IKCA基因項(xiàng)目立項(xiàng)

PIK3CA、TP53基因項(xiàng)目立項(xiàng)報(bào)告分子診斷部徐正紅20150302PIK3CA基因項(xiàng)目TP53基因項(xiàng)目技術(shù)方法風(fēng)險(xiǎn)評(píng)估一.PIK3CA基因項(xiàng)目1.項(xiàng)目背景吉非替尼厄洛替尼西妥昔單抗帕尼單抗FDA要求對(duì)擬采用易瑞沙、特羅凱等EGFR-TKI治療的患者,進(jìn)行EGFR基因突變檢測(cè)FDA要求使用西妥昔單抗、帕尼單抗治療結(jié)直腸癌前，必須進(jìn)行KRAS基因檢測(cè)耐藥PIK3CA基因定位于3q26.3，長(zhǎng)34kb，包含21個(gè)外顯子，編碼1068種氨基酸，該組氨基酸產(chǎn)生一組長(zhǎng)124kD的蛋白。PIK3CA編碼I類(lèi)磷脂酰肌醇-3-激酶(phosphatidylinositol3-kinases,PI3Ks)的p110催化亞單位，即PI3Kp110a。I類(lèi)PI3K為異源二聚體，由一個(gè)調(diào)節(jié)亞基（p85）和一個(gè)催化亞基（p110）組成。1.項(xiàng)目背景PIK3CA的突變約4/5發(fā)生在螺旋區(qū)(exon9)和激酶區(qū)(exon20)這兩個(gè)熱點(diǎn)區(qū)域。PIK3CA基因突變引起p110a異常激活，進(jìn)而活化PI3Ks，激活下游Akt等信號(hào)分子，激活或抑制其下游靶蛋白進(jìn)而調(diào)節(jié)細(xì)胞的增殖、分化、凋亡以及遷移等生物學(xué)效應(yīng)，最終導(dǎo)致腫瘤的發(fā)生。1.項(xiàng)目背景CFDA注冊(cè)產(chǎn)品：產(chǎn)品名稱生產(chǎn)單位批準(zhǔn)日期人PIK3CA基因突變檢測(cè)試劑盒（熒光PCR法）北京雅康博生物科技有限公司2014.06.17人類(lèi)PIK3CA基因突變檢測(cè)試劑盒（流式熒光雜交法）益善生物技術(shù)股份有限公司2014.07.07人類(lèi)PIK3CA基因突變檢測(cè)試劑盒（熒光PCR法）廈門(mén)艾德生物醫(yī)藥科技有限公司2013.04.021.項(xiàng)目背景其他的PIK3CA基因突變檢測(cè)試劑盒：產(chǎn)品名稱公司一種PIK3CA基因突變熒光定量PCR分型檢測(cè)試劑盒及檢測(cè)方法廣州達(dá)健一種檢測(cè)人PIK3CA基因突變的方法和試劑盒蘇州科貝用于檢測(cè)直腸癌PIK3CA基因熱點(diǎn)突變位點(diǎn)杭州艾迪康醫(yī)學(xué)檢驗(yàn)PIK3CA

基因突變檢測(cè)試劑盒(PCR-熒光探針?lè)?北京鑫諾美迪基因檢測(cè)技術(shù)有限公司1.項(xiàng)目背景國(guó)內(nèi)相關(guān)專(zhuān)利：專(zhuān)利名稱申請(qǐng)（專(zhuān)利權(quán)）人申請(qǐng)?zhí)柣蚴跈?quán)公告號(hào)PIK3CA基因驅(qū)動(dòng)突變的檢測(cè)探針、引物及試劑盒廈門(mén)艾德生物醫(yī)藥科技有限公司CN102453765BPIK3CAH1047R敲入非人動(dòng)物乳腺癌模型霍夫曼-拉羅奇有限公司CN201280046858一種PIK3CA基因突變檢測(cè)液相芯片廣州益善生物技術(shù)股份有限公司CN201010160769PIK3CA基因突變的檢測(cè)探針、液相芯片及其檢測(cè)方法廣州益善生物技術(shù)股份有限公司CN101445832B一種PIK3CA基因突變熒光定量PCR檢測(cè)試劑盒及檢測(cè)方法寧波有成生物醫(yī)藥科技有限公司CN201310260412一種用于檢測(cè)PIK3CA基因突變的試劑盒武漢友芝友醫(yī)療科技有限公司CN201410734805引物及該引物質(zhì)譜檢測(cè)PIK3CA基因熱點(diǎn)突變的方法武漢康圣達(dá)醫(yī)學(xué)檢驗(yàn)所有限公司CN201210574584一種PIK3CA基因突變熒光定量PCR分型檢測(cè)試劑盒及其檢測(cè)方法廣州達(dá)健生物科技有限公司

CN201210330990用于檢測(cè)結(jié)直腸癌PIK3CA基因熱點(diǎn)突變位點(diǎn)的試劑盒杭州艾迪康醫(yī)學(xué)檢驗(yàn)中心有限公司

CN201210234133一種檢測(cè)人PIK3CA基因突變的方法和試劑盒蘇州科貝生物技術(shù)有限公司

CN2010106085001.項(xiàng)目背景cancer%PIK3CAmutationSamplesource(primarytissue

vscancercellline)ExonmutatedFunctionaldomainReferenceLiver35.6(26/73)Primary9and20HelicalandkinaseLee

etal

(2004)Totalliver36%(26/73)

Breast33.3(4/12)Celllines9and20HelicalandkinaseBachman

etal(2004)Breast21.4(9/42)Primary1,9and20p85,helicalandkinaseBachman

etal(2004)Breast18.1(13/72)Primary9and20HelicalandkinaseLevine

etal(2005)Breast40.0(28/70)Primary6,7,9and20C2,helicalandkinaseCampbell

etal(2004)Breast20.7(19/92)Primary9and20HelicalandkinaseWu

etal

(2005a)Breast8.3(1/12)Primary20kinaseSamuels

etal(2004)Breast33.3(5/15)Celllines9and20HelicalandkinaseWu

etal

(2005a)Breast26.9(25/93)Primary9and20HelicalandkinaseLee

etal

(2004)Breast28.0(14/50)Celllines1,9and20p85,helicalandkinaseSaal

etal

(2005)Breast26.4(77/292)Primary1,4,7,9,13,18,20p85,C2,helicalandkinaseSaal

etal

(2005)Totalbreast26%(195/750)

Colon31.6(74/234)Primary1,2,4,7,9,18and20P85,C2,helicalandKinaseSamuels

etal(2004)Colon13.6(14/103)Primary9and20HelicalandkinaseVelho

etal(2005)Colon18.8(6/32)Primary9and20HelicalandkinaseCampbell

etal(2004)Totalcolon25%(94/369)2.臨床意義---突變率Ovarian12.1(24/198)Primary9and20HelicalandkinaseLevine

etal(2005)Ovarian6.0(11/182)Primary9and20HelicalandkinaseCampbell

etal(2004)Totalovarian9%(35/380)Gastric25.0(3/12)Primary18and20KinaseSamuels

etal(2004)Gastric10.6(5/47)Primary9and20HelicalandkinaseVelho

etal(2005)Gastric6.5(12/185)Primary9and20HelicalandkinaseLee

etal

(2004)Gastric4.3(4/94)Primary9and20HelicalandkinaseLi

etal

(2005)Totalgastric7%(24/338)

Brain26.7(4/15)Primary4,5and13C2andhelicalSamuels

etal(2004)Brain4.6(13/285)Primary9and20HelicalandkinaseBroderick

etal(2004)Totalbrain6%(17/300)

Lung1.3(3/229)Primary9and20HelicalandkinaseLee

etal

(2004)Lung4.2(1/24)Primary9HelicalSamuels

etal(2004)Totallung2%(4/253)

Leukaemia1.1(1/88)Primary9HelicalLee

etal

(2004)Totalleukaemia1%(1/88)Totalcancersreported15%(382/2551)[*]2.臨床意義義---突變率藥物檢測(cè)項(xiàng)目檢測(cè)意義易瑞沙特羅凱EGFR基因突變預(yù)測(cè)療效KRAS基因突變預(yù)測(cè)療效西妥昔單抗帕尼單抗KRAS基因突變預(yù)測(cè)療效BRAF基因突變預(yù)測(cè)療效威羅菲尼BRAF基因突變預(yù)測(cè)療效易瑞沙特羅凱西妥昔單抗帕尼單抗PIK3CA基因突變耐藥相關(guān)2.臨床意義義---耐藥PI3K作為EGFR下游信信號(hào)分子子被激活活，可導(dǎo)導(dǎo)致腫瘤瘤細(xì)胞對(duì)對(duì)EGFR-TKI藥藥物的耐耐藥，例例如PIK3CA基因的突突變可導(dǎo)導(dǎo)致西妥昔單單抗（愛(ài)愛(ài)必妥）），帕尼尼單抗（（維克替替比）對(duì)對(duì)轉(zhuǎn)移性性結(jié)直腸腸癌治療療的耐受受，導(dǎo)致致吉非替替尼，厄厄洛替尼尼對(duì)非小小細(xì)胞肺肺癌和食食道癌晚晚期患者者的治療療耐受[1-4]。K-ras，BRAF和PIK3CA基因因突變率率約占結(jié)結(jié)直腸癌癌總體患患者的56％。。KRAS，BRAF，PIK3CA任何何一個(gè)或或多個(gè)突突變對(duì)個(gè)個(gè)體化靶靶向藥物物西妥昔單單抗（愛(ài)愛(ài)必妥）），帕尼單抗抗（維克克替比））治療無(wú)效效，全部部為野生生型治療療有效[4]。PIK3CA突突變對(duì)于于乳腺癌癌個(gè)體化化靶向藥藥物曲妥珠單單抗（赫赫賽汀））治療無(wú)效效，野生生型治療療有效[5-6]。2.臨床意義義---耐藥COSMICIDNMAAChange突變率775c.3140A>Gp.H1047R80%Commonmutations776c.3140A>Tp.H1047L760c.1624G>Ap.E542K763c.1633G>Ap.E545K765c.1635G>Tp.E545D3.項(xiàng)目?jī)?nèi)容容PIK3CAmutationorgeneamplificationwasdetectedin30.5%ofallovariancancersand45%oftheendometrioidandclearcellsubtypes,andE542K,E545K,E545D,H1047RandH1047Lmutationsweredetected[7].PIK3CAE542K,E545K,andE545Dmutationinexon9,H1047RandH1047Lmutationinexon20arethecommonmutations,andmaybeassociatedwiththeefficacyofEGFR-TKItherapy[8-9].Mostofthemutationsoccurattwohotspots,namelyE545KinthehelicaldomainandH1047Rinthecatalyticdomain,andE545KH1047Rareassociatedwithinvasionandmetastasis[10-12].continuedactivationofPI3KsignalingbythePIK3CAoncogenicmutant,p110alphaE545K,wassufficienttoabrogategefitinib-inducedapoptosis.

[13]PIK3CAE542KmayaffecttheefficacyofEGFR-TKItherapyinlungcancer[14].3.項(xiàng)目?jī)?nèi)容引文[1]Sartore-BianchiA,MartiniM,MolinariF,etal.PIK3CAmutationsincolorectalcancerareassociatedwithclinicalresistancetoEGFR-targetedmonoclonalantibodies[J].Cancerresearch,2009,69(5):1851-1857[2]PaezJG,J?nnePA,LeeJC,etal.EGFRmutationsinlungcancer:correlationwithclinicalresponsetogefitinibtherapy[J].Science,2004,304(5676):1497-1500.[3]JanmaatML,Gallegos-RuizMI,RodriguezJA,etal.PredictivefactorsforoutcomeinaphaseIIstudyofgefitinibinsecond-linetreatmentofadvancedesophagealcancerpatients[J].JournalofClinicalOncology,2006,24(10):1612-1619.[4]FidlerMJ,MorrisonLE,BasuS,etal.PTENandPIK3CAgenecopynumbersandpooroutcomesinnon-smallcelllungcancerpatientswithgefitinibtherapy[J].Britishjournalofcancer,2011,105(12):1920-1926.[5]SuetaA,

YamamotoY,

Yamamoto-IbusukiM.Anintegrativeanalysisof

PIK3CA

mutation,PTEN,andINPP4Bexpressionintermsof

trastuzumab

efficacyinHER2-positivebreastcancer.PLoSOne.

2014Dec26;9(12):e116054.doi:10.1371/journal.pone.0116054.eCollection2014.[6]YuanK,WuH,WangY,etal.Phospho-PRAS40Thr246predictstrastuzumabresponseinpatientswithHER2-positivemetastaticbreastcancer[J].OncologyLetters,2015,9(2):785-789.[7]CampbellIG,RussellSE,ChoongDYH,etal.[J].Cancerresearch,2004,64(21):7678-7681.MutationofthePIK3CAgeneinovarianandbreastcancer[8]BroderickDK,DiC,ParrettTJ,etal.MutationsofPIK3CAinanaplasticoligodendrogliomas,high-gradeastrocytomas,andmedulloblastomas[J].Cancerresearch,2004,64(15):5048-5050.[9]ZhangL,ShiL,ZhaoX,etal.PIK3CAgenemutationassociatedwithpoorprognosisoflungadenocarcinoma[J].OncoTargetsandtherapy,2013,6:497.[10]YamaguchiH,YoshidaS,MuroiE,etal.Phosphoinositide3-kinasesignalingpathwaymediatedbyp110αregulatesinvadopodiaformation[J].TheJournalofcellbiology,2011,193(7):1275-1288.[11]Isakoff,

S.J.,

J.A.

Engelman,

H.Y.

Irie,

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Luo,

S.M.

Brachmann,

R.V.

Pearline,

L.C.

Cantley,J.S.

Brugge.

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Breastcancer-associatedPIK3CAmutationsareoncogenicinmammaryepithelialcells.

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65:10992–11000.

doi:10.1158/0008-5472.CAN-05-2612[12]

Kang,

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102:802807.doi:10.1073/pnas.0408864102[13]EngelmanJA,MukoharaT,ZejnullahuK,etal.AllelicdilutionobscuresdetectionofabiologicallysignificantresistancemutationinEGFR-amplifiedlungcancer[J].JournalofClinicalInvestigation,2006,116(10):2695.[14]HanJY,KimSH,LeeYS,etal.Comparisonoftargetednext-generationsequencingwithconventionalsequencingforpredictingtheresponsivenesstoepidermalgrowthfactorreceptor-tyrosinekinaseinhibitor(EGFR-TKI)therapyinnever-smokerswithlungadenocarcinoma[J].LungCancer,2014,85(2):161-167.3.項(xiàng)目?jī)?nèi)容二.TP53基因項(xiàng)目TP53(tumorprotein53)基因定位位于17p13.1，約20kb長(zhǎng)，由11個(gè)外顯子和和10個(gè)內(nèi)含子組組成。編碼碼393個(gè)氨基酸蛋蛋白，分子子量53kD，是一種半半衰期短的的核內(nèi)磷酸酸化蛋白，，能通過(guò)自自身修飾或或和其他蛋蛋白相互作作用來(lái)調(diào)節(jié)節(jié)細(xì)胞周期期及細(xì)胞凋凋亡。1.項(xiàng)目背景當(dāng)細(xì)胞在正正常狀態(tài)時(shí)時(shí)，TP53維持一個(gè)較較低的蛋白白水平，不不干擾正常常細(xì)胞的運(yùn)運(yùn)行，當(dāng)細(xì)細(xì)胞在有損損傷或在應(yīng)應(yīng)激情況下下，TP53蛋白的水平平迅速升高高，使之很很快的激活活并啟動(dòng)對(duì)對(duì)正常細(xì)胞胞的修復(fù)功功能。而當(dāng)當(dāng)細(xì)胞DNA損傷嚴(yán)重而而不能完全全修復(fù)時(shí)，，TP53蛋白將引發(fā)發(fā)細(xì)胞凋亡亡，發(fā)揮其其抑制細(xì)胞胞生長(zhǎng)的作作用?！盎蚪M衛(wèi)衛(wèi)士”1.項(xiàng)目背景CFDA注冊(cè)產(chǎn)品：：無(wú)產(chǎn)品名稱生產(chǎn)單位TP53基因6種突變檢測(cè)試劑盒（僅供科研使用）廈門(mén)艾德生物醫(yī)藥科技有限公司人腫瘤蛋白p53(TP53)檢測(cè)試劑盒上海信裕生物技術(shù)有限公司科研類(lèi)產(chǎn)品品：1.項(xiàng)目背景相關(guān)專(zhuān)利：：發(fā)明名稱申請(qǐng)（專(zhuān)利權(quán)）人申請(qǐng)/公開(kāi)號(hào)一種檢測(cè)P53基因SNP位點(diǎn)的試劑及其應(yīng)用中國(guó)醫(yī)學(xué)科學(xué)院腫瘤醫(yī)院CN104131101Ap53基因突變的快速檢測(cè)方法上海賽安生物醫(yī)藥科技有限公司CN103436593A一種檢測(cè)P53基因突變的引物和探針遼寧琦潤(rùn)生物科技有限公司CN102533957B基于納米探針直接檢測(cè)肺癌樣品中P53基因突變的方法中國(guó)科學(xué)院上海微系統(tǒng)與信息技術(shù)研究所CN101392286A一種鑒定外周血DNA肝癌相關(guān)P53基因249位密碼子突變的多重PCR的試劑盒四川大學(xué)華西醫(yī)院CN101067153A1.項(xiàng)目背景-相關(guān)產(chǎn)品專(zhuān)專(zhuān)利TP53是迄今發(fā)現(xiàn)現(xiàn)與人類(lèi)腫腫瘤相關(guān)性性最高的基基因（50%以上人類(lèi)腫腫瘤中發(fā)生TP53突變），同同時(shí)TP53的突變率在在所有的腫腫瘤中也是是最高的，，尤其是在在上皮組織織發(fā)生的腫腫瘤中，TP53的突變率可可以高達(dá)50%-90%。野生型TP53能夠抑制多多藥耐藥蛋蛋白基因轉(zhuǎn)轉(zhuǎn)錄，減少少多藥耐藥藥蛋白生成成。突變型的TP53是一種腫瘤瘤促進(jìn)因子子，可引起起腫瘤形成成或細(xì)胞轉(zhuǎn)轉(zhuǎn)化，增強(qiáng)多藥耐耐藥基因表表達(dá)。TP53變異與腫瘤瘤細(xì)胞對(duì)鉑類(lèi)化療藥的耐耐藥性相關(guān)關(guān)，但不影影響紫杉醇類(lèi)藥的敏感性性，所以TP53作為一個(gè)新新的耐藥基基因，其突突變檢測(cè)可可用于指導(dǎo)導(dǎo)臨床腫瘤瘤患者化療療的個(gè)體化化用藥。臨床意義鉑類(lèi)藥的作作用機(jī)理：：作用于DNA，引起DNA復(fù)制障礙，，從而抑制制癌細(xì)胞的的分裂。與與其他抗癌癌藥一樣，，鉑類(lèi)抗癌癌藥影響DNA合成的作用用是非特異異性的。但但腫瘤細(xì)胞胞比正常細(xì)細(xì)胞增殖快快，合成DNA迅速，并且且DNA受損后的修修復(fù)功能不不完善，因因此，腫瘤瘤細(xì)胞對(duì)抗抗癌藥的細(xì)細(xì)胞毒作用用更為敏感感，從而顯顯示出藥物物的抗癌作作用。紫杉醇類(lèi)藥藥物作用機(jī)機(jī)理：抗微管藥物物,通過(guò)促進(jìn)微微管蛋白聚聚合抑制解解聚,保持微管蛋蛋白穩(wěn)定,抑制細(xì)胞有有絲分裂。。體外實(shí)驗(yàn)驗(yàn)證明紫杉杉醇具有顯顯著的放射射增敏作用用，可能是是使細(xì)胞中中止于對(duì)放放療敏感的的G2和M期。臨床意義COSMICIDNMAAChangeIARCTP53Database:Numberofreports10648c.524G>Ap.R175H1210Hotspots;commonmutations;themostfrequentmutations10656c.742C>Tp.R248W73410662

c.743G>Ap.R248Q93310659c.817C>Tp.R273C70610660c.818G>Ap.R273H851項(xiàng)目?jī)?nèi)容Codons248,273,and175aretheTP53mutationhotspotsfoundinmosthumancancers.Mostmutationsathotspots,includingR248W,R248Q,R273C,R273H,R175H,arenonfunctional[1].p53mutantssuchasR248WandR273HcanbindtheMre11-Rad5-NBS1(MRN)complexandinterferewithitsabilitytorecruittheataxia–telangiectasia-mutated(ATM)kinasetoDNAdouble-strandbreaks,ultimatelycausinggeneticinstability[2].p53His175andp53His273exertedverysimilareffectsonthecellularresponsetocisplatin;bothconferredincreasedresistancetolowconcentrationsofthedrug[3].Theexpressionofallformsofmutantp53proteinexceptp53His273enhancedsensitivitytocisplatinanddoxorubicin[4].R175HandR273H,exhibitsignificantlygreaterresistancetoanumberofantitumordrugs,includingdoxorubicin,cisplatin,etoposide,and5-FU[5].Reconstitutionofamutantp53(R248W)inthesecellsinhibitedthesensitivitytocisplatintreatment[6].ThetransformantofR248Qmutationgainedhigheractivityofinvasion,whileitsanti-cancerdrugsensitivityalsoincreased[7].R273Hand

R273Cincreaseresistancetocisplatintreatment[8].項(xiàng)目?jī)?nèi)容Q:廈門(mén)艾德的的TP53基因檢測(cè)試試劑盒可以以6種突變（R175H,R175C,R248W,R248Q,R273H,R273C)，為什么我我們只檢測(cè)測(cè)5種突變，不不檢測(cè)R175C突變點(diǎn)？A：R175C突變?yōu)榉菬釤狳c(diǎn)突變，，其突變蛋蛋白具有部部分功能，，且無(wú)文獻(xiàn)獻(xiàn)支持該突突變點(diǎn)與藥藥物的相關(guān)關(guān)作用關(guān)系系。項(xiàng)目?jī)?nèi)容ThemutantCys175exhibitswild-typeproperties.ItisonlyrecognizedbyPAb1620anddoesnotbindtohsp7O.Indeed,italsobehavesasawild-typeintransactivationassay[9].InvitroevidenceforsuchdifferenceswasprovidedbythesystematicmutagenesisoftheR175hotspotcodoninhumanp53:theR175Cmutantwaswild-typeinitsphenotype[1,10].FunctionalclassificationbasedontheoveralltranscriptionalactivityofR175CisclassifiedaspartiallyfunctionalinIARCTP53database.R175Cisafalse-positivedetection,andthep.R175Cmutant(c.523C>T)isnotimpairedforanyTP53function.Consideringthisbodyofevidence,wehypothesizethatp.R175Cmaybeapassengermutationcoselectedduringneoplastictransformation[11].項(xiàng)目?jī)?nèi)容[1]Xu-MonetteZY,WuL,ViscoC,etal.MutationalprofileandprognosticsignificanceofTP53indiffuselargeB-celllymphomapatientstreatedwithR-CHOP:reportfromanInternationalDLBCLRituximab-CHOPConsortiumProgramStudy[J].Blood,2012,120(19):3986-3996.[2]SongH,HollsteinM,XuY.

p53gain-of-functioncancermutantsinducegeneticinstabilitybyinactivatingATM.NatureCellBiol2007;

9:573––580[3]BlandinoG,LevineAJ,OrenM.Mutantp53gainoffunction:differentialeffectsofdifferentp53mutantsonresistanceofculturedcellstochemotherapy[J].Oncogene,1999,18(2):477-485.[4]CHANGFULIN,LAIMD.Variousformsofmutantp53confersensitivitytocisplatinanddoxorubicininbladdercancercells[J].TheJournalofurology,2001,166(1):304-310.[5]Martinez-RiveraM,SiddikZH.Resistanceandgain-of-resistancephenotypesincancersharboringwild-typep53[J].Biochemicalpharmacology,2012,83(8):1049-1062.[6]LiuK,LingS,LinWC.TopBP1mediatesmutantp53gainoffunctionthroughNF-Yandp63/p73[J].Molecularandcellularbiology,2011,31(22):4464-4481.[7].KamiyaY,OhshimaT.Theindividualcellpropertiesoforalsquamouscellcarcinomaandp53tumorsuppressorgenemutation[J].OralScienceInternational,2005,2(2):104-117.[8]LiJ,YangL,GaurS,etal.MutantsTP53p.R273Handp.R273Cbutnotp.R273GEnhanceCancerCellMalignancy[J].Humanmutation,2014,35(5):575-584.[9]GohAM,CoffillCR,LaneDP.Theroleofmutantp53inhumancancer[J].TheJournalofpathology,2011,223(2):116-126.[10]LeroyB,AndersonM,SoussiT.TP53mutationsinhumancancer:databasereassessmentandprospectsforthenextdecade[J].Humanmutation,2014,35(6):672-688.[11]OryK,LegrosY,AuguinC,etal.Analysisofthemostrepresentativetumour-derivedp53mutantsrevealsthatchangesinproteinconformationarenotcorrelatedwithlossoftransactivationorinhibitionofcellproliferation[J].TheEMBOjournal,1994,13(15):3496.項(xiàng)目?jī)?nèi)容引文三.技術(shù)方法PCR技術(shù)：castPCR?technologyTaqMan?MutationDetectionAssays技術(shù)原理Highspecificity—mutantalleledetectionisbasedonanallele-specificprimer,whilewildtypebackgroundissuppressedbytheproprietaryMGBblockeroligonucleotideHighsensitivity—assayscandetectdownto0.1%mutationinabackgroundofwildtypeDNA,asdemonstratedinspikingexperimentsWidedynamicrangeandexcellentPCRefficiency—assaysdemonstrateatleast4logsofdynamicrangeandanaveragePCRefficiencyof100%±10%Fast,simplework?ow—likeotherTaqMan?Assays,typicallyrequires3hoursfromsampletoresults,withminimumhands-ontime技術(shù)優(yōu)勢(shì)樣本類(lèi)型：：gDNA,FFPEtissues,freshfrozentissues,celllines適合機(jī)型：：樣本和機(jī)型型時(shí)間4月5-7月8-10月11-12月內(nèi)容采購(gòu)相關(guān)試劑，質(zhì)粒，開(kāi)始預(yù)實(shí)驗(yàn)反應(yīng)體系優(yōu)化，靈敏度，精密度，穩(wěn)定性等試驗(yàn)臨床試驗(yàn)，測(cè)序?qū)嶒?yàn)數(shù)據(jù)整理，相關(guān)材料的編寫(xiě)項(xiàng)目進(jìn)程項(xiàng)目預(yù)算產(chǎn)品價(jià)格使用次數(shù)單次檢測(cè)TaqMan?mutationdetectionassay8130.0075108.4TaqMan?GenotypingMasterMix,2?8600.0010008.6IPCMix2767.005005.5每孔合計(jì)122.5使用ABI試劑研發(fā)的的預(yù)算：項(xiàng)目預(yù)算項(xiàng)目?jī)r(jià)格（萬(wàn)元）試劑3.0耗材1.0測(cè)序0.6其他0.4合計(jì)5.0自己研發(fā)的的預(yù)算：試劑和PCR技術(shù)的穩(wěn)定性性實(shí)驗(yàn)過(guò)程中的的不確定性（（實(shí)驗(yàn)室、試試劑等污染情情況）技術(shù)路線調(diào)整整和時(shí)間臨床樣本的收收集、樣本的的質(zhì)量試劑盒生產(chǎn)市場(chǎng)推廣四.風(fēng)險(xiǎn)評(píng)估Thankyou!9、靜夜四無(wú)鄰鄰，荒居舊業(yè)業(yè)貧。。1月-231月-23Wednesday,January4,202310、雨中黃葉葉樹(shù)，燈下下白頭人。。。22:00:1822:00:1822:001/4/202310:00:18PM11、以以我我獨(dú)獨(dú)沈沈久久，，愧愧君君相相見(jiàn)見(jiàn)頻頻。。。。1月月-2322:00:1822:00Jan-2304-Jan-2312、故人江海別別，幾度隔山山川。。22:00:1822:00:1822:00Wednesday,January4,202313、乍見(jiàn)見(jiàn)翻疑疑夢(mèng)，，相悲悲各問(wèn)問(wèn)年。。。1月-231月-2322:00:1822:00:18January4,202314、他鄉(xiāng)生生白發(fā)，，舊國(guó)見(jiàn)見(jiàn)青山。。。04一一月202310:00:18下下午22:00:181月-2315、比不了得就就不比，得不不到的就不要要。。。一月2310:00下下午1月-2322:00January4,202316、行動(dòng)出成果果，工作出財(cái)財(cái)富。。2023/1/422:00:1822:00:1804January2