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目的在实时荧光定量PCR技术的基础上建立一种诊断效果较好、简便、经济的快速诊断唐氏综合征的技术。方法以D21s1411,D21s1259为检测位点,D19s222为参照位点,用SYBR GreenⅠ作为荧光染料,采用荧光定量PCR技术检测37例唐氏综合征及131例非唐氏综合征胎儿羊水标本的STR片段,分析唐氏综合征组及非唐氏综合征组的4对△Ct值。结果 1.非唐氏征组的△Ct1411及△Ct1259的均数、标准差及95%置信区间均为正值,唐氏征组的△Ct1411及△Ct1259的均数及95%置信区间均为负值;两组间95%置信区间互不重叠。2.非唐氏征组与唐氏征组位点D21s1411及D21s1259△Ct值差异均有统计学意义,分别为t=12.088,P=0.000及t=13.884,P=0.000。3.用D21s1411进行诊断时,ROC曲线下的面积为0.9291,将△Ct14110.1327为截止值时的灵敏度与特异度之和最大;用D21s1259进行诊断时,ROC曲线下的面积为0.985,将△Ct1259=-0.2016作为截止值时的灵敏度与特异度之和最大。结论 1.以D21s1411和D21s1259为检测位点,应用荧光定量PCR方法检测羊水标本的STR片段,可有效判断胎儿是否为唐氏综合征,检出率达91%以上。2.用SYBRGreenⅠ作为染料进行荧光定量PCR检测,操作步骤简单,成本较低,效果良好。
Objective To establish a rapid, simple and economical method for the diagnosis of Down’s syndrome based on real-time fluorescence quantitative PCR. Methods D21s1411 and D21s1259 were used as detection sites and D19s222 as reference site. SYBR Green Ⅰ was used as a fluorescent dye, and STR fragments of 37 Down’s syndrome and 131 non-Down’s syndrome fetal amniotic fluid samples were detected by fluorescence quantitative PCR. Four pairs of △ Ct values were analyzed in Down Syndrome group and non-Down Syndrome group. Results 1. The mean, standard deviation and 95% confidence intervals of △ Ct1411 and △ Ct1259 in non-Down’s syndrome group were all positive. The mean and 95% confidence intervals of △ Ct1411 and △ Ct1259 in Down’s syndrome group were Negative values; 95% confidence intervals between the two groups do not overlap. The differences of △ Ct values of D21s1411 and D21s1259 in non-Down’s syndrome group and Down’s syndrome group were statistically significant (t = 12.088, P = 0.000, t = 13.884, P = 0.000.3, respectively) with D21s1411 At the time of diagnosis, the area under the ROC curve was 0.9291, and the sum of the sensitivity and the specificity when △ Ct14110.1327 was the cut-off value was the largest. When D21s1259 was used for diagnosis, the area under the ROC curve was 0.985 and △ Ct1259 = -0.2016 was taken as The sum of the sensitivity and the specificity at the cut-off is the largest. Using D21s1411 and D21s1259 as detection sites, using fluorescent quantitative PCR method to detect STR fragments of amniotic fluid samples can effectively determine whether the fetus is Down Syndrome, the detection rate of 91% or more. 2. With SYBRGreen Ⅰ as a dye for quantitative PCR detection, the operation steps are simple, the cost is lower, the effect is good.