中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2012年
11期
1000-1005
,共6页
李怡%刘国彦%张换敬%郑立谋%曾骥孟
李怡%劉國彥%張換敬%鄭立謀%曾驥孟
리이%류국언%장환경%정립모%증기맹
乳腺肿瘤%基因,erbB-2%受体,erbB-2%聚合酶链反应
乳腺腫瘤%基因,erbB-2%受體,erbB-2%聚閤酶鏈反應
유선종류%기인,erbB-2%수체,erbB-2%취합매련반응
Breast neoplasms%Genes,erbB-2%Receptor,erbB-2%Polymerase chain reaction
目的 研究建立一种基于实时荧光定量PCR技术和新型环状引物的简捷、准确、廉价、易于标准化检测乳腺癌组织人表皮生长因子受体2(HER2)基因mRNA表达水平的方法,为临床上肿瘤个性化分子靶向药物治疗提供用药指导.方法 设计HER2和内参基因的新型特异性环状引物,通过Excel在乳腺癌组织标本中随机抽取5份标本检测候选内参基因的表达,同时用geNorm、NormFinder和BestKeeper软件筛选和评估乳腺癌组织中稳定表达的内参基因,设置Mg2+浓度和引物浓度梯度对PCR体系进行优化,建立乳腺癌HER2基因mRNA表达水平的新型环状引物Eva Green染料实时荧光定量逆转录(FQ RT)-PCR检测方法(以下简称“自建FQ RT-PCR”法),并对其灵敏度、特异性、稳定性进行评价；同时用自建FQ RT-PCR法与免疫组织化学(IHC)法平行检测2008至2012年厦门大学附属中山医院普外科收集的55份乳腺癌组织和其中32份同一患者匹配的正常组织(距癌组织≥5 cm)中HER2基因的表达水平,并评价自建FQ RT-PCR法对乳腺癌的诊断效能.结果 自建FQ RT-PCR法对HER2基因和核糖体蛋白L37a(RPL37A)基因的最低检测限均为101拷贝/μl,线性范围均为101～ 106拷贝/μl(r=0.997)；HER2和RPL37A基因的高、低浓度标准品(106拷贝/μl和101拷贝/μl)批内变异系数(CV)分别为(5.93±0.57)％和(5.11±0.59)％、(2.49±0.81)％和(2.98±0.97)％；批间CV为(5.76±0.58)％和(7.71±0.61)％、(3.75±0.76)％和(4.40±0.96)％.FQ RT-PCR法与IHC法平行检测87份乳腺癌组织标本HER2基因表达水平,FQ RT-PCR法的敏感度为96.36％(53/55),特异度为78.13％ (25/32),阳性预测值为88.33％ (53/60),阴性预测值为92.59％ (25/27),与IHC检测结果总符合率为89.66％(78/87).且与HIC法具有较高的一致性(Kappa=0.770,P＞0.05).结论 成功建立了FQ RT-PCR检测乳腺癌HER2基因mRNA表达水平的方法,该方法敏感度高、特异度好且快速、价廉,适合临床检验实验室进行肿瘤标本的HER2基因表达检测.
目的 研究建立一種基于實時熒光定量PCR技術和新型環狀引物的簡捷、準確、廉價、易于標準化檢測乳腺癌組織人錶皮生長因子受體2(HER2)基因mRNA錶達水平的方法,為臨床上腫瘤箇性化分子靶嚮藥物治療提供用藥指導.方法 設計HER2和內參基因的新型特異性環狀引物,通過Excel在乳腺癌組織標本中隨機抽取5份標本檢測候選內參基因的錶達,同時用geNorm、NormFinder和BestKeeper軟件篩選和評估乳腺癌組織中穩定錶達的內參基因,設置Mg2+濃度和引物濃度梯度對PCR體繫進行優化,建立乳腺癌HER2基因mRNA錶達水平的新型環狀引物Eva Green染料實時熒光定量逆轉錄(FQ RT)-PCR檢測方法(以下簡稱“自建FQ RT-PCR”法),併對其靈敏度、特異性、穩定性進行評價；同時用自建FQ RT-PCR法與免疫組織化學(IHC)法平行檢測2008至2012年廈門大學附屬中山醫院普外科收集的55份乳腺癌組織和其中32份同一患者匹配的正常組織(距癌組織≥5 cm)中HER2基因的錶達水平,併評價自建FQ RT-PCR法對乳腺癌的診斷效能.結果 自建FQ RT-PCR法對HER2基因和覈糖體蛋白L37a(RPL37A)基因的最低檢測限均為101拷貝/μl,線性範圍均為101～ 106拷貝/μl(r=0.997)；HER2和RPL37A基因的高、低濃度標準品(106拷貝/μl和101拷貝/μl)批內變異繫數(CV)分彆為(5.93±0.57)％和(5.11±0.59)％、(2.49±0.81)％和(2.98±0.97)％；批間CV為(5.76±0.58)％和(7.71±0.61)％、(3.75±0.76)％和(4.40±0.96)％.FQ RT-PCR法與IHC法平行檢測87份乳腺癌組織標本HER2基因錶達水平,FQ RT-PCR法的敏感度為96.36％(53/55),特異度為78.13％ (25/32),暘性預測值為88.33％ (53/60),陰性預測值為92.59％ (25/27),與IHC檢測結果總符閤率為89.66％(78/87).且與HIC法具有較高的一緻性(Kappa=0.770,P＞0.05).結論 成功建立瞭FQ RT-PCR檢測乳腺癌HER2基因mRNA錶達水平的方法,該方法敏感度高、特異度好且快速、價廉,適閤臨床檢驗實驗室進行腫瘤標本的HER2基因錶達檢測.
목적 연구건립일충기우실시형광정량PCR기술화신형배상인물적간첩、준학、렴개、역우표준화검측유선암조직인표피생장인자수체2(HER2)기인mRNA표체수평적방법,위림상상종류개성화분자파향약물치료제공용약지도.방법 설계HER2화내삼기인적신형특이성배상인물,통과Excel재유선암조직표본중수궤추취5빈표본검측후선내삼기인적표체,동시용geNorm、NormFinder화BestKeeper연건사선화평고유선암조직중은정표체적내삼기인,설치Mg2+농도화인물농도제도대PCR체계진행우화,건립유선암HER2기인mRNA표체수평적신형배상인물Eva Green염료실시형광정량역전록(FQ RT)-PCR검측방법(이하간칭“자건FQ RT-PCR”법),병대기령민도、특이성、은정성진행평개；동시용자건FQ RT-PCR법여면역조직화학(IHC)법평행검측2008지2012년하문대학부속중산의원보외과수집적55빈유선암조직화기중32빈동일환자필배적정상조직(거암조직≥5 cm)중HER2기인적표체수평,병평개자건FQ RT-PCR법대유선암적진단효능.결과 자건FQ RT-PCR법대HER2기인화핵당체단백L37a(RPL37A)기인적최저검측한균위101고패/μl,선성범위균위101～ 106고패/μl(r=0.997)；HER2화RPL37A기인적고、저농도표준품(106고패/μl화101고패/μl)비내변이계수(CV)분별위(5.93±0.57)％화(5.11±0.59)％、(2.49±0.81)％화(2.98±0.97)％；비간CV위(5.76±0.58)％화(7.71±0.61)％、(3.75±0.76)％화(4.40±0.96)％.FQ RT-PCR법여IHC법평행검측87빈유선암조직표본HER2기인표체수평,FQ RT-PCR법적민감도위96.36％(53/55),특이도위78.13％ (25/32),양성예측치위88.33％ (53/60),음성예측치위92.59％ (25/27),여IHC검측결과총부합솔위89.66％(78/87).차여HIC법구유교고적일치성(Kappa=0.770,P＞0.05).결론 성공건립료FQ RT-PCR검측유선암HER2기인mRNA표체수평적방법,해방법민감도고、특이도호차쾌속、개렴,괄합림상검험실험실진행종류표본적HER2기인표체검측.
Objective Based on real-time PCR technique and ring primers,to establish a simple,accurate,cost-effective and easily standardized quantitative assay for quantification of HER2 mRNA,and apply to provide medication guidance for clinical tumor personalized molecular targeted therapy.Methods Screening reference gene which was stable expression in breast cancer,and optimizing the PCR reaction system.Then a real-time PCR with Eva Green for quantification of the mRNA expression levels of HER2 gene was developed.The specificity,sensitivity and reproducibility of the method were evaluated 87 specimens including 55 liquid nitrogen-frozen breast cancer tissues and 32 normal tissues were detected by the real-time quantitative reverse transcription (FQ RT)-PCR and immunohistochemistry(IHC).Results The standard curve of the method indicated a good linear relationship between the Ct value and the template concentration with the correlation coefficient being 0.997.The linear range of the system was from 101 to 106 copies/μl and the lower detection limit was 101 copies/μl.It had a high sensitivity and good specificity.The inter-assay coefficients of variation of HER and RPL37A genes were (5.93 ± 0.57)％ and (5.11 ± 0.59)％,(2.49 ±0.81)％ and (2.98 ±0.97)％ respectively.The intra-assay coefficients of variation were (5.76 ±0.58)％and (7.71 ±0.61)％,(3.75 ±0.76)％ and (4.40 ±0.96)％ respectively.Using the optimized FQ RTPCR system,HER2 gene of 87 specimens was quantificated.The sensitivity of the assay was 96.36％ (53/55),the specificity was 78.13％ (25/32),the positive predictive value was 88.33％ (53/60),the negative predictive value was 92.59％ (25/27),and the total coincidence rate between FQ RT-PCR and IHC was 89.66％ (78/87).The correlation of the results between the FQ RT-PCR and IHC was good (Kappa =0.770,P ＞ 0.05).Conclusions The method can quantify the mRNA expression levels of HER2 gene rapidly and cost-effictively with high sensitivity and specificity.It can be applied to clinical molecular diagnosis with attractive prospect.
