Within this contribution we introduce a 3D-printed cartridge system enabling the convenient and cost-efficient sample preparation from sputum for subsequent PCR based detection schemes. The developed fluidic system operates is...
We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.
With this study, an innovative and
convenient enrichment and detection
strategy for eight clinically relevant pneumonia pathogens, namely,
Acinetobacter baumannii
,
Escherichia
coli
,
Haemophilus influenzae
,
Klebsiella pneumoniae
,
Moraxella catarrhalis
,
Pseudomonas
aeruginosa
,
Staphylococcus aureus,
and
Streptococcus pneumoniae
is introduced.
Bacteria were isolated from sputum samples with amine-modified particles
exploiting pH-dependent electrostatic interactions between bacteria
and the functionalized particle surface. Following this, an asymmetric
polymerase chain reaction as well as subsequent stringent array-based
hybridization with specific complementary capture probes were performed.
Finally, results were visualized by an enzyme-induced silver nanoparticle
deposition, providing stable endpoint signals and consequently an
easy detection possibility. The assay was optimized using spiked samples
of artificial sputum with different strains of the abovementioned
bacterial species. Furthermore, actual patient sputum samples with
S. pneumoniae
were successfully analyzed. The presented
approach offers great potential for the urgent need of a fast, specific,
and reliable isolation and identification platform for important pneumonia
pathogens, covering the complete process chain from sample preparation
up to array-based detection within only 4 h.
AbstractRaman spectroscopy and its variants allow for the investigation of a wide range of biological and biomedical samples, i. e. tissue sections, single cells and small molecules. The obtained information is on a molecular level. By making use of databases and chemometrical approaches, the chemical composition of complex samples can also be defined. The measurement procedure is straight forward, however most often sample preparation protocols must be implemented. While pure samples, such as high purity powders or highly concentrated chemicals in aqueous solutions, can be directly measured without any prior sample purification step, samples of biological origin, such as tissue sections, pathogens in suspension or biofluids, food and beverages often require pre-processing steps prior to Raman measurements. In this book chapter, different strategies for handling and processing various sample matrices for a subsequent Raman microspectroscopic analysis were introduced illustrating the high potential of this promising technique for life science and medical applications. The presented methods range from standalone techniques, such as filtration, centrifugation or immunocapture to innovative platform approaches which will be exemplary addressed. Therefore, the reader will be introduced to methods that will simplify the complexity of the matrix in which the targeted molecular species are present allowing direct Raman measurements with bench top or portable setups.
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