Waqtiga-dhabta ah ee fluorescence-ga PCR waa hab lagu cabbiro wadarta guud ee alaabta ka dib wareeg kasta oo falcelinta silsiladda polymerase (PCR) ee falcelinta-weynida DNA-da iyadoo la isticmaalayo fluorophore.Habka waxaa loo isticmaalaa in lagu qiyaaso taxane DNA oo gaar ah oo ku jira muunada si loogu tijaabiyo hababka tixraaca gudaha ama dibadda.Tan iyo markii la aasaasay, imtixaannada PCR ee tirooyinka fluorescent ayaa si isa soo taraya caan ugu noqday macallimiinta shaybaadhka.
Mabda'a PCR Fluorescence: Fluorescence PCR, oo marka hore loo yaqaan TaqManPCR ka dibna sidoo kale Real-TimePCR, waa farsamo qiyaaseed cusub oo nucleic acid ah oo uu sameeyay PE (PerkinElmer) ee USA 1995. Farsamadu waxay ku salaysan tahay kordhinta baaritaanka fluorescently calaamadeysan ama u dhigma. dheeha fluorescent ee PCR-ga caadiga ah si loo gaaro hawsheeda qiyaaseed.Mabda'a: marka falcelinta PCR-gu sii socoto, alaabada falcelinta PCR-ga ayaa urura oo xoogga isha fluorescent ayaa kordhaysa si siman.Wareeg kasta, calaamada xoojinta iftiinka fluorescence ayaa la ururiyaa si aan ula socono isbeddelka qadarka alaabta isbeddelka xoojinta fluorescence oo sidaas awgeed loo helo garaaf qalooca cod-weyneeye.
Guud ahaan, qalooca cod-weyneysiinta fluorescence waxa loo qaybin karaa saddex weji: wejiga calaamada asalka ah ee fluorescence, muuqalka jibbaarada ee fluorescence iyo wejiga sare.Inta lagu jiro marxaladda calaamadda asalka, calaamada dheesha ee la xoojiyey waxaa lagu daboolaa calaamada asalka ah ee fluorescence oo isbeddelada qaddarka alaabta lama go'aamin karo.Marxaladda dhul-balaadhan, badeecada kordhintu si xad dhaaf ah uma korodho, ma jiro xidhiidh toosan oo ka dhexeeya qaddarka badeecadda dhammaadka ah iyo qaddarka qaabka bilawga ah, lambarka nuqulka DNA-ga bilaabmaya lama xisaabin karo iyadoo lagu salaynayo qaddarka alaabta PCR ee u dambaysa.Kaliya marka lagu jiro wejiga weynaynta jibbaarada ee calaamadda fluorescent ayaa jira xiriir toos ah oo u dhexeeya logarithm ee qaddarka alaabta PCR iyo qaddarka template bilowga, waxaanan dooran karnaa inaan tan ku cabbirno heerkan.Si ay ugu habboonaato tirooyinka iyo isbarbardhigga, laba fikradood oo aad muhiim u ah ayaa lagu soo bandhigay farsamada tirada PCR ee waqtiga-dhabta ah: heerka fluorescence-ka iyo qiimaha CT-ga.
Xadku waa qiimo si macmal ah loo dejiyay oo ku yaal qalooca cod-weyneysiinta fluorescence.wajiga jibbaaran ee kordhinta PCR.
Qiimaha Ct: waa tirada wareegyada uu calaamada fluorescence ee tuubo kasta oo falcelin ah uu maray si uu u gaadho qiimaha domain ee go'an.
Xidhiidhka ka dhexeeya qiimaha Ct iyo qaabka bilawga ah: daraasaduhu waxay muujiyeen in qiimaha Ct ee template kasta uu xidhiidh toos ah la leeyahay logarithm ee lambarka nuqulka bilawga ah ee templatekaas, koobiyada badan ee nambarka koobiga bilawga ah, yaraynta Ct qiimaha.Qiimayaasha Ct waa kuwo deggan.Qalooca caadiga ah waxaa lagu samayn karaa iyadoo la isticmaalayo halbeeg leh nambarka koobiga bilawga ah ee la yaqaan, halkaas oo iskudubarid toosan ay ka dhigan tahay logarithm ee nambarka koobiga bilawga iyo iskudubarid toosan oo u taagan qiimaha Ct sida ku cad shaxanka hoose.
Sidaa darteed, marka la helo qiimaha Ct ee muunad aan la garanayn, nambarka koobiga bilawga ah ee muunaddaas waxaa laga xisaabin karaa qalooca caadiga ah.
Qiimaha Ct ma aha mid joogto ah oo waxaa saameyn kara muunado kala duwan iyo qalabyo kala duwan, xitaa haddii isla muunadda lagu soo celiyo 2 jeer isla qalab isku mid ah, qiimaha Ct wuu kala duwanaan karaa.
Qiimaynta fluorescence ee tirade: Qiimaynta fluorescence ee tirade waxa loo qaybin karaa baadhida fluorescent iyo midabada fluorescent iyadoo ku xidhan calamadaha la isticmaalo.Baadhitaannada Fluorescent waxaa ka mid ah tignoolajiyada Beacon (teknoolojiyadda iftiinka molecular, oo uu matalo American Tagyi), Baaritaannada TaqMan (oo ay matasho ABI) iyo tignoolajiyada FRET (oo ay matasho Roche);Midabaynta fluorescent waxaa ka mid ah midabyo dhaadheer oo buuxa iyo midabyo fluorescent ah oo aan dheregsanayn, wakiilka caadiga ah ee midabada fluorescent-ka ee aan dheregsanayn waa SYBRGreen I, oo sida caadiga ah hadda la isticmaalo;saturated Wakiilka caadiga ah ee midabada fluorescent-ka ee aan dheregsanayn waa SYBRGreenⅠ;Midabada dhaadheer ee buuxa waa EvaGreen, LCGreen, iwm.
SYBRGreenI waa dheeha DNA-da oo si caadi ah loo isticmaalo oo loogu talagalay PCR fluorescent, kaas oo si aan gaar ahayn ugu xidha DNA-ga laba-geesoodka ah.Xaaladeeda xorta ah, SYBRGreenI waxay soo saartaa iftiin daciif ah, laakiin marka lagu xiro DNA-da laba-geesoodka ah, fluorescence waxay kordhisaa 1000-laab.Sidaa darteed, wadarta guud ee calaamada fluorescence ee ay soo saarto fal-celintu waxay la mid tahay tirada DNA-da laba-geesoodka ah ee hadda jirta oo waxay korodhaa marka alaab-weynayntu korodho.
Faa'iidooyinka dheeha DNA-da ee laba-geesoodka ah: Naqshad tijaabo ah oo fudud, kaliya 2 asal ayaa loo baahan yahay, looma baahna in la naqshadeeyo shaybaarro, looma baahna in la naqshadeeyo baaritaanno badan oo si degdeg ah loogu tijaabiyo hiddo-wadaha badan, awoodda lagu sameeyo falanqaynta qalooca barta dhalaalka, tijaabi gaarnimada falcelinta kordhinta, qiimaha bilowga ah oo hooseeya, guud ahaan wanaagsan oo sidaas darteed inta badan loo isticmaalo cilmi-baarista gudaha iyo dibedda.
Habka baaritaanka Fluorescent (Farsamada Taqman): Marka PCR-weyneynta la sameeyo, labo ka mid ah furayaasha ayaa lagu daraa iyada oo ay weheliso baaritaan gaar ah oo fluorescent ah.Marka baaritaanka uu yahay mid sax ah, calaamada fluorescence ee ay sii daayaan kooxda wariyaha waxaa nuuga kooxda la demiyay oo laguma arko qalabka PCR;inta lagu guda jiro kor u qaadida PCR (wajiga fidinta), 5'-3' nashaada jeexjeexa ee Taq enzyme waxay hoos u dhigtaa baaritaanka enzymatically, taasoo ka dhigaysa kooxda fluorescence weriyaha iyo kooxda fluorescence demi.
Codsiyada PCR ee tirada fluorescent.
Cilmi-baarista bayoolajiga molecular:
1. Falanqaynta aashitada nukleic ee tirada.Falanqaynta tirade iyo tayada ee cudurada faafa, ogaanshaha jeermiska cudurada faafa ama fayrasyada, sida hargabkii ugu dambeeyay ee hargabka A (H1N1), ogaanshaha tirooyinka nuqulka hiddasidaha ee dhirta iyo xayawaanka transgenic, ogaanshaha heerarka firfircoonida hidda-wadaha RNAi, iwm.
2. Falanqaynta muujinta hidde-sidaha kala duwan.Isbarbardhigga farqiga u dhexeeya muujinta hidda-wadaha ee u dhexeeya shaybaarrada la daweeyay (tusaale, daaweynta daroogada, daaweynta jirka, daaweynta kiimikaad, iwm.), kala duwanaanshaha muujinta hiddo-wadaha gaarka ah ee wejiyada kala duwan iyo xaqiijinta microarray cDNA ama natiijooyinka muujinta kala duwan
3. Ogaanshaha SNP.Ogaanshaha hal nucleotide polymorphisms ayaa muhiim u ah daraasadda u nuglaanshaha shakhsi ahaaneed ee cudurrada kala duwan ama jawaabta shakhsi ahaaneed ee daroogooyinka gaarka ah, iyo sababta oo ah qaab-dhismeedka xariifnimada ee calaamadaha molecular, marka xogta isku xigta ee SNP la ogaado, way fududahay oo sax ah u isticmaal farsamadan ogaanshaha SNP-ta sare leh.
4. Ogaanshaha Methylation.Methylation waxay la xiriirtaa cuduro badan oo bini'aadamka ah, gaar ahaan kansarka, Laird wuxuu soo sheegay farsamo la yiraahdo Methylight, taas oo daaweysa DNA ka hor inta aan la kordhin si ay u noqoto cytosine unmethylated uracil iyo methylated cytosine aan saameyn ku yeelan, iyada oo la isticmaalayo qalab gaar ah iyo baaritaan Taqman si loo kala saaro DNA-da methylated iyo unmethylated. .dareen badan.
Cilmi-baadhis caafimaad:
1. Baadhitaanka dhalmada ka hor: dadku ma daweyn karaan cudurrada la iska dhaxlo ee ay sababaan maadada hidde-sidaha oo la beddelo, ilaa hadda, waxay yareyn karaan oo keliya tirada carruurta xanuunsan ee ku dhasha la socodka dhalmada ka hor si looga hortago cudurrada kala duwan ee la iska dhaxlo.Tani waa hab aan fiicneyn oo ay si fudud u aqbalaan haweenka uurka leh.
2. Ogaanshaha cudur-sidaha: Baadhitaanka PCR-ga ee fluorescent-ka ayaa ogolaanaya in la ogaado tirada cudurada sida gonococcus, Chlamydia trachomatis, Mycoplasma solium, fayraska papilloma ee aadanaha, fayraska herpes simplex, fayraska difaaca jirka, fayraska cagaarshow, fayraska hargabka, Mycobacterium tuberculosis iyo cytomevirus, EB.Waxay leedahay faa'iidooyinka dareenka sare, cabbirka muunada hoose, degdegga iyo fudaydka marka loo eego hababka tijaabada dhaqameed.
3. Qiimaynta waxtarka dawooyinka: falanqaynta tirada ee fayraska cagaarshow B (HBV) iyo fayraska cagaarshow C (HCV) waxay tusinaysaa xidhiidhka ka dhexeeya culayska fayraska iyo waxtarka dawooyinka qaarkood.Haddii heerka serum ee HBV-DNA uu hoos u dhaco inta lagu jiro daawaynta lamivudine ka dibna uu mar kale kordho ama ka bato heerkii hore, waxay muujinaysaa isbeddelka fayraska.
4. Tijaabada Oncogenetic: Inkasta oo habka horumarinta burooyinka aan weli caddayn, waxaa si weyn loo aqbalay in isbeddellada hidde-sideyaasha khuseeya ay yihiin sababta asaasiga ah ee isbeddelka oncogenic.Kordhinta muujinta iyo isbeddelka unugyada oncogenes ayaa lagu arki karaa marxaladaha hore ee burooyinka badan.Waqtiga-dhabta ah ee fluorescence-ga PCR ma aha oo kaliya wax ku ool ah in la ogaado isbeddellada hidda-wadaha, laakiin sidoo kale waxay si sax ah u ogaan kartaa muujinta oncogenes.Habkan ayaa loo isticmaalay in lagu ogaado muujinta hiddo-wadaha kala duwan oo ay ka mid yihiin hidda-wadaha telomerase hTERT, hidda-wadaha granulocytic leukemia WT1 ee daba-dheeraada, hidda-wadaha ER-ka oncogenic, hidda-wadaha PSM-ka ee kansarka qanjirka 'prostate', iyo hiddo-wadaha fayraska ee buro-xiriirka leh.
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Waqtiga boostada: Jun-21-2022