多重荧光定量PCR方法的建立及其在快速产前诊断中的应用

Establishment of multiple quantitative fluorescent polymerase chain reaction assay and its application in rapid prenatal diagnosis of common chromosome aneuploidies

目的 探讨用于快速产前诊断的多重荧光定量PCR（QF-PCR）方法的建立并评价其临床应用价值.方法 2008年5月到2009年7月在北京协和医院产前诊断中心进行产前诊断的孕妇170例,其中收集羊水123例,新鲜绒毛9例,脐血20例,自然流产绒毛18例.孕妇均为汉族,平均年龄（34.1±4.6）岁,平均孕周为（19.6±1.0）周.采用基因组DNA提取试剂盒提取羊水、绒毛及脐血标本中DNA.采用3种荧光素标记的引物,针对人类染色体中的短串联重复序列（STR）位点参照基因库（GenBank）和文献资料设计并合成20对引物,其中21号染色体6对引物,18号染色体4对引物,13号染色体4对引物,X和Y染色体1对通用引物,另有X染色体4对引物,Y染色体1对引物.设计检测方案为每份标本均进行两套8重QF-PCR（8×QF-PCR）,共检测21、18、13号染色体及性染色体各4个位点;如果无法达到诊断要求,再追加第3套4个位点.同时与染色体核型分析结果进行对照.结果 （1）核型分析：170例标本均成功进行了核型分析,其中正常核型151例（89%,151/170）,异常核型19例（11%,19/170）.（2）QF-PCR检测：170例标本中,QF-PCR成功检测167例（98%）,失败3例,QF-PCR检测均在2～3 d得出结果.QF-PCR检测结果正常134例,均与核型分析结果一致;核型分析异常的19例中,QF-PCR检测出异常18例（其中8例21三体及3例18三体）.167例QF-PCR成功检测标本中,第1套＋第2套引物组合共确诊150例（90%,150/167）,加用第3套引物组合共检测3例（2%,3/167）,另有14例不提供信息（8%,14/167）.（3）QF-PCR诊断效率：QF-PCR用于常见非整倍体异常产前诊断的敏感度为95%（18/19）,特异度为100%（134/134）,假阳性率0（0/134）,假阴性率5%（1/19）,阳性预测值为100%（18/18）,阴性预测值为99%（134/135）.（4）QF-PCR检测常染色体及性染色体结果：21号常染色体STR位点中D21S1270和D21S1411杂合度最高,性染色体中DXS8377杂合度最高,扩增较稳定.结论 多重QF-PCR技术能成功用于常见非整倍体异常的快速产前诊断,检测结果准确,适合于规模较大的产前诊断中心进行大样本检测.

Objective To establish the multiple quantitative fluorescent polymerase chain reaction （QF-PCR）assay and evaluate its clinical application in prenatal diagnosis.Methods Totally 170 samples Were collected between May 2008 and July 2009 in prenatal center of Peking Union Medical College Hospital：123 of them were amniotic fluid,9 were chofionic villous samples,20 were fetal blood and 18 were villi from aborted fetuses.All samples were from women of Han nationality,with mean age of （34.1±4.6） years old,and with mean gestational age of（19.6±1.0）weeks.Cytogenetic cultures and karyotyping were made to every sample.Genomic DNA wag extracted from the samples.The sequences of twenty short tandem repeat （STR） markers were designed according to the GenBank and references,including 6 STR markers in chromosome 21.4 in chromosome 18.4 in chromosome 13,4 in chromosome X,1 in chromosome Y and 1 universal marker in both X and Y chromosome.Each sample was amplified by two sets of multiple QF-PCR,which included 4 STR markers in each of 21,18,13 and sex chromosomes. If the result was uninformative,the third set of anotherd 4 STR markers was added. Results （ 1 ） Karyotyping. Cytogenetic analysis were made for all the 170 samples, 151 （89%） of which were normal, and 19 （11% ） were abnormal （2）QF-PCR assay. 167（98% ） samples were detected by QF-PCR. The results were obtained within 2 -3 days after sampling. 134 samples were proved normal by QF-PCR, which was consistent with karyotyping. Among the 19 abnormal karyotype samples, 18 were detected as abnormal（ eight were 21-trisomy, three were 18-trisomy）by QF-PCR. Among the 167 samples, 150（90% ） were detected using the first and second set of STR mixtures, and 3（2% ） were detected when the third set of STR was added. The remain 14（8% ） were uninformative. （3） The diagnostic efficiency of QF-PCR. The sensitivity of QF-PCR in prenatal diagnosis of common aneuploidities was 95%, the specificity, the false positive rate, the false negative rate, the positive predictive value and negative predictive value were 100% ,0,5%, 100% and 99% , respectively. （4）Autusome and sex chromosome detection by QF-PCR. Among all the STR markers,D21S1270 and D21S1411 had the highest heterozygosifies in chromosome 21, and DXS8377 had the highest in sex chromosome. The amplifications were stable. Conclusion Multiple QF-PCR assay is a valid alternative in rapid prenatal diagnosis of common chromosome aneuploidies. With high accuracy, it can be used for numerous sample test in large-scale laboratories.