Construction and application of quality control materials for Chlamydia trachomatis polymerase chain reaction detection / 中华检验医学杂志

ABSTRACT

Objective To construct quality control materials for chlamydia trachomatis (CT) polymerase chain reaction detection and evaluate the stability of the material.Methods The reference regarding the target sequence for CT PCR detection has been reviewed.The ovedap extension technique and molecular cloning techniques were used to construct a recombinant lasmid.Then the recombinant plasmid pTARGETTM-CT was transfected into a HTB-SiHa cells.The cuhured epithelia cells,vere collected as quality control material.Then we evaluated the stability of this material with domestic kits for CT PCR detection.The stability in different conditions were summarized and evaluated.The EQA samples for CT test survey ere prepared from the above prepared cells and distributed to the EQA participants nationwidely.Results Five fragments from CT(178-610),(1219-1993),(2471-3260),(5239-5864),(6722-7499) were cloned into pTARGET TM.The recombinant plasmid was transfected into mammalian cells as a final form for the quality control materials.Real-time PCR analysis howed the original material was positive with domestic chlamydia trachomatis kit(3.21×108 copies/ml).A Series of dilution resulted in the decreased result .The stability testing indicated the quality control materials were stable at least for one month when stored at 4℃,room temperate or 37℃.Conclusions We used several kinds of molecular iology methods such as ovedap PCR and enzyrnatie digest to construct a recombinant plasmid which contained several fragments.The quality control materials for chlamydia trachomatis PCR detection was developed successfully.