Kuona mashandiro emaprimers uye probes muchikamu chekutanga chePCR reagents uye kuona akanyanya akakodzera maitiro ekuita ndizvo zvinodikanwa kuti uve nechokwadi chekufambira mberi kwakanaka kwekuyedzwa kwepamutemo.
Saka isu tinoda sei kusimbisa iyo yekutanga probe munhanho yekutanga?
Iwo makuru maratidziro ndiwo ekutanga, amplification curve, ct kukosha, amplification kunyatsoita, yakaderera-concentration sampuli yekuona, CV, nezvimwe.
Baseline
Iyo yekutanga ndiyo mutsara wakatwasuka muPCR amplification curve.Mumatanho mashoma ekutanga ePCR amplification reaction, iyo fluorescence chiratidzo haichinje zvakanyanya uye inoumba mutsara wakatwasuka.Mutsetse wakatwasuka uyu ndiwo wekutanga.
Paunenge uchiongorora PCR primer probes, tarisa kuti yekutanga iri nhanho.Kuchena kweiyo primer probe concentration inozokanganisa iyo yekutanga, sekuita kuti yekutanga kusimuka kana kudonha.Iyo yekutanga zvakare iri intuitive chiratidzo.
Amplification Curve
Imwe intuitive chiratidzo chimiro cheiyo amplification curve.Zvakanakisa kuve neS-yakaita curve kudzivirira chechipiri amplification kana mamwe abnormal amplification curves.
Ct Kukosha
Huwandu hwema cycles anoenderana neinflection point kubva kune yekutanga kusvika kune exponential kukura ndiyo Ct kukosha.
Kune imwecheteyo sampuli, akasiyana primer probes anokonzera akasiyana amplification curves, uye inoenderana Ct kukosha ichakanganiswa neamplification kunyatsoshanda uye kupindira degree.Muchirevo, idiki iyo Ct kukosha kweiyo primer probe yatinosarudza, zviri nani.
Amplification Kubudirira
Imwe yedzakanyanya kuvimbika uye yakagadzikana nzira dzekuongorora PCR kukwidziridzwa kwekuita ndiyo yakajairwa curve, iyo inozivikanwa zvakare zvakanyanya nevaongorori.Iyo nzira inosanganisira kugadzira nhevedzano yemasampuli kudzora huwandu hwehukama hwematemplate ekunangwa.Aya masampuli anowanzo gadzirwa ne serial dilutions ye concentrated stock solutions, inonyanya kushandiswa ndeye gumi-kupeta dilution.Uchishandisa nhevedzano yemasampuli akacheneswa, uchishandisa yakajairwa qPCR chirongwa kukwidziridza kuwana kukosha kweCq, uye pakupedzisira kudhirowa curve yakajairwa zvichienderana nekusangana kwesample yega yega uye inoenderana Cq kukosha kuti uwane mutsara equation Cq= -klgX0+b, uye amplification kunyatsoshanda E=10(-1 /k) -1.Paunenge uchishandisa qPCR yehuwandu hwekuongorora, iyo amplification kunyatsoshanda kunodiwa kuve mukati me90% -110% (3.6>k> 3.1).
Low-concentration Samples Detection
Kana iyo sampuli yekumisikidza yakadzikira, mazinga ekuona eakasiyana primer probe akasiyana.Isu tinosarudza makumi maviri epasi-concentration samples kuti tidzokorore, uye iyo primer-probe system ine yakanyanya kucherechedzwa mwero ndiyo yakanyanya kunaka.
Coefficient of Variation (CV)
10 duplicate samples inogona kuwonekwa iine akasiyana primer probes zvinoenderana nemutsara chiyero cheiyo reagent ye nucleic acid amplification yekuona.
Quantitative Reagents:
Kururama
Kurongeka mukati mebatch imwe chete kunofanira kusangana: iyo coefficient of variation (CV,%) yelogarithmic value yetest concentration ndeye ≤5%.Kana iyo sampuli yekumisikidza yakadzikira, iyo coefficient yekusiyana (CV,%) yelogarithm yekuongororwa yekuona ndeye ≤10%
Qualitative reagents:
Kururama
Kurongeka mukati mebatch imwe kunofanirwa kusangana:
(1) Coefficient yekusiyana kweCt kukosha (CV,%) ≤5%
Sample imwe chete inoedzwa mukuenderana kwegumi nguva, uye mhinduro dzebvunzo dzinofanira kuenderana
Nguva yekutumira: Sep-18-2021
