沙眼衣原体聚合酶链反应测定质控物的构建及其应用研究

目的 研制能够模拟真实临床样本且无生物传染危险性的沙眼衣原体(chlamydia trachomatis,CT)PCR检测质摔物,并进行稳定性研究.方法 在充分查阅文献的基础上,明确国内外已有文献报道的CT:PCR检测的靶序列,选择最普遍应用的CT质粒序列,采用重叠(overlap)PCR、分子克隆等技术构建含有所选择的常用检测目的 CT质粒序列的重组真核表达载体,即pTARGETTM-CT 质粒.然后,将载体以脂质体转染的方式转染入宫颈上皮细胞(HTB-SiHa细胞)中,收集细胞,经含20%小牛血清的细胞培养液稀释,即成为能模拟CT检测临床样本的细胞质控物.最后观察得到的质控物对目前国内常用商品试剂盒的适用性及在4℃、37℃及室温条件下的稳定性.结果 成功构建了含CT质粒(178-610)、(1219-1993)、(2471-3260)、(5239-5864)、(6722-7499)等5个片段的重组真核表达载体,并转入HTB-SiHa细胞中,得到了可模拟临床样本的CT PCR检测的质控物.采用深圳匹基生物工程有限公司、中山大学达安基因股份有限公司商品CT PCR检测试剂盒对转染后样本进行检测,得到阳性结果;定量为3.21×108拷贝/ml;倍比稀释后较高浓度样本检测结果呈梯度下降、10倍稀释循环阈值相差约3.3;对不同浓度稀释后在4℃、37℃及室温条件下保存1个月的样本的检测结果进行随机区组的两因素方差分析后发现,在各温度下保存的样本经检测后的结果问差异无统计学意义,所构建的质控样本至少可以稳定保存1个月.结论 利用重叠PCR与双酶切的方法,建立了一种将多间隔片段连接并构建入同一载体的简便有效的方法.成功地得到了可模拟临床标本的CT PCR检测质控物.

Construction and application of quality control materials for Chlamydia trachomatis polymerase chain reaction detection

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.