I-Real Time PCR, eyaziwa nangokuthi i-quantitative PCR noma i-qPCR, iyindlela yokuqapha nokuhlaziya ngesikhathi sangempela imikhiqizo yokukhulisa i-PCR.
Ngenxa yokuthi i-PCR yobuningi inezinzuzo zokusebenza okulula, okusheshayo nokulula, ukuzwela okuphezulu, ukuphindaphinda okuhle, kanye nezinga lokungcola okuphansi, isetshenziswa kabanzi ekuhlolweni kwezokwelapha, ukuhlola ukusebenza kahle kwezidakamizwa, ucwaningo lwesisho sezakhi zofuzo, ucwaningo lwe-transgenic, ukutholwa kwezakhi zofuzo, ukutholwa kwe-pathogen, ukutholwa kwezilwane nezitshalo., ukuhlolwa kokudla kanye neminye imikhakha.
Ngakho-ke, kungakhathaliseki ukuthi wenza ucwaningo oluyisisekelo kwisayensi yezempilo, noma abasebenzi bezinkampani zemithi, izinkampani ezifuywayo, izinkampani zokudla, noma ngisho nabasebenzi bokuhlola ukuphuma nokuhlukaniswa kwabantu, iminyango yokuqapha imvelo, izibhedlela kanye namanye amayunithi, uzovezwa kancane noma kancane Noma udinga ukwazi ulwazi lokuthola i-PCR yobuningi.

Isimiso Sesikhathi Sangempela PCR

I-Real Time PCR iyindlela lapho izinto ze-fluorescent zengezwa kusistimu yokusabela kwe-PCR, futhi ukuqina kwesiginali ye-fluorescence kunqubo yokusabela kwe-PCR kubhekwa ngesikhathi sangempela ithuluzi le-PCR lobuningi, futhi ekugcineni idatha yokuhlola iyahlaziywa futhi icutshungulwe.

【Ijika lokukhulisa】iyikhevu echaza inqubo eguquguqukayo ye-PCR.Ijika lokukhulisa le-PCR empeleni aliyona ijika elivamile elichazayo, kodwa ijika le-sigmoid.

[Isigaba seplatifomu sejika lokukhulisa]Ngokukhula kwenani lemijikelezo ye-PCR, ukungasebenzi kwe-DNA polymerase, ukuncipha kwama-dNTP nama-primers, kanye nokuvinjwa kokusabela kokuhlanganiswa ngokusabela ngomkhiqizo we-pyrophosphate, njll., i-PCR ayihlali ikhula ngokuqhubekayo., futhi ekugcineni izongena ethafeni.

[Isifunda sokukhula esibonakalayo se-Amplification Curve]Nakuba isigaba sethafa sihluka kakhulu, endaweni ethile yesifunda sokukhula okunamandla kwejika lokukhulisa, ukuphindaphinda kuhle kakhulu, okubaluleke kakhulu ekuhlaziyweni komthamo we-PCR.

[Threshold value kanye ne-Ct value]Sisetha inani elilinganiselwe lokutholwa kwe-fluorescence endaweni efanele endaweni yokukhula kwe-exponential yejika lokukhulisa, okuyivelu ye-threshold (Threshold).Ukuphambana kwenani lomkhawulo kanye nejika lokukhulisa inani le-Ct, okungukuthi, inani le-Ct libhekisela enanini lemijikelezo (I-Threshold Cycle) lapho inani lomkhawulo lifinyelelwa.

Igrafu engezansi ikhombisa ngokusobala ubudlelwano phakathi komugqa we-threshold kanye nekhevu yokukhulisa izwi, i-threshold kanye nevelu ye-Ct.

【Indlela yokulinganisa?】

Kufakazelwe ngetiyori yezibalo ukuthi inani le-Ct linobudlelwano bomugqa ophambene ne-logarithm yenani lezifanekiso zokuqala.I-Real Time PCR iqapha imikhiqizo yokukhulisa i-PCR ngesikhathi sangempela futhi ilinganisela phakathi nesigaba sokukhulisa ulwazi.

Ngomjikelezo ngamunye we-PCR, i-DNA yanda ngokuphindaphindiwe izikhathi ezingu-2, futhi ngokushesha yafinyelela ithafa.

Kucatshangwa ukuthi inani lokuqala i-DNA ngu-A₀ , ngemva kwemijikelezo engu-n, inani lethiyori lomkhiqizo we-DNA lingavezwa kanje:

A _n =A ₀ ×2ⁿ

Bese, lapho inani lokuqala le-DNA liyi-A 0, ngokushesha inani lomkhiqizo okhulisiwe lifinyelela inani lokuthola u-A, futhi inani lemijikelezo lapho ufinyelela ku-An yinani le-Ct.Okusho ukuthi, uma inani lokuqala le-DNA elingu-A 0 liba likhulu, kulapho ijika lokukhulisa i-amplification lifika phezulu, futhi ngokuhambisana nenani elidingekayo lemijikelezo n lincane.

Senza ukuhlanjululwa kwe-gradient kwezinga lokugxilisa ingqondo okwaziwayo futhi sikusebenzise njengesifanekiso se-Real Time PCR, futhi uchungechunge lwamajika okukhulisa ulwazi luzotholakala ngezikhathi ezilinganayo ngokulandelana kokuqala inani le-DNA ukusuka kokuningi kuye kokuncane.Ngokobudlelwano bomugqa phakathi kwevelu ye-Ct kanye ne-logarithm yenani lezifanekiso eziqalayo, a[ijika elivamile] lingadalwa .

Ngokufaka esikhundleni senani le-Ct lesampula ngokugxilisa okungaziwa kujika elijwayelekile, inani lesifanekiso sokuqala sesampula esinokugxilisa okungaziwa singatholwa, okuwumgomo wobuningi be-Real Time PCR.

Indlela yokutholwa ye-Real Time PCR

I-Real Time PCR ithola imikhiqizo yokukhulisa i-PCR ngokuthola amandla e-fluorescence ohlelweni lokusabela.

Umgomo Wendlela Yokushumeka Udayi We-Fluorescent】

Udayi we-fluorescent, njenge-TB Green ® , ingabophezela ngokungaqondile ku-DNA enezintambo ezimbili ezinhlelweni ze-PCR kanye ne-fluoresce lapho ibopha.

Amandla e-fluorescence ohlelweni lokusabela anyuke kakhulu ngokwanda kwemijikelezo ye-PCR.Ngokuthola ukushuba kwe-fluorescence, inani lokukhulisa i-DNA ohlelweni lokusabela lingagadwa ngesikhathi sangempela, bese inani lesifanekiso sokuqala kusampula lingalinganiswa ngokuhlehla.

【Isimiso se-fluorescent probe method】

i-fluorescent probeiwukulandelana kwe-nucleic acid eneqembu le-fluorescent ekugcineni kwe-5 kanye neqembu lokucisha ekugcineni kwe-3, elingabophezela ngokuqondile kusifanekiso.Uma i-probe iqinile, i-fluorescence ekhishwa yi-fluorophore icinywa yiqembu lokucisha futhi ayikwazi ukuvutha.Lapho i-probe ibola, into ye-fluorescent izohlukana futhi ikhiphe i-fluorescence.

I-fluorescent probe yengezwa kwisisombululo sokusabela kwe-PCR.Phakathi nenqubo yokuthungatha, i-fluorescent probe izobophezela endaweni ethile yesifanekiso.Phakathi nenqubo yokunwetshwa, umsebenzi we-5′→3′ exonuclease we-enzyme ye-PCR ungabolisa i-fluorescent probe ehlanganiswe nesifanekiso, futhi into ye-fluorescent iyahlukaniswa ukuze ikhiphe i-fluorescence.Ngokuthola ukuqina kwe-fluorescence ye-probe ohlelweni lokusabela, inhloso yokuqapha inani lokukhulisa umkhiqizo we-PCR ingafinyelelwa.

【Ukukhetha Indlela Yokuthola I-Fluorescence】

Uma isetshenziselwa ukuhlukanisa ukulandelana nge-homology ephezulu futhi yenze ukutholwa kwe-PCR ephindaphindiwe njengokuhlaziywa kokuthayipha kwe-SNP, indlela ye-fluorescent probe ayinakushintshwa.
Kokunye ukuhlola kwe-PCR ye-Real Time, indlela ye-chimera ye-fluorescent elula, elula futhi eshibhile ingasetshenziswa.

Ukucaciswa okuqinile, okukwazi ukwenza i-multiplex PCR

Ukushoda

Izidingo ezicacile eziphezulu zokukhulisa;

I-multiplex PCR ayikwazi ukwenziwaIsidingo sokuklama ama-probe athile, izindleko eziphezulu;

ngezinye izikhathi umklamo we-probe unzima

Imikhiqizo Ehlobene: