Ixesha lokwenyani i-PCR, ekwabizwa ngokuba yi-PCR yobungakanani okanye i-qPCR, yindlela yokubeka iliso ngexesha lokwenyani kunye nohlalutyo lweemveliso zokukhulisa i-PCR.
Ngenxa yokuba i-PCR yobungakanani inenzuzo yokusebenza okulula, ngokukhawuleza kwaye kulula, uvakalelo oluphezulu, ukuphindaphinda okulungileyo, kunye nezinga lokungcoliseka eliphantsi, lisetyenziswa ngokubanzi kuvavanyo lwezonyango, uvavanyo lokuphumelela kweziyobisi, uphando lwe-gene expression, uphando lwe-transgenic, ukufumanisa i-gene, ukufumanisa i-pathogen, ukufunyanwa kwezilwanyana kunye nezityalo., uvavanyo lokutya kunye neminye imimandla.
Ke ngoko, nokuba ubandakanyeka kuphando olusisiseko kwisayensi yobomi, okanye abaqeshwa beenkampani zamachiza, iinkampani ezifuywayo, iinkampani zokutya, okanye abasebenzi bokuhlolwa kokuphuma kunye ne-quarantine bureaus, amasebe okubeka iliso kokusingqongileyo, izibhedlele kunye nezinye iiyunithi, uya kuba ngaphezulu okanye ngaphantsi kokuvezwa okanye kuya kufuneka wazi ulwazi lokulawula ubungakanani bePCR.

Umgaqo wexesha lokwenyani PCR

I-Real Time PCR yindlela apho izinto ze-fluorescent zongezwa kwi-PCR reaction system, kunye nokuqina komqondiso we-fluorescence kwinkqubo yokusabela kwe-PCR ibekwe esweni ngexesha lokwenyani ngesixhobo se-PCR sobungakanani, kwaye ekugqibeleni idatha yovavanyo iyahlalutywa kwaye iqhutywe.

【Igophe lokukhulisa】ligophe elichaza inkqubo eguqukayo yePCR.Igophe lokukhulisa le-PCR ayisiyiyo igophe eliqhelekileyo, kodwa lijiko le-sigmoid.

[Inqanaba leqonga legophe lokukhulisa]Ngokunyuka kwenani lemijikelezo ye-PCR, ukungasebenzi kwe-DNA polymerase, ukuchithwa kwe-dNTPs kunye ne-primers, kunye nokuvinjelwa kwe-synthesis reaction by-product pyrophosphate, njl., i-PCR ayisoloko ikhula ngokukhawuleza., kwaye ekugqibeleni izakungena kwithafa.

[INgingqi yokuKhula okubonakalayo kwe-Amplification Curve]Nangona isigaba se-plateau sihluka kakhulu, kwindawo ethile yommandla wokukhula okubonakalayo kwijika le-amplification, ukuphindaphinda kuhle kakhulu, okubaluleke kakhulu kuhlalutyo lobungakanani be-PCR.

[Ixabiso lomqobo kunye nexabiso le-CT]Sibeka ixabiso elilinganiselweyo lokufumanisa i-fluorescence kwindawo efanelekileyo kwindawo yokukhula ye-exponential ye-curve yokukhulisa, okuyixabiso le-threshold (i-Threshold).I-intersection ye-threshold value kunye ne-curve yokukhulisa ixabiso le-Ct, oko kukuthi, ixabiso le-Ct libhekisela kwinani lemijikelezo (i-Threshold Cycle) xa ixabiso le-threshold lifinyelelwe.

Igrafu engezantsi ibonisa ngokucacileyo ubudlelwane phakathi komgca we-threshold kunye ne-amplification curve, i-threshold kunye nexabiso le-Ct.

【Indlela yokwenza ubungakanani?】

Kuye kwangqinwa ngethiyori yemathematika ukuba ixabiso le-Ct linobudlelwane bomgca ophambeneyo kunye nelogarithm yenani leetemplates zokuqala.Ixesha lokwenyani I-PCR ibeka iliso kwimveliso yokukhulisa i-PCR ngexesha lokwenyani kwaye izilinganise ngexesha lesigaba sokukhulisa ulwazi.

Ngomjikelezo ngamnye we-PCR, i-DNA yanda ngokukhawuleza ngamaxesha e-2, kwaye ngokukhawuleza yafikelela kwi-plateau.

Ukucinga ukuba isixa sokuqalisa i-DNA ngu-A₀ , emva kwemijikelo ye-n, inani lethiyori yemveliso ye-DNA inokubonakaliswa ngolu hlobo:

A _n =A ₀ ×2ⁿ

Emva koko, inani lokuqala le-DNA i-A 0, ngokukhawuleza inani lemveliso ekhulisiwe lifikelela kwixabiso lokubona An , kunye nenani lemijikelezo xa ufikelela kwi-An yixabiso le-Ct.Oko kukuthi, i-DNA yokuqala isixa A 0, kokukhona incopho yegophe lokukhulisa, kwaye ngokuhambelanayo inani elifunekayo lemijikelo n lincinci.

Senza ukuhlanjululwa kwe-gradient yomgangatho woxinaniso owaziwayo kwaye sisebenzise njengethemplate ye-Real Time PCR, kwaye uchungechunge lwee-curves zokukhulisa ziya kufumaneka ngezithuba ezilinganayo ngokomyalelo wokuqalisa inani le-DNA ukusuka ngaphezulu ukuya ngaphantsi.Ngokobudlelwane bomgca phakathi kwexabiso le-Ct kunye nelogarithm yenani leetemplates zokuqala, a[igophe eliqhelekileyo] linokwenziwa .

Ngokufaka endaweni yexabiso le-Ct yesampuli kunye nogxininiso olungaziwayo kwi-curve esemgangathweni, isixa sokuqala se-template yesampuli kunye noxinaniso olungaziwayo lunokufumaneka, oluyi-quantitative principle ye-Real Time PCR.

Indlela yokubona ye-PCR yexesha lokwenyani

Ixesha lokwenyani I-PCR ibona iimveliso zokukhulisa i-PCR ngokubona ubunzulu be-fluorescence kwinkqubo yokusabela.

Umgaqo weFluorescent Dye embedding Method】

Idayi zeFluorescent, njenge-TB Green ®, inokubophelela ngokungaqhelekanga kwi-DNA ephindwe kabini kwiinkqubo ze-PCR kunye ne-fluoresce ekubopheni.

Ubunzulu be-fluorescence kwinkqubo yokusabela bunyuke ngokukhawuleza ngokunyuka kwemijikelo ye-PCR.Ngokubona ubunzulu be-fluorescence, isixa se-DNA yokukhulisa inkqubo yokusabela sinokujongwa ngexesha langempela, kwaye ke isixa sethemplethi yokuqalisa kwisampulu sinokuqikelelwa umva.

【Umgaqo wendlela ye-fluorescent probe】

iprobe ye-fluorescentulandelelwano lwe-nucleic acid kunye neqela le-fluorescent ekupheleni kwe-5 kunye neqela lokucima ekupheleni kwe-3, elinokuthi libophe ngokuthe ngqo kwi-template.Xa i-probe intact, i-fluorescence ekhutshwe yi-fluorophore icinywa liqela lokucima kwaye ayikwazi ukukhupha i-fluoresce.Xa i-probe ibolile, i-fluorescent substance iya kuhlukana kwaye ikhuphe i-fluorescence.

Iprobe yefluorescent yongezwa kwisisombululo se-PCR reaction.Ngexesha lenkqubo yokucoca, iprobe ye-fluorescent iya kubophelela kwindawo ethile yethemplethi.Ngexesha lenkqubo yokwandiswa, umsebenzi we-5′→3′ exonuclease we-enzyme ye-PCR unokubolisa i-fluorescent probe exutywe netemplate, kwaye into yefluorescent iyahlukaniswa ukuze ikhuphe i-fluorescence.Ngokubona ubunzulu be-fluorescence beprobe kwinkqubo yokusabela, injongo yokubeka iliso kwixabiso lokukhulisa imveliso ye-PCR inokufezekiswa.

【Ukukhethwa kweNdlela yokuFumana iFluorescence】

Ukuba isetyenziselwa ukwahlula ulandelelwano nge-homology ephezulu kunye nokwenza ubhaqo lwe-PCR oluphindaphindayo olufana nohlalutyo lokuchwetheza lwe-SNP, indlela yokuhlola i-fluorescent ayinakubuyiselwa.
Ngolunye uvavanyo lwe-PCR lweXesha lokwenyani, indlela elula, elula kwaye ephantsi ye-fluorescent chimera ingasetyenziswa.

probesIzinto ezithile eziqinileyo, ezikwaziyo ukuphindaphinda i-PCR

Ukusilela

Iimfuno ezicacileyo eziphezulu zokukhulisa;

i-multiplex PCR ayinakwenziwaKufuneka ukuyila iiprobe ezithile, ixabiso eliphezulu;

ngamanye amaxesha uyilo lophando lunzima

Iimveliso ezinxulumeneyo: