Immobilized Capture Probes Research Articles

Quantitation of dopamine D 2 receptor mRNA in a mesencephalic cell culture using a nonradioactive competitive reverse transcription polymerase chain reaction method

Studies on gene expression during differentiation and maturation processes have to cope with determinations of extremely low steady state levels of specific mRNA. Using the experimental model of dopamine D 2 receptor (D 2R) expression in a primary mesencephalic cell culture we worked out a quantitative reverse transcription polymerase chain reaction method which allows to analyze and quantify mRNA levels of cells present in a few wells of the culture. The method uses an internal cRNA standard which shares both primer binding sites and PCR product length with the target sequence. The amplicons are quantitated in microplates by hybridization with immobilized capture probes that allow for the distinction of internal standard and target sequences followed by the chemiluminescent detection of hybridized DNA. Applying this method the levels of D 2 receptor mRNA of the mesencephalic cell culture on day in vitro 1 amounted to about 250 fg/μg RNA and increased to about 1200 fg/μg RNA on day in vitro 13–15.

Quantification ofCoxiella burnetii by polymerase chain reaction (PCR) and a colorimetric microtiter plate hybridization assay (CMHA)

A colorimetric microtiter plate hybridization assay (CMHA) for the quantitative determination of Coxiella burnetii DNA after amplification by externally controlled polymerase chain reaction (PCR) is described. The quantification assay is based on an enzyme linked immunosorbent assay (ELISA) format. Cloned DNA, representing a sequence complementary to an internal part of the diagnostic amplicon, was noncovalently attached to the wells of a microtiter plate. Biotinylated PCR product was hybridized to the immobilized capture probe. Bound product was detected via streptavidin horse-radish peroxidase. The devised nonisotopic technique allows specific, rapid, and convenient quantification of C. burnetii DNA. Additionally, it is compatible with standard laboratory ELISA equipment, making this assay amenable to automation and permitting processing of large sample numbers.

Multiplex PCR amplification and immobilized capture probes for detection of bacterial pathogens and indicators in water

Detection of pathogens ( Legionella species) and indicator bacteria (coliform bacteria) was achieved by multiplex (simultaneous) PCR amplification of diagnostic gene sequences and by hybridization to immobilized poly-dT-tailed capture probes using a dot- or slot-blot approach. Complex manipulations of primer concentrations and staggered additions of primers were required in order to achieve equal amplification of multiple genes. Multiplex PCR amplification of two different Legionella genes, one specific for L. pneumophila ( mip) and the other for the genus Legionella (5S rRNA), was achieved by staggered amplification. Multiplex PCR amplification using differing amounts of primers specific for lacZ and lamB genes permitted the detection of coliform bacteria and those associated with human faecal contamination, including the indicator bacterial species E. coli and enteric pathogens Salmonella and Shigella. Hybridization of biotin-labelled amplified DNA, in which the biotin was incorporated during PCR amplification from biotinylated-dUTP, to immobilized 400-dT-tailed capture probes permitted specific and sensitive detection of target gene sequences. The sensitivity of colorimetric detection achieved by PCR amplification of target DNA was at a level equivalent to 1–2 bacterial cells, which is the same level of sensitivity obtained with radioactive detection. The simultaneous amplification of several genes and hybridization to immobilized capture probes with colorimetric detection is an effective, efficient and rapid detection method for various human bacterial pathogens.