Ukuqinisekisa ukusebenza kwama-primer kanye nama-probes esigabeni sokuqala sama-reagents e-PCR kanye nokuthola izimo zokusabela ezifanele kakhulu kuyizimfuneko zokuqinisekisa ukuqhubeka kahle kokuhlolwa okusemthethweni.
Ngakho-ke sidinga kanjani ukuqinisekisa uphenyo lokuqala esigabeni sokuqala?
Izinkomba eziyinhloko ziyisisekelo, ijika lokukhulisa, inani le-ct, ukusebenza kahle kokukhulisa, ukutholwa kwesampula yokugxila okuphansi, i-CV, njll.
Isisekelo
Isisekelo umugqa ovundlile ku-PCR amplification curve.Emijikelezweni embalwa yokuqala yokusabela kokukhulisa i-PCR, isignali ye-fluorescence ayishintshi kakhulu futhi yenza umugqa oqondile.Lo mugqa oqondile uyisisekelo.
Lapho uhlola ama-PCR primer probe, naka ukuthi isisekelo sisezingeni yini.Ukuhlanzeka kokugxilisa kwe-primer probe kuzothinta isisekelo, njengokubangela ukuthi isisekelo sikhuphuke noma siwe.Isisekelo futhi siyinkomba enembile kakhulu.
Ijika Lokukhulisa
Enye inkomba enembile ukuma kwejika lokukhulisa.Kungcono kakhulu ukuba nejika elimise okuka-S ukuze ugweme ukukhuliswa kwesibili noma amanye amajika okukhulisa angajwayelekile.
Ct Inani
Inombolo yemijikelezo ehambisana nephoyinti lokuguquguquka ukusuka kusisekelo ukuya ekukhuleni komchazi inani le-Ct.
Kusampula efanayo, ama-primer probe ahlukene aphumela kumajika ahlukene okukhulisa, futhi inani elihambisanayo le-Ct lizothinteka ngokusebenza kahle kokukhulisa kanye nedigri yokuphazamiseka.Ngokombono, inani elincane le-Ct le-primer probe esiyikhethayo, lingcono.
I-Amplification Efficiency
Enye yezindlela ezithembeke kakhulu nezizinzile zokuhlola ukusebenza kahle kokukhulisa i-PCR ijika elijwayelekile, eliqashelwa kabanzi abacwaningi.Indlela ibandakanya ukwenza uchungechunge lwamasampuli ukulawula inani elihlobene lezifanekiso eziqondiwe.Lawa masampuli ngokuvamile enziwa ngokuhlanjululwa kwe-serial kwezixazululo zesitoko esigxilile, okuvame ukusetshenziswa kakhulu ukuhlanjululwa okuphindwe ka-10.Kusetshenziswa uchungechunge lwamasampuli ahlanjululiwe, kusetshenziswa uhlelo olujwayelekile lwe-qPCR ukuze kukhuliswe ukuze kutholwe inani le-Cq, futhi ekugcineni zidwebe ijika elijwayelekile ngokuya ngokugxilisa isampula ngayinye kanye nenani elihambisanayo le-Cq ukuze kutholwe isibalo somugqa Cq= -klgX0+b, kanye nokusebenza kahle kokukhulisa u-E=10(-1 /k) -1.Uma usebenzisa i-qPCR ekuhlaziyeni inani, ukusebenza kahle kokukhulisa kuyadingeka ukuthi kube kububanzi obungu-90% -110% (3.6>k>3.1).
Ukutholwa kwamasampuli agxile kancane
Uma ukugxiliswa kwesampula kuphansi, amazinga okuthola ama-primer probe ahlukene ayahluka.Sikhetha amasampuli angu-20 agxile kancane ukuze siziphindaphinde, futhi isistimu ye-primer-probe enezinga eliphezulu lokutholwa ingcono kakhulu.
I-Coefficient of Variation (CV)
Amasampula ayi-10 ayimpinda angatholwa ngama-primer probes ahlukene ngokuya ngendinganiso yomugqa we-reagent yokutholwa kwe-nucleic acid amplification.
Ama-reagents amaningi:
Ukunemba
Ukunemba phakathi kwenqwaba eyodwa kufanele kuhlangabezane: i-coefficient of variation (CV,%) yenani le-logarithmic lokugxila kokuhlolwa ngu-≤5%.Uma ukugxiliswa kwesampula kuphansi, i-coefficient of variation (CV,%) ye-logarithm yokugxila kokutholwa ngu-≤10%
Ama-reagents afanelekile:
Ukunemba
Ukunemba phakathi kwenqwaba eyodwa kufanele kuhlangabezane:
(1) I-Coefficient of variation of Ct value (CV,%) ≤5%
Isampula efanayo ihlolwa ngokuhambisana izikhathi ezingu-10, futhi imiphumela yokuhlolwa kufanele ihambisane
Isikhathi sokuthumela: Sep-18-2021