I n v i t r o diagnostic test systems. Qualitative nucleic acid-based i n v i t r o examination procedures for detection and identification of microbial pathogens

Microbial infections are recognized by the innate immune system through germline-encoded pattern recognition receptors (PRRs). As most microbial pathogens contain DNA and/or RNA during their life cycle, nucleic acid sensing has evolved as an essential strategy for host innate immune defense. Pathogen-derived nucleic acids with distinct features are recognized by specific host PRRs localized in endolysosomes and the cytosol. Activation of these PRRs triggers signaling cascades that culminate in the production of type I interferons and proinflammatory cytokines, leading to induction of an antimicrobial state, activation of adaptive immunity, and eventual clearance of the infection. Here, we review recent progress in innate immune recognition of nucleic acids upon microbial infection, including pathways involving endosomal Toll-like receptors, cytosolic RNA sensors, and cytosolic DNA sensors. We also discuss the mechanisms by which infectious microbes counteract host nucleic acid sensing to evade immune surveillance.

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Higher Cytokine and Opsonizing Antibody Production Induced by Bovine Serum Albumin (BSA)-Conjugated Tetrasaccharide Related to Streptococcus pneumoniae Type 3 Capsular Polysaccharide

Frontiers in Immunology ◽

10.3389/fimmu.2020.578019 ◽

2020 ◽

Vol 11 ◽

Author(s):

Ekaterina A. Kurbatova ◽

Nelli K. Akhmatova ◽

Anton E. Zaytsev ◽

Elina A. Akhmatova ◽

Nadezhda B. Egorova ◽

...

Keyword(s):

Bovine Serum Albumin ◽

Serum Albumin ◽

Diagnostic Test ◽

Bovine Serum ◽

Capsular Polysaccharide ◽

Binding Capacity ◽

Immunological Study ◽

Gm Csf ◽

Test Systems ◽

Type 3

A number of studies have demonstrated the limited efficacy of S. pneumoniae type 3 capsular polysaccharide (CP) in the 13-valent pneumococcal conjugate vaccine against serotype 3 invasive pneumococcal diseases and carriage. Synthetic oligosaccharides (OSs) may provide an alternative to CPs for development of novel conjugated pneumococcal vaccines and diagnostic test systems. A comparative immunological study of di–, tri–, and tetra–bovine serum albumin (BSA) conjugates was performed. All oligosaccharides conjugated with biotin and immobilized on streptavidin-coated plates stimulated production of IL-1α, IL-2, IL-4, IL-5, IL-10, IFNγ, IL-17A, and TNFα, but not IL-6 and GM-CSF in monocultured mice splenocytes. The tetrasaccharide–biotin conjugate stimulated the highest levels of IL-4, IL-5, IL-10, and IFNγ, which regulate expression of specific immunoglobulin isotypes. The tetra–BSA conjugate adjuvanted with aluminum hydroxide elicited high levels of IgM, IgG1, IgG2a, and IgG2b antibodies (Abs). Anti-CP-induced Abs could only be measured using the biotinylated tetrasaccharide. The tetrasaccharide ligand possessed the highest binding capacity for anti-OS and antibacterial IgG Abs in immune sera. Sera to the tetra–BSA conjugate promoted greater phagocytosis of bacteria by neutrophils and monocytes than the CRM197-CP-antisera. Sera of mice immunized with the tetra–BSA conjugate exhibited the highest titer of anti-CP IgG1 Abs compared with sera of mice inoculated with the same doses of di– and tri–BSA conjugates. Upon intraperitoneal challenge with lethal doses of S. pneumoniae type 3, the tri– and tetra–BSA conjugates protected mice more significantly than the di–BSA conjugate. Therefore, it may be concluded that the tetrasaccharide ligand is an optimal candidate for development of a semi-synthetic vaccine against S. pneumoniae type 3 and diagnostic test systems.

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Detection and Identification of Postharvest Microbial Pathogens

Postharvest Pathogens and Disease Management ◽

10.1002/0471751987.ch2 ◽

2005 ◽

pp. 9-78

Keyword(s):

Microbial Pathogens ◽

Detection And Identification

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Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

Canadian Journal of Microbiology ◽

10.1139/m90-113 ◽

1990 ◽

Vol 36 (9) ◽

pp. 664-669 ◽

Cited By ~ 2

Author(s):

David R. Preston ◽

G. Rasul Chaudhry ◽

Samuel R. Farrah

Keyword(s):

Tissue Culture ◽

Nucleic Acid ◽

Environmental Samples ◽

Nucleic Acid Hybridization ◽

Dot Blot ◽

Polio Virus ◽

Detection And Identification ◽

Specificity And Sensitivity ◽

Hybridization Procedure ◽

Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

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