We have develoPed a simPle and economical method f0r Chlamydia trachomatisdetecting, called microtiter plate hybridization (PCR-MPH), which may replace stan-dard PCR. This method is similar to that of an ELISA. Brithe...We have develoPed a simPle and economical method f0r Chlamydia trachomatisdetecting, called microtiter plate hybridization (PCR-MPH), which may replace stan-dard PCR. This method is similar to that of an ELISA. Brithe, the PCR productslabeled at the 5'termini with biotin were hybridized with probes immobilized on a mi-crotiter well, and the bound PCR products were detected by streptavidin-c0njugatedenzymes followed by color development. Two inprovements have been made in immobi-lizing the probe to the microtiter wells, in terms of increasing both immobility and hy-bridization deciency. One is that singleustranded (ss )DNA, without the complemen-tary strand, is used. The other is that instead of a single copy, a tandem array of theprobe is used for immobilization and hybridization. Using of ssDNA containing abouta 5O-rePeat array of a relevant sequence as an immobilized probe, the sensitivity in-creased 1O-fold over that of a single oligonucleotide unit. We also found that the hy-brldizatlon condltions such as time, temPerature, and solution composition could be simplthed. The advantages of this microtiter plate-hybridization method for routinepathogens detecting are a short time assay, easy processing of large numbers of sansples, and the potential for automation.展开更多
文摘We have develoPed a simPle and economical method f0r Chlamydia trachomatisdetecting, called microtiter plate hybridization (PCR-MPH), which may replace stan-dard PCR. This method is similar to that of an ELISA. Brithe, the PCR productslabeled at the 5'termini with biotin were hybridized with probes immobilized on a mi-crotiter well, and the bound PCR products were detected by streptavidin-c0njugatedenzymes followed by color development. Two inprovements have been made in immobi-lizing the probe to the microtiter wells, in terms of increasing both immobility and hy-bridization deciency. One is that singleustranded (ss )DNA, without the complemen-tary strand, is used. The other is that instead of a single copy, a tandem array of theprobe is used for immobilization and hybridization. Using of ssDNA containing abouta 5O-rePeat array of a relevant sequence as an immobilized probe, the sensitivity in-creased 1O-fold over that of a single oligonucleotide unit. We also found that the hy-brldizatlon condltions such as time, temPerature, and solution composition could be simplthed. The advantages of this microtiter plate-hybridization method for routinepathogens detecting are a short time assay, easy processing of large numbers of sansples, and the potential for automation.
