摘要
目的探索滤纸固定艾滋病病毒Ⅰ型核酸(HIV-1 DNA)的稳定性、均一性,评价滤纸片干血斑HIV-1DNA检测方法的敏感性、特异性。方法套式PCR扩增HIV-1的env、gag、pol三区。不同环境条件下保存的滤纸片干血斑,分别在不同的时间段检测;滤纸片干血斑的中心及圆斑的上下左右4个边缘,随机打取5个2.5mm圆片用于检测。采自新疆现场的94份阴性样本和90份阳性样本,分别制成滤纸片干血斑样本和全血样本,比较两者的HIV-1DNA检测结果。结果滤纸片干血斑-20℃或4℃放置1年或室温放置3个月,或在37℃放置两天或冻融2次,实验的敏感性未见下降;37℃放置3天或冻融3次,已经有样本不能检出。滤纸干血斑不同位点随机打取圆片扩增,4个样本中,中点均阳性,但在上点、左点、右点分别出现一份阴性结果。滤纸干血斑法和全血法检测HIV-1前病毒DNA比较,敏感性是96.1%,特异性是98.9%。结论滤纸干血斑在室温条件下运输是可行的,推荐尽量靠近滤纸干血斑的中间位点打取圆片扩增;该方法操作简便、经济、敏感、特异。
Objective To explore HIV-1DNA homogeneous and stable distribution on filter paper and to evaluate the sensitivity and specificity of detection method of human immunodeficiency virus type 1 （HIV-1） DNA on dried blood spots （DBSs）. Methods HIV-1 env,gag and pol regions were amplified by nested PCR respectively. The DBSs were stored under different conditions and at different periods of time to test the storage expiration of BDSs. A punch was used to punch five discs φ02.5mm of filter paper from the blood spot at random, and then were amplified. Ninety-four HIV-1 uninfected samples and ninety HIV-1 infected samples were tested. A single individual had one whole blood specimen and one dried blood spot specimen, each of which was amplified by three optimized primer sets respectively. The results of these two detections were compared. Results The sensitivity of the assay did not decrease after storage of the blood on filter paper for 1 year at -20℃ or 4℃ ,for 3 months at room temperature or for 2 days after incubation at 37℃ for 2 times after freezing ＆ melting. There were a few samples that could not be tested after incubation at 37℃ , for 3 days or after freezing ＆ melting 3 times. Among 4 samples the random punched discs were positive at the middle of DBS,but negative at up,left and right position of DBS respectively. The assay detected HIV-1 DNA on dried blood spots with a sensitivity of 96.1% and a specificity of 98.9 %, as compared to PCR of HIV-1 DNA extracted from the whole blood with QIAamp DNA Blood Mini Kit. Conclusions It is applicable that the DBSs be transported at room temperature. It is optimal to punch a disc at the middle of the BDS. The detection method of HIV-1 DNA on dried blood spots is simple, cost effective, sensitive and specific.
出处
《中国艾滋病性病》
CAS
2008年第2期118-120,共3页
Chinese Journal of Aids & STD
