A Simple Method of Detecting Chlamydia Trachomatis Using Enzymatically Amplified DNA and Immobilized Probes on Microtiter Plate

We have developed a simple and economical method for Chlamydia trachomatis detecting, called microtiter plate hybridization (PCR-MPtt) , which may replace standard PCR. This method is similar to that of an ELISA. Briefly, the PCR products labeled at the 5‘ termini with biotin were hybridized with probes immobilized on a microtiter well

A Simple Method of Detecting Chlamydia Trachomatis Using Enzymatically Amplified DNA and Immobilized Probes on Microtiter Plate

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.