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Extraction of Cell Wall-Bound Teichoic Acids and Surface Proteins from Listeria monocytogenes

Gram-positive bacteria contain a cell wall consisting of a thick peptidoglycan layer decorated with surface proteins and polysaccharide-based polymers. The latter include the wall teichoic acids (WTAs), which are anionic glycopolymers covalently linked to the peptidoglycan ...

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In Vitro Peptidoglycan Synthesis Assay with Lipid II Substrate

Bacterial cell wall peptidoglycan is synthesized from lipid II precursor by two reactions. Glycosyltransferases polymerize the glycan chains and transpeptidases form the peptide cross-links. The bifunctional class A penicillin-binding proteins catalyze both of these rea ...

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Quantitative and Qualitative Preparations of Bacterial Outer Membrane Vesicles

Gram-negative bacterial outer membrane vesicle production and function have been studied using a variety of quantitative and qualitative methods. These types of analyses can be hampered by the use of impure vesicle preparations. Here we describe a set of techniques that are useful for the q ...

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The Outer Membrane of Gram-Negative Bacteria: Lipid A Isolation and Characterization

The isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) are important methodologies utilized to gain understanding of the Gram-negative cell envelope. Here, we describe protocols often employed by our laboratory for small- and large-scale isolation of ...

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Assembly of Bacterial Outer Membrane Proteins

Various methods that are routinely used to study the subcellular localization of membrane proteins in wild-type Gram-negative bacteria fall short in genetic studies addressing the biogenesis of outer membrane proteins (OMPs). Here, we describe three biochemical methods that can be ...

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Production and Crystallization of Bacterial Type V Secretion Proteins

X-ray crystallography has become the most powerful approach to determine the three dimensional structures of proteins. The major bottleneck issues in protein crystallography are the availability of high-quality protein samples and the production of diffracting crystals. Sin ...

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Isolation of Bacterial Type IV Machine Subassemblies

The bacterial type IV secretion systems (T4SSs) deliver DNA and protein substrates to bacterial and eukaryotic target cells generally by a mechanism requiring direct contact between donor and target cells. Recent advances in defining the architectures of T4SSs have been made through i ...

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Pore Formation by T3SS Translocators: Liposome Leakage Assay

Gram-negative bacteria utilize a dedicated membrane-embedded apparatus, the type III secretion system (T3SS), to inject proteins into host cells. The passage of the proteins across the target membrane is accomplished by a proteinaceous pore—the translocon—formed within the host ...

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Fluorescence Microscopy and Proteomics to Investigate Subcellular Localization, Assembly, and Function of the Type II Secretion System

Investigation of secretion systems is often critical to understanding the virulence mechanisms of bacterial pathogens. With estimates as high as 30–40% of proteins secreted or localized to the cell envelope, information about the subcellular localization and organization of sec ...

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Quality in the Molecular Microbiology Laboratory

In the clinical microbiology laboratory advances in nucleic acid detection, quantification, and sequence analysis have led to considerable improvements in the diagnosis, management, and monitoring of infectious diseases. Molecular diagnostic methods are routinely used to ...

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Overcoming Inhibition in Real-Time Diagnostic PCR

PCR is an important and powerful tool in several fields, including clinical diagnostics, food analysis, and forensic analysis. In theory, PCR enables the detection of one single cell or DNA molecule. However, the presence of PCR inhibitors in the sample affects the amplification efficiency ...

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Multiplex Real-Time PCR (MRT-PCR) for Diarrheagenic

Diarrheagenic Escherichia coli strains are important causes of diarrhea in children from the developing world and are now being recognized as emerging enteropathogens in the developed world. Current methods of detection are too expensive and labor-intensive for routine detecti ...

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Rapid Detection of the Escherichia coli Genospecies in Water by Conventional and Real-Time PCR

The presence of Escherichia coli has long been established as the most reliable microbiological indication of fecal contamination in water. Current recommended culture-based methods for assessing water quality by the detection of E. coli are lengthy and lack ubiquity (ability to det ...

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Real-Time Quantitative PCR, Pathogen Detection and MIQE

Nucleic acids are the ultimate biomarker and real-time PCR (qPCR) is firmly established as the method of choice for nucleic acid detection. Together, they allow the accurate, sensitive and specific identification of pathogens, and the use of qPCR has become routine in diagnostic laborator ...

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PCR Detection of Helicobacter pylori in Clinical Samples

Helicobacter pylori is an important pathogen whose primary niche is the human stomach. H. pylori is etiologically associated with gastric inflammation (gastritis), peptic ulcer disease, and gastric cancer. Both noninvasive (e.g., urea breath and stool antigen tests) and invasive (ga ...

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Sensitive and Rapid Detection of Campylobacter jejuni and Campylobacter coli Using Loop-Mediated Isothermal Amplification

Loop-mediated isothermal amplification (LAMP) is an established nucleic acid amplification method offering rapid, accurate, and cost-effective diagnosis of infectious diseases. From the beginning of DNA extraction to final detection of Campylobacter jejuni and Campylob ...

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Detection of Pathogenic Leptospira spp. Through Real-Time PCR (qPCR) Targeting the LipL32 Gene

Rapid diagnosis of leptospirosis, through culture and/or serology, can be difficult without proper expertise and is often delayed due to the length of time required to obtain results. Polymerase chain reaction (PCR), more specifically the real-time detection of the amplified PCR produ ...

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Diagnosis of Clostridium difficile Infection Using Real-Time PCR

Clostridium difficile is known to cause antibiotic-associated diarrhea and pseudomembranous colitis. Toxinogenic strains of the bacterium produce toxins A (TcdA) and B (TcdB), which are associated with the pathogenicity. The standard methods for diagnosis of C. difficile infec ...

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Pre-analytical Sample Treatment and DNA Extraction Protocols for the Detection of Bacterial Pathogens from Whole Blood

Molecular diagnostics is an increasing popular approach for the direct detection and identification of pathogenic bacteria in clinical samples. Conventional culture techniques are time-consuming and therefore causing a delay in the diagnosis of the patient. Alternative tec ...

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Detection of Bacterial Contamination in Platelet Concentrates Using Flow Cytometry and Real-Time PCR Methods

Despite considerable advances in the safety of blood components based on the application of highly sensitive and specific screening methods to minimize the viral infection risk, the prevention of transfusion-associated bacterial infection remains a major challenge in transfu ...

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