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PubMed2005

PCR microfluidic devices for DNA amplification.

The miniaturization of biological and chemical analytical devices by micro-electro-mechanical-systems (MEMS) technology has posed a vital influence on such fields as medical diagnostics, microbial detection and other bio-analysis. Among many miniaturized analytical devices, the polymerase chain reaction (PCR) microchip/microdevices are studied extensively, and thus great progress has been made on aspects of on-chip micromachining (fabrication, bonding and sealing), choice of substrate materials, surface chemistry and architecture of reaction vessel, handling of necessary sample fluid, controlling of three or two-step temperature thermocycling, detection of amplified nucleic acid products, integration with other analytical functional units such as sample preparation, capillary electrophoresis (CE), DNA microarray hybridization, etc. However, little has been done on the review of above-mentioned facets of the PCR microchips/microdevices including the two formats of flow-through and stationary chamber in spite of several earlier reviews [Zorbas, H. Miniature continuous-flow polymerase chain reaction: a breakthrough? Angew Chem Int Ed 1999; 38 (8):1055-1058; Krishnan, M., Namasivayam, V., Lin, R., Pal, R., Burns, M.A. Microfabricated reaction and separation systems. Curr Opin Biotechnol 2001; 12:92-98; Schneegabeta, I., Köhler, J.M. Flow-through polymerase chain reactions in chip themocyclers. Rev Mol Biotechnol 2001; 82:101-121; deMello, A.J. DNA amplification: does 'small' really mean 'efficient'? Lab Chip 2001; 1: 24N-29N; Mariella, Jr. R. MEMS for bio-assays. Biomed Microdevices 2002; 4 (2):77-87; deMello AJ. Microfluidics: DNA amplification moves on. Nature 2003; 422:28-29; Kricka, L.J., Wilding, P. Microchip PCR. Anal BioAnal Chem 2003; 377:820-825]. In this review, we survey the advances of the above aspects among the PCR microfluidic devices in detail. Finally, we also illuminate the potential and practical applications of PCR microfluidics to some fields such as microbial detection and disease diagnosis, based on the DNA/RNA templates used in PCR microfluidics. It is noted, especially, that this review is to help a novice in the field of on-chip PCR amplification to more easily find the original papers, because this review covers almost all of the papers related to on-chip PCR microfluidics.

DNA↗
PubMed1992

Interleukin 6 determination in the detection of microbial invasion of the amniotic cavity.

A growing body of evidence suggests a role for cytokines in the mechanisms responsible for preterm parturition associated with intrauterine infection. Interleukin 6, a polyfunctional cytokine that is secreted by tissues in the feto-maternal interface in response to microbial products, has been implicated in the host response to intrauterine infection. The purpose of this study was to establish whether measurement of amniotic fluid concentrations of interleukin 6 could be of value in the diagnosis of microbial invasion of the amniotic cavity. Fluid was obtained by transabdominal amniocentesis from patients with preterm labour and intact chorioamniotic membranes and cultured for aerobic and anaerobic bacteria and mycoplasmas. Interleukin 6 concentrations were determined by an ELISA validated for human amniotic fluid. An interleukin 6 concentration above 11.2 ng/ml had a 93.7% sensitivity and a 92.3% specificity in the diagnosis of intra-amniotic infection. Moreover, patients with an amniotic fluid interleukin 6 level above 11.2 ng/ml and a negative amniotic fluid culture failed to respond to tocolysis, delivered a preterm infant and showed histological evidence of chorioamnionitis, and their neonates were at risk for congenital infections.

Amniotic Fluid↗
PubMed2003

Possible quorum sensing in the rumen microbial community: detection of quorum-sensing signal molecules from rumen bacteria.

The bioluminescence assay using Vibrio harveyi BB170 was used to examine quorum-sensing autoinducer 2 (AI-2) activity from cell-free culture fluids of rumen bacteria. The assay showed that the culture fluids of four species of rumen bacteria, Butyrivibrio fibrisolvens, Eubacterium ruminantium, Ruminococcus flavefaciens, and Succinimonas amylolytica, contained AI-2-like molecules. Furthermore, homologues for luxS genes were detected in rumen fluids collected from three cows and in bacterial cells of P. ruminicola subsp. ruminicola and R. flavefaciens. These findings suggest that the quorum-sensing system mediated by AI-2 is present in the rumen.

Animals↗
PubMed1994

Bacteriophages presence in human faeces of healthy subjects and patients with gastrointestinal disturbances.

The variation of the content of enteric bacteria and their bacteriophages in faeces from the different types of diarrhoeal processes has been studied. A total of 122 samples of human faeces from both healthy individuals and patients with diarrhoeal diseases of functional or infectious origin were tested. Detection rates for all microbial parameters tested decreased in the faeces of individuals with functional gastrointestinal disturbances. On the contrary, no significant differences of the microbial detection frequency was observed in faeces containing pathogenic microorganisms compared to faeces of healthy subjects. Human faeces were a poor source of F-specific, Salmonella, and Bacteroides bacteriophages, whereas specific Escherichia coli phages were isolated in most samples tested. Coliphage concentrations in faeces of healthy individuals were not directly correlated with levels of faecal coliforms. On the basis of their high correlation, faecal streptococci and coliphages were the most adequate indicators of the intestinal ecosystem variations in subjects with diarrhoeal processes.

Bacteriophages↗
PubMed2001

The extremely rapid oligonucleotide hybridization and high throughput detection of microbial gene sequences using fluorescence polarization.

The hybridization of oligonucleotide sequences complementary to the genes of Shiga toxins (verotoxins) types 1 and 2 of enterohaemorrhagic Escherichia coli (EHEC) and human hepatitis C virus (HCV) was monitored using fluorescence polarization under the reaction condition of high salt concentration (0.8 M NaCl), which was optimized to obtain a higher rate of hybridization. The time courses of hybridization of fluorescently labeled oligomers (probe DNAs) with the amplified DNA or RNA of the genes were recorded. Two methods, the asymmetric PCR and NASBA, were used to amplify the genetic DNA of Shiga toxins and that of RNA in HCV, respectively. Probe DNA sequences were designed which hybridized extremely rapidly with amplicons of the genes of Shiga toxins types 1 and 2 and that of HCV. In the cases using the three different DNA probes, the hybridization was 90% complete in about 1 min, considerably faster than that of the 3 min reported previously. The rapidity of this hybridization could not be explained by the melting temperature or the G+C content of the probe sequences but its relationship with high order structure of the single stranded DNA or RNA of the amplicons in the solution was strongly suggested.

Base Sequence↗