Ukuqinisekisa ukusebenza kwee-primers kunye ne-probes kwinqanaba lokuqala le-PCR reagents kunye nokumisela ezona meko zokusabela ezifanelekileyo ziyimfuneko yokuqinisekisa inkqubela phambili egudileyo yovavanyo olusesikweni.
Ke kufuneka siqinisekise njani i-primer probe kwinqanaba lokuqala?
Iimpawu eziphambili zisisiseko, i-curve ye-amplification, ixabiso le-ct, ukusebenza kakuhle kwe-amplification, ukubonwa kwesampuli ephantsi-concentration, i-CV, njl.
Isiseko
Isiseko ngumgca othe tye kwi-curve yokukhulisa i-PCR.Kwimijikelo embalwa yokuqala ye-PCR yokukhulisa i-amplification, isignali ye-fluorescence ayitshintshi kakhulu kwaye yenza umgca othe ngqo.Lo mgca uthe tye usisiseko.
Xa uhlola i-PCR primer probes, qaphela ukuba isiseko sisezingeni.Ukucoceka koxinzelelo lwe-primer probe kuya kuchaphazela isiseko, njengokubangela ukuba isiseko siphakame okanye siwe.Isiseko sikwasisalathisi esicacileyo.
Ijika Lokukhulisa
Esinye isalathisi esibonakalayo sisimo segophe lokukhulisa.Kungcono ukuba negophe elimise okwe-S ukunqanda ulwandiso lwesibini okanye ezinye iigophe zokukhulisa ezingaqhelekanga.
Ct Ixabiso
Inani lemijikelezo ehambelana ne-inflection point ukusuka kwisiseko ukuya ekukhuleni kwe-exponential yi-Ct value.
Kwisampuli efanayo, iiprobes ezahlukeneyo ze-primer ziphumela kwii-curves zokukhulisa ezahlukeneyo, kwaye ixabiso le-Ct elihambelanayo liya kuchaphazeleka ngobuchule bokukhulisa kunye ne-interference degree.Kwithiyori, i-Ct encinci yexabiso le-primer probe esiyikhethayo, ngcono.
Ukwandiswa ngokuSebenzayo
Enye yeendlela ezithembekileyo nezizinzileyo zokuvavanya ukusebenza kakuhle kokukhulisa i-PCR yigophe eliqhelekileyo, elikwavunywa ngokubanzi ngabaphandi.Indlela ibandakanya ukwenza uthotho lweesampulu ukulawula inani leetemplates ekujoliswe kuzo.Ezi sampuli zihlala zenziwe ngokuxutywa kwe-serial yezisombululo zesitokhwe esigxininisiweyo, eyona nto isetyenziswa kakhulu yi-10-fold dilution.Ukusebenzisa uchungechunge lweesampulu ezihlanjululweyo, usebenzisa inkqubo ye-qPCR eqhelekileyo ukukhulisa ukufumana ixabiso le-Cq, kwaye ekugqibeleni udwebe ijika eliqhelekileyo ngokugxininiswa kwesampulu nganye kunye nexabiso le-Cq elihambelanayo lokufumana i-equation yomgca Cq = -klgX0 + b, kunye nokusebenza kakuhle kwe-amplification E = 10 (-1 / k) -1.Xa usebenzisa i-qPCR kuhlalutyo lobungakanani, ukusebenza kakuhle kokukhulisa kufuneka kube kuluhlu lwe-90% -110% (3.6>k>3.1).
Ukufunyanwa kweeSampuli ezikwiqondo eliphantsi
Xa i-concentration yesampulu iphantsi, amaxabiso okubona ama-primer probes ahlukeneyo ahlukile.Sikhetha iisampulu ze-20 ezisezantsi zokuxilonga, kunye nenkqubo ye-primer-probe enezinga eliphezulu lokufumanisa eyona nto ingcono.
I-Coefficient of Variation (CV)
Iisampulu eziphindwe kabini ze-10 zingabonwa nge-primer probes ezahlukeneyo ngokwemigangatho yomgca we-reagent yokufumanisa i-nucleic acid amplification.
IiReagents zobungakanani:
Ukuchaneka
Ukuchaneka ngaphakathi kwebhetshi enye kufuneka kuhlangane: i-coefficient of variation (CV,%) yexabiso lelogarithmic yoxinaniso lovavanyo ngu-≤5%.Xa isampuli yoxinaniso iphantsi, i-coefficient of variation (CV,%) ye-logarithm yoxinaniso lokufumanisa ≤10%
Ii-reagents ezisemgangathweni:
Ukuchaneka
Ukuchaneka kwibhetshi enye kufuneka kuhlangane:
(1) I-Coefficient of variation of Ct value (CV,%) ≤5%
Isampuli efanayo ivavanywa ngokuhambelana ngamaxesha e-10, kwaye iziphumo zovavanyo kufuneka zihambelane
Ixesha lokuposa: Sep-18-2021
