实验基础

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is an economically significant veterinary pathogen that causes Johne’s disease in cattle and sheep. There is a critical need for improved diagnostic tests to detect M. paratuberculosis infection in these ...

A microimmunofluorescence technique for the diagnosis of Q fever is described. Although this method is useful for serological diagnosis of Q fever, some technical difficulties are associated with it. First, the test antigens must be produced by a cell culture method in a level-3 biohazard fa ...

Isolation of Mycobacterium bovis from suspected cases of bovine tuberculosis demands laborious and time-consuming procedures. Also, direct PCR procedures on tissue samples show poor sensitivity, whereas radiometric and fluorescence-based identification procedures ...

Nucleic acid-based diagnostics gradually are replacing or complementing culture-based, biochemical, and immunological assays in routine microbiology laboratories. Similar to conventional tests, the first-generation deoxyribonucleic acid assays determined on ...

In this chapter, a protocol called on-chip polymerase chain reaction (PCR) is presented for the deoxyribonucleic acid (DNA) microarray-based detection of bacterial target sequences. On-chip PCR combines, in a single step, the conventional amplification of a target with a simultaneo ...

Direct deoxyribonucleic acid (DNA)-based detection methods are crucial for future environmental monitoring and clinical diagnosis. In this chapter, we provide an overview of of the various sample preparation approaches for bacteria for direct analyses (i.e., without culturing) ...

The polymerase chain reaction (PCR) is a fundamental part of modern molecular biology. Fluorescence-based PCR methods also are now available, which enable rapid, specific, and sensitive assays for the amplification and analysis of deoxyribonucleic acid (DNA). These methods are perf ...

The Naval Research Laboratory has developed an array-based biosensor system capable of detecting multiple pathogenic and toxic species in complex matrices. Sandwich fluoroimmunoassays are performed on the surface of a patterned microscope slide that acts as an optical waveguid ...

The rapid and accurate identification of methicillin-resistant Staphylococcus aureus (MRSA) is of great importance for the affected patient, the involved ward, and the microbiological laboratory. Resistance to methicillin is encoded by the mecA gene in S. aureus. Because routine l ...

Escherichia coli O157:H7 is a highly virulent pathogen that causes severe food poisoning in humans. Many outbreaks involving this pathogen have been linked to minced beef. A protocol is presented here to detect E. coli O157:H7 in minced beef. The method consists of an enrichment step in modified tr ...

Use of high copy number bacterial RNA offers several advantages in a diagnostics context compared with current deoxyribonucleic acid-based assays. The opportunity to only detect viable cells by targeting labile RNA transcripts may create an opportunity for “real-time” monitoring ...

Quencher extension is a novel single-step closed tube real-time method to quantify single nucleotide polymorphisms (SNPs) in combination with primer extension. A probe with a 5′-reporter is single-base extended with a dideoxy nucleotide containing a quencher if the target SNP allele is ...

The membrane glycoproteins (Gn and Gc) of viruses in the family Bunyaviridae form projections on the virion envelope and are involved in virus entry and eliciting protective immunity. The glycoproteins are modified by N-linked glycosylation and accumulate in the Golgi complex where vi ...

Sequences derived from the respiratory syncytial virus (RSV) fusion (F) protein were expressed in insect cells as recombinant glutathione-S-transferase (GST)-tagged proteins. The sequence covering the F2 subunit (GST-F2), and a truncated form of the F protein in which the transmembr ...

The full-length and truncated forms of recombinant envelope (E) glycoprotein from Dengue virus type 1, Singapore strain S275/90 were expressed in the yeast, Pichia pastoris, using a secretory vector. A truncated form of the E protein in which the transmembrane domain was deleted was secreted ...

Hepatitis C virus (HC V) infection is a major cause of severe chronic liver disease including cirrhosis and hepatocellular carcinoma. HCV has been classified into six major genotypes that exhibit extensive genetic variability, particularly in the envelope glycoproteins E1 and E2. Kno ...

Although many virus proteins are glycosylated, the pattern of glycosylation that is exhibited can be highly variable, and it is largely dependent on how a specific virus protein is processed by the host cell during infection. However, irrespective of their glycosylation pattern, many vir ...

Glycosaminoglycans (GAGs), including heparan sulfate (HS), are expressed on the surface of nearly all cells, linked to transmembrane proteins. These GAGs are sulfated to varying extents, lending a negative charge, and are used by a large number of viruses to initiate infection of immortali ...

The Bac-to-Bac Baculovirus expression system was used to generate a recombinant baculovirus capable of expressing the severe acute respiratory syndrome (SARS)-coronavirus (CoV) 3a protein. Using the same expression system, two structural proteins, membrane (M) and envelope (E), ...

The respiratory syncytial virus fusion (F) protein is initially expressed as a single polypeptide chain (F0). The F0 subsequently undergoes posttranslational cleavage-by-cell protease activity to produce the F1 and F2 subunits. Each of the two subunits within the mature F protein is mod ...