Bagheri ◽  
P. Khodaygan
Keyword(s):  
Date Palm ◽  
Multiple Infections ◽  
Infection Frequency ◽  
Pcr Assay ◽  
Specific Primers ◽  
Consensus Sequences ◽  
Pcr Products ◽  
Bacterial Endosymbionts ◽  
Different Populations ◽  
Polymerase Chain

AbstractThe date palm hopper,Ommatissus lybicus, is a key pest of the date palm, which is expected to be comprised of many allopatric populations. The current study was carried out to determine bacterial endosymbiont diversity in the different populations of this pest. Ten date palm hopper populations were collected from the main date palm growing regions in Iran and an additional four samples from Pakistan, Oman, Egypt and Tunisia for detection of primary and secondary endosymbionts using polymerase chain reaction (PCR) assay with their specific primers. The PCR products were directly sequenced and edited using SeqMan software. The consensus sequences were subjected to a BLAST similarity search. The results revealed the presence of ‘CandidatusSulcia muelleri’ (primary endosymbiont) andWolbachia,ArsenophonusandEnterobacter(secondary endosymbionts) in all populations. This assay failed to detect ‘CandidatusNasuia deltocephalinicola’ andSerratiain these populations. ‘Ca. S. muelleri’ exhibited a 100% infection frequency in populations andWolbachia,ArsenophonusandEnterobacterdemonstrated 100, 93.04 and 97.39% infection frequencies, respectively. The infection rate ofArsenophonusandEnterobacterranged from 75 to 100% and 62.5 to 100%, respectively, in different populations of the insect. The results demonstrated multiple infections by ‘Ca. Sulcia muelleri’,Wolbachia,ArsenophonusandEnterobacterin the populations and may suggest significant roles for these endosymbionts on date palm hopper population fitness. This study provides an insight to endosymbiont variation in the date palm hopper populations; however, further investigation is needed to examine how these endosymbionts may affect host fitness.

scholarly journals Specific DNA identification of Pheretima in the Naoxintong capsule

2019 ◽  
Vol 14 (1) ◽  
Author(s):  
Xiaoxiao Zhu ◽  
Hoi-Yan Wu ◽  
Pang-Chui Shaw ◽  
Wei Peng ◽  
Weiwei Su
Keyword(s):  
Pcr Amplification ◽  
Cerebrovascular Diseases ◽  
Coi Gene ◽  
Chemical Marker ◽  
Dna Identification ◽  
Specific Primers ◽  
Blast Search ◽  
Pcr Products ◽  
Crude Drugs ◽  
Nucleotide Database

Abstract Background Pheretima is a minister drug in Naoxintong capsule (NXTC), a well-known traditional Chinese medicine (TCM) formula for the treatment of cardiovascular and cerebrovascular diseases. Owing to the loss of morphological and microscopic characteristics and the lack of recognized chemical marker, it is difficult to identify Pheretima in NXTC. This study aims to evaluate the feasibility of using DNA techniques to authenticate Pheretima, especially when it is processed into NXTC. Methods DNA was extracted from crude drugs of the genuine and adulterant species, as well as nine batches of NXTCs. Based on mitochondrial cytochrome c oxidase subunit I (COI) gene, specific primers were designed for two genera of genuine species, Metaphire and Amynthas, respectively. PCR amplification was performed with the designed primers on crude drugs of Pheretima and NXTCs. The purified PCR products were sequenced and the obtained sequences were identified to species level with top hit of similarity with BLAST against GenBank nucleotide database. Results Primers MF2R2 and AF3R1 could amplify specific DNA fragments with sizes around 230–250 bp, both in crude drugs and NXTC. With sequencing and the BLAST search, identities of the tested samples were found. Conclusion This study indicated that the molecular approach is effective for identifying Pheretima in NXTC. Therefore, DNA identification may contribute to the quality control and assurance of NXTC.

scholarly journals A 16S rDNA-based nested PCR protocol to detect Campylobacter gracilis in oral infections

2003 ◽  
Vol 17 (2) ◽  
pp. 142-146 ◽  
Author(s):  
José Freitas Siqueira Júnior ◽  
Isabela das Neves Rôças
Keyword(s):  
16S Rdna ◽  
High Sensitivity ◽  
High Specificity ◽  
Cross Reactivity ◽  
Nested Polymerase Chain Reaction ◽  
Oral Infections ◽  
Root Canals ◽  
Infected Root ◽  
Pcr Products ◽  
Species Specific

The aim of this study was to describe a 16S rDNA-based nested polymerase chain reaction (nPCR) assay to investigate the occurrence of Campylobacter gracilis in oral infections. Samples were collected from ten infected root canals, ten cases of acute periradicular abscesses and eight cases of adult marginal periodontitis. DNA extracted from the samples was initially amplified using universal 16S rDNA primers. A second round of amplification used the first PCR products to detect C. gracilis using oligonucleotide primers designed from species-specific 16S rDNA signature sequences. The nPCR assay used in this study showed a detection limit of 10 C. gracilis cells and no cross-reactivity was observed with nontarget bacteria. C. gracilis was detected in the three types of oral infections investigated - 4/10 infected root canals; 2/10 acute periradicular abscesses; and 1/8 subgingival specimens from adult periodontitis. The method proposed in this study showed both high sensitivity and high specificity to directly detect C. gracilis in samples from root canal infections, abscesses, and subgingival plaque. Our findings confirmed that C. gracilis may be a member of the microbiota associated with distinct oral infections, and its specific role in such diseases requires further clarification.

scholarly journals Molecular beacons allow specific RT-LAMP detection of B.1.1.7 variant SARS-CoV-2

2021 ◽  
Author(s):  
Scott Sherrill-Mix ◽  
Gregory D. Van Duyne ◽  
Frederic D. Bushman
Keyword(s):  
Genetic Variants ◽  
Molecular Beacons ◽  
Spike Protein ◽  
Detection Methods ◽  
Gene Encoding ◽  
Detection And Identification ◽  
Rapid Spread ◽  
The World ◽  
Primer Sets ◽  
Current Detection

AbstractOver the course of the COVID-19 pandemic, several SARS-CoV-2 genetic variants of concern have appeared and spread throughout the world. Detection and identification of these variants is important to understanding and controlling their rapid spread. Current detection methods for a particularly concerning variant, B.1.1.7, require expensive qPCR machines and depend on the absence of a signal rather than a positive indicator of variant presence. Here we report an assay using a pair of molecular beacons paired with reverse transcription loop mediated amplification to allow isothermal amplification from saliva to specifically detect B.1.1.7 and other variants which contain a characteristic deletion in the gene encoding the viral spike protein. This assay is specific, affordable and allows multiplexing with other SARS-CoV-2 LAMP primer sets.

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