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Journal of Microbiological Methods

He Ren, Xiaoqing Wu, Yuping Lyu, Hongzi Zhou, Xueying Xie, Xinjian Zhang, Hetong Yang
Botrytis cinerea is an important plant pathogen causing grey mold disease in a wide range of plant species. The aim of this study was to identify reliable reference genes that can be used for the analysis of relative gene expression in B. cinerea with quantitative real-time reverse transcription PCR (qRT-PCR). Six commonly used housekeeping genes including actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), ubiquitin-conjugating enzyme (UCE), α-tubulin (α-TUB) and β-tubulin (β-TUB) were selected to test their expression stabilities in B...
September 13, 2017: Journal of Microbiological Methods
Takahito Watanabe, Koichi Yoshioka, Ayako Kido, Junseok Lee, Hikari Akiyoshi, Takashi Watanabe
The functions and properties of fungal sheath, an extracellular polysaccharide produced by many white-rot fungi, have been studied. However, the strong adherence of the sheath to fungal hyphae had been a major impediment in preparing intracellular proteins from the fungi and analyzing their cellular responses. To overcome this issue, we developed a rapid and easy method to remove the polysaccharide sheath using a selective lignin degrader, Ceriporiopsis subvermispora, which produces large sheath amounts in the presence of a lignin-derived aromatic compound...
September 12, 2017: Journal of Microbiological Methods
Wenxia Sun, Shishan Yuan, Haowen Huang, Ning Liu, Yunhong Tan
A biosensor based on localized surface plasmon resonance (LSPR) was developed to detect the antibody of Mycobacterium tuberculosis using the fusion protein CFP10-ESAT6 as an antigen. To explore the diagnostic potential of the biosensor for tuberculosis (TB), the fusion protein CFP10-ESAT6 was immobilized on gold nanorods (Au NRs) by chemical modification, and the functionalized Au NRs were subsequently incubated with serums collected from TB patients, non-tuberculous pulmonary disease patients or healthy individuals...
September 11, 2017: Journal of Microbiological Methods
Tepyuda Sritrakul, Supachai Nitipan, Worawidh Wajjwalku, Anchalee La-Ard, Chattip Suphatpahirapol, Wimol Petkarnjanapong, Boonsong Ongphiphadhanakul, Siriwan Prapong
Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein...
September 11, 2017: Journal of Microbiological Methods
Isabel Cruz-Lachica, Isidro Marquez-Zequera, Raul Allende-Molar, Josefa Adriana Sañudo-Barajas, Josefina Leon-Felix, Raymundo Saul Garcia-Estrada
Gilbertella persicaria is an important phytopathogen that is confused with Mucor spp. and Rhizopus spp. The main distinguishing characteristic of G. persicaria is the presence of appendages in sporangiospores, and their observation by conventional staining techniques generally fails. A technique is described using light microscopy for fast and reliable diagnosis.
September 9, 2017: Journal of Microbiological Methods
Carolina S Santos, Juliana N Ramos, Veronica V Vieira, Carina S Pinheiro, Roberto Meyer, Neuza M Alcantara-Neves, Rommel T Ramos, Artur Silva, Raphael Hirata, Liza Felicori, Carlos Ruiz de Alegría Puig, Jesús Navas, Vasco Azevedo, Ana L Mattos-Guaraldi, Luis G C Pacheco
A multiplex-PCR (mPCR) assay was designed with species-specific primers which generate amplicons of 226bp, 434bp and 106bp for differentiating the species C. striatum, C. amycolatum, and C. xerosis, respectively. mPCR results were 100% in agreement with identifications achieved by 16S rRNA and rpoB gene sequencing and by VITEK-MS.
September 6, 2017: Journal of Microbiological Methods
Nathita Phumthanakorn, Pattrarat Chanchaithong, Nuvee Prapasarakul
Staphylococcus pseudintermedius commonly colonizes the skin of dogs, whilst nasal carriage may occur in humans who are in contact with dogs or the environment of veterinary hospitals. Genes encoding cell wall-associated (CWA) proteins have been described in Staphylococcus aureus but knowledge of their occurrence in S. pseudintermedius is still limited. The aim of the study was to develop a method to detect S. pseudintermedius surface protein genes (sps) encoding CWA proteins, and to examine the distribution of the genes in isolates from different sources...
September 6, 2017: Journal of Microbiological Methods
Saskia Zimmermann, Lorena Tuchscherr, Jürgen Rödel, Bettina Löffler, Jürgen A Bohnert
Real-time fluorescent efflux assays are commonly used for measuring the efflux of bacterial pumps. Here we describe an optimized protocol for the NorA efflux pump in S. aureus using DiOC3 instead of ethidium bromide. Glucose and sodium formate were tested as energy carriers. This novel method is fast and reproducible.
September 5, 2017: Journal of Microbiological Methods
G F Dall, S T J Tsang, P J Gwynne, A J Wilkinson, A H R W Simpson, S J B Breusch, M P Gallagher
In vitro biofilm assays are a vital first step in the assessment of therapeutic effectiveness. Current biofilm models have been found to be limited by throughput, reproducibility, and cost. We present a novel in vitro biofilm model, utilising a sodium alginate substratum for surface biofilm colony formation, which can be readily dissolved for accurate evaluation of viable organisms. The dissolving bead biofilm assay was evaluated using a range of clinically relevant strains. The reproducibility and responsiveness of the assay to an antimicrobial challenge was assessed using standardised methods...
September 1, 2017: Journal of Microbiological Methods
Lissa Cruz-Saavedra, Marina Muñoz, Cielo León, Manuel Alfonso Patarroyo, Gabriela Arevalo, Paula Pavia, Gustavo Vallejo, Julio César Carranza, Juan David Ramírez
Metacyclic trypomastigotes are essential for the understanding of the biology of Trypanosoma cruzi, the agent of Chagas disease. However, obtaining these biological stages in axenic medium is difficult. Techniques based on charge and density of the parasite during different stages have been implemented, without showing a high efficiency in the purification of metacyclic trypomastigotes. So far, there is no protocol implemented where sepharose-DEAE is used as a resin. Therefore, herein we tested its ability to purify metacyclic trypomastigotes in Liver Infusion Triptose (LIT) medium cultures...
September 1, 2017: Journal of Microbiological Methods
Maria Cryskely A Batinga, Jaíne C Dos Santos, Julia T R Lima, Maria Fernanda D Bigotto, Kerstin Muner, Thalita Faita, Rodrigo M Soares, David A V da Silva, Trícia M F S Oliveira, Helena L Ferreira, Jaqueline A Diniz, Lara B Keid
Brucella canis, a gram-negative, facultative intracellular and zoonotic bacterium causes canine brucellosis. Direct methods are the most appropriate for the detection of canine brucellosis and bacterial isolation from blood samples has been employed as gold-standard method. However, due to the delay in obtaining results and the biological risk of the bacterial culturing, the polymerase chain reaction (PCR) has been successfully used as an alternative method for the diagnosis of the infection. Sample preparation is a key step for successful PCR and protocols that provide high DNA yield and purity are recommended to ensure high diagnostic sensitivity...
August 29, 2017: Journal of Microbiological Methods
E Okada, M Nisenbaum, J Martínez Arca, S E Murialdo
To our knowledge, this communication is the first report of chemotaxis towards chlorophenols by any bacteria. We used a recently published method based on the agarose in-plug assay combined with video processing analysis and we also present a new index of bacterial mean speed for these assays.
August 24, 2017: Journal of Microbiological Methods
Daniel Weiß, Ines Engelmann, Sascha D Braun, Stefan Monecke, Ralf Ehricht
A novel multiplex real-time PCR was designed to detect the clinically most important carbapenemase genes, blaKPC, blaVIM, blaNDM and blaOXA-48. The multiplex assay was verified testing genomic DNA of 24 carbapenemase-producing strains. It was validated using a blinded panel of 82 carbapenemase-producing and 50 non-producing isolates by direct colony PCR.
August 24, 2017: Journal of Microbiological Methods
Pichayanut Poolperm, Thanya Varinrak, Yasushi Kataoka, Khajornsak Tragoolpua, Takuo Sawada, Nattawooti Sthitmatee
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 seroconversion in vaccinated poultry, but are not suitable for diagnosis. Commercial ELISA kits are available for chickens and turkeys, but not for ducks. The present study reports development and standardization of an in-house indirect ELISA for detection of duck antibody to fowl cholera...
August 24, 2017: Journal of Microbiological Methods
Ana C Ramos, Ana C Gales, Jussimara Monteiro, Suzane Silbert, Thomás Chagas-Neto, Antonia M O Machado, Cecilia G Carvalhaes
The rapid detection of KPC-producing Enterobacteriaceae by microbiology laboratories has been required for infectious control programs. Herein we evaluated the performance of a novel immunochromatographic test for detecting KPC-2-, KPC-3-, KPC-4-, KPC-6-, KPC-7-, KPC-8-, and KPC-11-producing isolates and the influence of different growth media on the test performance.
August 24, 2017: Journal of Microbiological Methods
Suhad A A Al-Salihi, Thomas A Scott, Andy M Bailey, Gary D Foster
The basidiomycete fungi Hypholoma fasciculare and H. sublateritium are both prolific producers of sesquiterpenes and triterpenes, some of which have relevant pharmaceutical properties. Although H. sublateritium has been transformed in the past, the low reported efficiencies highlighted the need for establishing an effective simple transformation system for these valuable species. We have optimized Agrobacterium tumefaciens-mediated transformation through testing various parameters in these two Hypholoma species, showing that a mixture of homogenized mycelia and Agrobacterium (strain LBA4404) co-cultivated for 84h at 25°C is optimal for efficient transformation in these basidiomycetes...
August 24, 2017: Journal of Microbiological Methods
Denise Wohlmeister, Débora Renz Barreto Vianna, Virginia Etges Helfer, Luciane Noal Calil, Andréia Buffon, Alexandre Meneghello Fuentefria, Valeriano Antonio Corbellini, Diogo André Pilger
Pathogenic Candida species are detected in clinical infections. CHROMagar™ is a phenotypical method used to identify Candida species, although it has limitations, which indicates the need for more sensitive and specific techniques. Infrared Spectroscopy (FT-IR) is an analytical vibrational technique used to identify patterns of metabolic fingerprint of biological matrixes, particularly whole microbial cell systems as Candida sp. in association of classificatory chemometrics algorithms. On the other hand, Soft Independent Modeling by Class Analogy (SIMCA) is one of the typical algorithms still little employed in microbiological classification...
August 16, 2017: Journal of Microbiological Methods
José M Marimón, María Morales, Susana Gamen, Ana Manrique, María Ercibengoa, Gustavo Cilla
The S. PneumoStrip test is a recently developed reverse hybridization strip-based commercial assay that allows for the identification of 76 pneumococcal serotypes, 42 individually and 34 in pairs, according to their specific gene sequences. The test was validated with reference strains of 92 different pneumococcal serotypes and with a selection of 75 clinical isolates representing 55 serotypes, showing 100% sensitivity and specificity. The test was also applied to 64 pneumococcal invasive isolates (23 different serotypes) consecutively collected between June 2016 and March 2017, with 60 (93...
August 14, 2017: Journal of Microbiological Methods
Mehmet Karakuş, Bahtiyar Yılmaz, Yusuf Özbel, Seray Töz
Molecular techniques are widely used in the field of parasitology to identify the genetic profile of the microbiological agents. Microsatellite typing studies are comprised of the amplification of polymorphic markers to analyze the fragment sizes using bioinformatics tools. Current methods need fluorescently labeled primers and size markers to obtain fragment peaks in ABI PRISM® systems and due to low discrimination power of gel-electrophoresis, it is not possible to differentiate primer-dimers from small fragments In the present study, we designed a new method for fragment analysis studies, which reduce the time by eliminating the classical PCR, the gel-electrophoresis and the preparation steps of fragment analysis...
August 14, 2017: Journal of Microbiological Methods
Zhibei Zheng, Wei Zheng, Haoqiu Wang, Jincao Pan, Xiaoying Pu
Currently, no protocols or commercial kits are available to determine the serotypes of Salmonella by using Luminex MAGPIX®. In this study, an xTAG assay for serotype determination of Salmonella suitable for Luminex MAGPIX® is described and 228 Salmonella isolates were serotype determined by this xTAG assay. The xTAG assay consists of two steps: 1) Multiplex PCR to amplify simultaneously O, H and Vi antigen genes of Salmonella, and 2) Magplex-TAG™ microsphere hybridization to identify accurately the specific PCR products of different antigens...
August 14, 2017: Journal of Microbiological Methods
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