中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2008年
4期
261-264
,共4页
陈凤萍%陈俐丽%沈默%陈伟%陶志华%吴秀玲%胡元平%李澄棣%陈占国%陈晓东
陳鳳萍%陳俐麗%瀋默%陳偉%陶誌華%吳秀玲%鬍元平%李澄棣%陳佔國%陳曉東
진봉평%진리려%침묵%진위%도지화%오수령%호원평%리징체%진점국%진효동
前列腺肿瘤%基因%聚合酶链反应%尿液
前列腺腫瘤%基因%聚閤酶鏈反應%尿液
전렬선종류%기인%취합매련반응%뇨액
Prostate neoplasms%Genes%Polymerase chain reaction%Urine
目的 建立双重实时荧光逆转录聚合酶链反应(RT-PCR)检测尿液DD3/PSA mRNA比值,评价其初步应用.方法 分别在前列腺特异性抗原(PSA)基因外显子1和2、差异显示编码3(DD3)基因外显子1和3之间设计一对引物和一条探针,PSA和DD3基因的TaqMan-MGB探针5'分别标记HEX和FAM荧光素,建立双重实时荧光RT-PCR检测尿液DD3/PSA mRNA比值的方法,并对方法学进行评价.对34例前列腺癌(Pca)、44例良性前列腺增生(BPH)患者前列腺按摩后尿液中的DD3/PSA mRNA比值进行检测,评价其临床应用价值.结果 扩增产物经测序证明为PSA和DD3特异性片段.以LNCaP细胞cDNA作模板,PSA mRNA和DD3 mRNA最低检测限分别为0.6细胞/反应和60细胞/反应.DD3/PSA mRNA比值批内、批间变异系数分别为3.8%～4.7%和4.1%～4.9%.Pca组尿液DD3/PSA mRNA比值明显高于BPH组(P＜0.01).尿液DD3/PSA mRNA比值诊断Pca的ROC曲线下面积(AUC)为0.746(95%CI:0.630～0.862),当截断值为0.254时其敏感度和特异度分别为64.7%和77.3%,其阳性率与临床和病理分级均无关.结论 成功建立了双重实时荧光RT-PCR检测尿液DD3/PSA mRNA比值的分子生物学诊断方法.该方法对Pca诊断具有较高特异度和敏感度,且节省操作时间、降低实验成本,有望成为Pca早期诊断的有效方法.
目的 建立雙重實時熒光逆轉錄聚閤酶鏈反應(RT-PCR)檢測尿液DD3/PSA mRNA比值,評價其初步應用.方法 分彆在前列腺特異性抗原(PSA)基因外顯子1和2、差異顯示編碼3(DD3)基因外顯子1和3之間設計一對引物和一條探針,PSA和DD3基因的TaqMan-MGB探針5'分彆標記HEX和FAM熒光素,建立雙重實時熒光RT-PCR檢測尿液DD3/PSA mRNA比值的方法,併對方法學進行評價.對34例前列腺癌(Pca)、44例良性前列腺增生(BPH)患者前列腺按摩後尿液中的DD3/PSA mRNA比值進行檢測,評價其臨床應用價值.結果 擴增產物經測序證明為PSA和DD3特異性片段.以LNCaP細胞cDNA作模闆,PSA mRNA和DD3 mRNA最低檢測限分彆為0.6細胞/反應和60細胞/反應.DD3/PSA mRNA比值批內、批間變異繫數分彆為3.8%～4.7%和4.1%～4.9%.Pca組尿液DD3/PSA mRNA比值明顯高于BPH組(P＜0.01).尿液DD3/PSA mRNA比值診斷Pca的ROC麯線下麵積(AUC)為0.746(95%CI:0.630～0.862),噹截斷值為0.254時其敏感度和特異度分彆為64.7%和77.3%,其暘性率與臨床和病理分級均無關.結論 成功建立瞭雙重實時熒光RT-PCR檢測尿液DD3/PSA mRNA比值的分子生物學診斷方法.該方法對Pca診斷具有較高特異度和敏感度,且節省操作時間、降低實驗成本,有望成為Pca早期診斷的有效方法.
목적 건립쌍중실시형광역전록취합매련반응(RT-PCR)검측뇨액DD3/PSA mRNA비치,평개기초보응용.방법 분별재전렬선특이성항원(PSA)기인외현자1화2、차이현시편마3(DD3)기인외현자1화3지간설계일대인물화일조탐침,PSA화DD3기인적TaqMan-MGB탐침5'분별표기HEX화FAM형광소,건립쌍중실시형광RT-PCR검측뇨액DD3/PSA mRNA비치적방법,병대방법학진행평개.대34례전렬선암(Pca)、44례량성전렬선증생(BPH)환자전렬선안마후뇨액중적DD3/PSA mRNA비치진행검측,평개기림상응용개치.결과 확증산물경측서증명위PSA화DD3특이성편단.이LNCaP세포cDNA작모판,PSA mRNA화DD3 mRNA최저검측한분별위0.6세포/반응화60세포/반응.DD3/PSA mRNA비치비내、비간변이계수분별위3.8%～4.7%화4.1%～4.9%.Pca조뇨액DD3/PSA mRNA비치명현고우BPH조(P＜0.01).뇨액DD3/PSA mRNA비치진단Pca적ROC곡선하면적(AUC)위0.746(95%CI:0.630～0.862),당절단치위0.254시기민감도화특이도분별위64.7%화77.3%,기양성솔여림상화병리분급균무관.결론 성공건립료쌍중실시형광RT-PCR검측뇨액DD3/PSA mRNA비치적분자생물학진단방법.해방법대Pca진단구유교고특이도화민감도,차절성조작시간、강저실험성본,유망성위Pca조기진단적유효방법.
Objective To establish a duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio,and to evaluate its effect in diagnosis of prostate cancer (PCa).Methods Urine samples were obtained from 34 patients with PCa and 44 patients with benign prostate hypertrophy (BPH) by prostate massage.A duplex TaqMan RT-PCR assay was developed:PCR primers were designed to amplify the fragments between the exon1 and exon2 in the PSA mRNA and between the exon1 and exon3 in the DD3 mRNA.and the PCR TaqMan-MGB probes were labeled with HEX and FAM respectively in 5'for PSA mRNA.LNCaP cells were used as template.DD3/PSA mRNA ratio was measured.Receiver operating characteristic curve (ROC) was drawn so as to evaluate its diagnostic efficacy.Results Sequencing showed that the PCR products were specific for PSA mRNA and DD3 mRNA.The minimum detection level was approximately 0.6 cells/reaction for PSA mRNA and was 60 cells/reaction for DD3 mRNA in the LNCaP cell cDNA.The intra-and inter-assay coefficients of variation of DD3/PSA mRNA were 3.8%～4.7% and 4.1%～4.9% respectively.Urine DD3/PSA mRNA ratio in PCa group was significantly higher than the BPH group(P＜0.01).When the cutoff value was defined as 0.254,the area under curve (AUC) of DD3/PSA mRNA ratio was 0.746(95% CI:0.630～0.862).and the sensitivity and specificity were 64.7% and 77.3% respectively.The urine DD3/PSA mRNA ratio positive rate was not correlated with clinical and pathological parameters.Conclusion A duplex TaqMan RT-PCR assay for urine DD3/PSA mRNA ratio has been established with an excellent clinical performance and specificity for PCa,saving time and reducing costs.It may be a promising method in the early diagnosis of PCa.
