人乳头瘤病毒16,18型核酸检测质控物的研制及应用

Development and application of quality control materials for human papillomavirus types 16 and 18 nucleic acid test

目的研制得到能模拟真实临床标本的人乳头瘤病毒(HPV)高危型16,18核酸检测质控物,并探讨其用于临床实验室室间质量评价的有效性。方法采用分子克隆方法得到含 HPV-16靶基因的重组质粒载体,转染已含 HPV-18的宫颈癌上皮细胞系(HeLa),获得含 HPV-16,18核酸的上皮细胞原液,经适当灭活后甲醇固定。将该细胞原液适当稀释后,组成含阴阳性共12份标本的样本盘,发至全国各开展临床 HPV-16,18检测的实验室,对回报结果进行分析。结果细胞原液经1:10,1:50,1:100,1:500稀释,实时荧光 PCR 检测,HPV-16 Ct 值分别为29.10,31.19,32.15和32.73,HPV-18 Ct 值分别为30.32,32.13,32.22和35.55。质控物在4℃可稳定40 d 以上,盲邮至新疆乌鲁木齐、内蒙古呼和浩特和厦门的样本,返回后检测无明显变化。44个临床实验室对高浓度样本的检测符合率平均为95.1%,对低浓度样本的检测符合率为57.4%。对阴性样本符合率为98.3%。结论获得可模拟临床标本的 HPV-16,18核酸检测质控物,质评结果表明可有效地用于临床实验室室间质评。

Objective To develop quality control materials for human papillomavirns （HPV） types 16 and 18 molecular detection and to evaluate the applicability of the materials in external quality assessment （EQA） of HPV-16,18 clinical detection. Methods The target gene from HPV16 was cloned into the pEGFP-C1 plasmid, then the recombinant plasmid pEGFP-C1-HPVI6 was transfected into the HPV18 carrying HeLa cells. The cultured epithelial cells carrying both HPV16 target gene and HPV18 were collected and fixed using methanol. EQA samples for HPV types 16 and 18 test diluted to the concentrations 1： 10, 1 ： 50, 1 ： 100, 1 ： 500 were prepared from the above prepared cells and distributed to 73 EQA participants nationwide to undergo RT-PCR. The reports from the participants were summarized and evaluated. Three samples were blindly mailed to Urnmqi, Hohhot, and Xiamen respectively to observe the influence of post. Results Forty-four of the 73 PCR laboratories sent feedback. For the quality control materials of the concentrations 1： 10, 1： 50, 1： 100, and 1：500 the corresponding Ct values were 29.10, 31.19, 32.15, and 32.73 respectively for HPV-16, and 30.32, 32.13, 32.22, and 35.55 respectively for HPV-18. Stability test indicated that the quality control materials were stable at least for 40 days when stored at 4℃. The EQA data from 44 participants showed, that the average fit rate was 95. 1% for the high concentration positive samples and was 57.4% for the low concentration samples, and was 98.3% for the negative samples. No significant changes were detected by real-time PCR in the returned EQA samples that were blindly mailed to Urnmqi, Hohhot, and Xiamen. Conclusion A quality control materials for HPV types 16 and 18 molecular detection has been developed, and quality assessment verifies its applicability in EQA.