PCR (聚合酶煉反應)

PCR(聚合酶煉反應)

分析

樣本

分析

控制樣本

評估樣本

為每個樣本選定合適的DNA提取方法

DNA提取

DNA評估

凝膠電泳法

分析結果

陳述結果s

分析樣本的步驟

(根據(jù)ENISO的標準)

1/13/2023BBCANonGMCitricAcidIPControlProgram分析程序PCR1/13/2023BBCANonGMCitricAcidIPControlProgramDNA提取

原材料加工食物難

易破壞細胞壁/膜

移走PCR抑制物質(zhì)

如：糖、可可

.提取足夠分量及合適的DNA1/13/2023BBCANonGMCitricAcidIPControlProgramDNA提取

無

有

卵磷脂

?脂肪

?油

?無

DNApelletwhite?有

hexane-提取

PCR以MicroSpin清洗CTAB-提取

PCR

如有需要，以MicroSpin或Wizard

清洗PCRQiagen提取

如有需要，以MicroSpin或Wizard清洗估計有大量

DNA是

否

1/13/2023BBCANonGMCitricAcidIPControlProgramTheprimeranneals~50°C

第二個循環(huán)

第一個循環(huán)

TheDNAmeltsat~95°CDNAPolymeraseoperatesat72°CPCR(聚合酶煉反應)的原理

1/13/2023BBCANonGMCitricAcidIPControlProgram

No.ofDoubleNumberofStrandedTargetPCRCyclesDNAMolecules102032

44

58

616

732

864

9128

10256

11512

121,024

132,048

144,096

158,192

1616,384

1732,768

1865,536

19131,072

20262,144

21524,288

221,048,576

232,097,152

244,194,304

258,388,608

2616,777,216

2733,554,432

2867,108,864

29134,217,728

30268,435,456

31536,870,912

321,073,741,824

332,147,483,648

344,294,967,296

358,589,934,592

3617,179,869,184

3734,359,738,368

3868,719,476,736

39137,438,953,472

40274,877,906,944

41549,755,813,888

421,099,511,627,776

432,199,023,255,552

444,398,046,511,104

458,796,093,022,208

4617,592,186,044,416

4735,184,372,088,832

4870,368,744,177,664

49140,737,488,355,328

50281,474,976,710,656

指數(shù)性增加DNA數(shù)量

實際上，PCR不能制造出右面的DNA數(shù)目情況

?有些DNA被破壞了?樣本含有禁忌物質(zhì)?沒有足夠的引物

PCR增加DNA數(shù)量?1/13/2023BBCANonGMCitricAcidIPControlProgramPCR測試想證明產(chǎn)品中有

GMO?35S啟動子

/NOS

想確認出現(xiàn)了哪種

GMO?RoundupReady大豆/Bt176玉米(Maximizer

)想確認GMO出現(xiàn)的數(shù)量

?定量測試

篩選測試

定性測試

*OurPCRbasedGMOtestingsystemsareprovenbynumerousringtrials,andsomebecamenationalreferencestandardsandarelistedintheCodexAlimentarius1/13/2023BBCANonGMCitricAcidIPControlProgram可提供的篩選測試

篩選測試

CaMV35S-promoter* FMV-34S-promoter*

nos-terminator*

nptII-gene* bar/pat-gene* pat(syn)-gene*Elementspecific

cryIIIa-gene*

(NewLeafpotato)

…能作超過50種GMO定性測試1/13/2023BBCANonGMCitricAcidIPControlProgram篩選檢驗

Detectsabout90%ofGMOsSystemsfornon35S/nonNOSGMOsonrequest1/13/2023BBCANonGMCitricAcidIPControlProgram可提供的定性檢驗大豆

: RoundupReady*玉米

: LibertyLink-T25* Bt176(Event176/Maximizer)* Bt11*

YieldGard(MON810/MaizeGard)* MON809*

SeedLink* RoundupReady(GA21)*

BtXtra(DBT418)* StarLink* 芥花籽

:

LibertyLink*

SeedLink*

RoundupReady*馬鈴薯

: allNewLeafvarieties*

allNewLeafvarieties

*

*綿花

: Bollgard*

*

BXN*

* RoundupReady*

**Elementspecific*Modification-andGMO-specific*Modification-/construct-specific,butcrossdetectionwithothers*Notfullyvalidated…及其他1/13/2023BBCANonGMCitricAcidIPControlProgram大豆定性測試1-4:Foodsamplescontainingsoyausing2 DNAextractions.Onlysample2ispositiveforRRSoyaLectinRoundupReadySoya190bp124bp1114bpM 1 2 3 4M 1 2 3 4 M1/13/2023BBCANonGMCitricAcidIPControlProgram*特別例子：芥花籽的篩選測試ElementspecificdetectiontodifferentiatethreevarietiesofGM-rapeseed:FMVpromoter

RoundupReadyNOS

SeedLinkpat/syn

LibertyLink(35SpromoterisalsopossibletodetectLibertyLink,butiftheresultispositive,youhavetoexcludeanaturalcontaminationwiththevirus(CaMV),becauserapeseedislikecaulifloweraplantofthefamilyBrassicacae)RoundupReady?--CornGA21LibertyLink?-CanolaBT176-CornRoundupReady?-SoyMON810/T25--Corn-BT11-CornLibertyLink?-CornFlavrSavr?-TomatoZeneca-TomatoRoundup-Ready?-CottonRoundup-Ready?-CanolaConventionalSoyl

35spromoterNOSTerminatorRoundupReady?,LibertyLink?andFlavrSavr?aretrademarksofMonsantocompanyandHoechstScheringArgEvoGmbHImportant!!1/13/2023BBCANonGMCitricAcidIPControlProgram定性

35S: 大豆,玉米,芥花籽,綿花,馬鈴薯大豆

: RoundupReady*玉米

: LibertyLink-T25*

* Bt176(Event176/Maximizer)* Bt11**

YieldGard(MON810/MaizeGard)** RoundupReady(GA21)*

BtXtra(DBT418)* StarLink*芥花籽

:

LibertyLink*

*

SeedLink*

*

RoundupReady*

*馬鈴薯

: AllNewLeafvarieties*

*

可提供的定量測試

*Elementspecific*Modification-andGMO-specific*Notfullyvalidated*Developmentofsystemsinnearfuture*Modification-/construct-specific,butcrossdetectionwithothers1/13/2023BBCANonGMCitricAcidIPControlProgram部分用PCR分析過的GMO+++=>100ng/μl.++=5-100ng/μl.+=<5ng/μl.(+)=DNAwasonlyextractableinsomecases.1/13/2023BBCANonGMCitricAcidIPControlProgramLane1)SizeMarker

(Phage-Lambda-DNA,HindIIIdigested)Lane2)Bovine-DNA,fresh,2μgLane3)Bovine-DNA,5min100°C,2μgLane4)Bovine-DNA,15min100°C,2μgLane5)Bovine-DNA,30min100°C,2μgLane6)Bovine-DNA,5min121°C,2μgLane7)Bovine-DNA,13min121°C,2μgLane8)SizeMarker(pUC18-DNA,HpaIIdigested)Lengthinbasepairs(bp)2313094616557436123222027564501/489404352242190

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