Ho netefatsa ts'ebetso ea li-primers le li-probes qalong ea li-reagents tsa PCR le ho fumana maemo a nepahetseng ka ho fetisisa a karabelo ke lintho tse hlokahalang ho netefatsa tsoelo-pele e ntle ea liteko tse hlophisitsoeng.
Joale re hloka ho netefatsa probe ea pele joang maemong a pele?
Lits'oants'o tsa mantlha ke li-baseline, amplification curve, ct value, amplification performance, tlhahlobo ea sampole e tlase haholo, CV, jj.
Baseline
Motheo ke mola o okametseng mokolokong oa amplification oa PCR.Lipotolohong tse 'maloa tsa pele tsa karabelo ea amplification ea PCR, lets'oao la fluorescence ha le fetohe haholo mme le etsa mola o otlolohileng.Mohala ona o otlolohileng ke oa motheo.
Ha u hlahloba li-primer probes tsa PCR, ela hloko hore na motheo ke boemo.Bohloeki ba "primer probe concentration" bo tla ama motheo, joalo ka ho etsa hore motheo o phahame kapa o theohe.Taba ea mantlha e boetse ke sesupo se hlakileng haholo.
Amplification Curve
Pontšo e 'ngoe e hlakileng ke sebopeho sa sekhahla sa amplification.Ho molemo ho ba le mothipa o bōpehileng joaloka S ho qoba ho hōlisa ha bobeli kapa li-curve tse ling tse sa tloaelehang tsa amplification.
Ct Boleng
Palo ea lipotoloho tse tsamaellanang le ntlha ea inflection ho tloha motheong ho ea ho kholo ea exponential ke boleng ba Ct.
Bakeng sa sampole e ts'oanang, li-primer probes tse fapaneng li etsa hore ho be le li-curve tse fapaneng tsa amplification, 'me boleng bo lumellanang ba Ct bo tla angoa ke katleho ea amplification le degree ea tšitiso.Ka khopolo, e nyane ea boleng ba Ct ea probe ea pele eo re e khethang, e molemo.
Amplification Katleho
E 'ngoe ea mekhoa e tšepahalang le e tsitsitseng ea ho lekola ts'ebetso ea matlafatso ea PCR ke mokhoa o tloaelehileng oa curve, oo hape o tsejoang haholo ke bafuputsi.Mokhoa ona o kenyelletsa ho etsa letoto la lisampole ho laola palo e lekanyelitsoeng ea litempele tse shebiloeng.Mehlala ena hangata e etsoa ka ho hlapolloa ha li-serial tsa tharollo ea setoko, e sebelisoang haholo ke ho hlapolloa ha makhetlo a 10.Ho sebelisa letoto la lisampole tse hlapollotsoeng, ho sebelisa lenaneo le tloaelehileng la qPCR ho holisa ho fumana boleng ba Cq, 'me qetellong u hule lekhalo le tloaelehileng ho latela mohopolo oa sampole e' ngoe le e 'ngoe le boleng bo lumellanang ba Cq ho fumana equation ea linear Cq= -klgX0+b, le matla a ho holisa E=10(-1 /k) -1.Ha o sebelisa qPCR bakeng sa tlhahlobo ea bongata, ho sebetsa hantle hoa matlafatso hoa hlokahala ho ba maemong a 90% -110% (3.6>k>3.1).
Ho fumanoa ha Mehlala e tlaase
Ha mohlala o le tlase, litekanyetso tsa ho lemoha ha li-primer probes tse fapaneng lia fapana.Re khetha lisampole tse 20 tse tlase haholo hore re ka li etsa hape, 'me sistimi ea "primer-probe" e nang le sekhahla se phahameng sa ho lemoha ke eona e ntle ka ho fetisisa.
Coefficient of Variation (CV)
Mehlala e 10 e kopitsoang e ka bonoa ka li-primer probes tse fapaneng ho latela maemo a mela ea reagent bakeng sa ho lemoha ha nucleic acid amplification.
Quantitative Reagents:
Ho nepahala
Ho nepahala ka har'a sehlopha se le seng ho lokela ho kopana: coefficient of variation (CV,%) ea boleng ba logarithmic ea mahloriso a teko ke ≤5%.Ha mohlala oa mahloriso a le tlase, coefficient of variation (CV,%) ea logarithm ea mahloriso a ho lemoha ke ≤10%
Li-reagents tsa boleng:
Ho nepahala
Ho nepahala ha sehlopha se le seng ho lokela ho kopana:
(1) Coefficient ea phapang ea boleng ba Ct (CV,%) ≤5%
Mohlala o tšoanang o lekoa ka ho bapisa makhetlo a 10, 'me liphetho tsa liteko li lokela ho lumellana
Nako ea poso: Sep-18-2021
