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Kutsimikizira magwiridwe antchito a ma primers ndi ma probes koyambirira kwa PCR reagents ndikuzindikira momwe zinthu ziliri zoyenera kuchita ndizofunika kuwonetsetsa kuti kuyesa kovomerezeka kukuyenda bwino.

Ndiye tifunika kutsimikizira bwanji kafukufuku woyambira kumayambiriro?

Zizindikiro zazikuluzikulu ndizoyambira, ma curve amplification, ct value, amplification performance, low-concentration zitsanzo, CV, etc.

Zoyambira

Choyambira ndi mzere wopingasa mu PCR amplification curve.M'mizere yoyambirira ya PCR amplification reaction, chizindikiro cha fluorescence sichisintha kwambiri ndikupanga mzere wowongoka.Mzere wowongoka uwu ndiwo maziko ake.

Mukawunika ma probe oyambira a PCR, samalani ngati mazikowo ali mulingo.Kuyera kwa ndende ya probe yoyambira kudzakhudza zoyambira, monga kupangitsa kuti choyambira chikwere kapena kugwa.Zoyambira nazonso ndi chizindikiro chodziwika bwino.
Kusanthula

Amplification Curve

Chizindikiro china chodziwikiratu ndi mawonekedwe a piritsi lokulitsa.Ndi bwino kukhala ndi mapindikira ooneka ngati S kuti mupewe kukulitsa kwachiwiri kapena ma curve ena achilendo.
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Mtengo wa CT

Chiwerengero cha zozungulira zomwe zimayenderana ndi malo osinthira kuchokera pazoyambira mpaka kukula kokulirapo ndi mtengo wa Ct.

Pachitsanzo chomwecho, ma probes oyambira osiyanasiyana amabweretsa ma curve osiyanasiyana, ndipo mtengo wofananira wa Ct umakhudzidwa ndi kuchuluka kwamphamvu komanso kusokoneza.Mwachidziwitso, mtengo wocheperako wa Ct wa kafukufuku woyambira womwe timasankha, ndibwino.

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Kuchita bwino kwa Amplification

Imodzi mwa njira zodalirika komanso zokhazikika zowunika momwe PCR ikukulirakulira ndi njira yokhotakhota, yomwe imadziwikanso ndi ofufuza.Njirayi imaphatikizapo kupanga zitsanzo zingapo kuti ziwongolere kuchuluka kwa ma tempulo omwe mukufuna.Zitsanzozi nthawi zambiri zimapangidwa ndi kusungunuka kwazinthu zokhazikika, zomwe zimagwiritsidwa ntchito kwambiri ndi dilution 10.Pogwiritsa ntchito zitsanzo zochepetsedwa, pogwiritsa ntchito pulogalamu ya qPCR kuti mukulitse kuti mupeze mtengo wa Cq, ndipo potsirizira pake mujambule mphira wokhazikika molingana ndi kuchuluka kwa chitsanzo chilichonse ndi mtengo wa Cq kuti mupeze mzere wofanana Cq= -klgX0+b, ndi kukulitsa mphamvu E = 10(-1 / k) -1.Mukamagwiritsa ntchito qPCR pakuwunika kachulukidwe, kukulitsa bwino kumafunika kukhala pakati pa 90% -110% (3.6>k> 3.1).

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Kuzindikira kwa Zitsanzo zotsika kwambiri

Pamene ndende yachitsanzo ili yotsika, mitengo yodziŵika ya ma probes osiyanasiyana amasiyana.Timasankha zitsanzo 20 zotsika kwambiri kuti tibwerezenso, ndipo makina ofufuzira omwe ali ndi chiwongoladzanja chapamwamba kwambiri ndi abwino kwambiri.

Kusanthula-5

Coefficient of Variation (CV)

Zitsanzo zobwereza 10 zitha kudziwika ndi ma probes osiyanasiyana oyambira molingana ndi mzere wa reagent wa nucleic acid amplification kuzindikira.

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Quantitative Reagents:
Kulondola
Kulondola kwapakati pa gulu limodzi kuyenera kukumana: coefficient of variable (CV,%) ya mtengo wa logarithmic wa mayeso a mayeso ndi ≤5%.Zitsanzo zikakhala zotsika, coefficient of variable (CV,%) ya logarithm ya ndende yodziwika ndi ≤10%


Ma reagents abwino:
Kulondola
Kulondola kwa gulu limodzi kuyenera kukwaniritsa:

(1) Coefficient of kusiyana kwa mtengo wa Ct (CV,%) ≤5%

Zitsanzo zomwezo zimayesedwa mofanana kwa nthawi 10, ndipo zotsatira zake ziyenera kukhala zofanana


Nthawi yotumiza: Sep-18-2021