Real Time PCR, inozivikanwawo sehuwandu PCR kana qPCR, inzira yechokwadi-nguva yekutarisa uye kuongororwa kwePCR amplification zvigadzirwa.
Nekuti kuwanda kwePCR ine zvakanakira kushanda kuri nyore, nekukurumidza uye kuri nyore, kunzwisiswa kwakanyanya, kudzokororwa kwakanaka, uye mwero wakaderera wekusvibiswa, inoshandiswa zvakanyanya mukuedzwa kwekurapa, kuongorora kushanda kwezvinodhaka, kutsvagisa gene expression, transgenic research, kucherechedzwa kwemajini, kuona pathogen, kuona mhuka nezvirimwa., kuongororwa kwezvokudya nemimwe minda.
Naizvozvo, kunyangwe iwe uri kuita tsvakiridzo yekutanga musainzi yehupenyu, kana vashandi vemakambani emishonga, makambani ekuchengeta mhuka, makambani ezvekudya, kana kunyange vashandi vekupinda-yekubuda-yekuongorora uye quarantine bureaus, madhipatimendi ekutarisa zvakatipoteredza, zvipatara uye mamwe mayuniti, iwe uchave wakanyanya kana kushoma kufumurwa Kana iwe unofanirwa kuziva ruzivo rwekugona kuwanda kwePCR.

Nheyo yeNguva Yechokwadi PCR

Real Time PCR inzira iyo fluorescent zvinhu zvinowedzerwa kuPCR reaction system, uye iyo fluorescence chiratidzo chekusimba mukuita kwePCR reaction inotariswa munguva chaiyo neiyo yakawanda PCR chiridzwa, uye pakupedzisira data rekuyedza rinoongororwa nekugadziriswa.

【Amplification curve】ndiyo curve inotsanangura dynamic process yePCR.Iyo amplification curve yePCR haisi iyo yakajairwa exponential curve, asi sigmoid curve.

[Platform chikamu cheamplification curve]Nekuwedzera kwenhamba yePCR cycles, kusashanda kweDNA polymerase, kupera kwedNTPs uye maprimers, uye kuvharidzirwa kwemaitiro ekugadzirisa nekuita ne-product pyrophosphate, nezvimwewo, PCR haigari ichiwedzera., uye pakupedzisira achapinda mubani.

[Exponential Growth Region yeAmplification Curve]Kunyangwe chikamu cheplateau chichisiyana zvakanyanya, mune imwe dunhu reiyo exponential yekukura curve yeamplification curve, iyo yekudzokorora yakanaka kwazvo, iyo yakakosha pakuwongorora kwehuwandu hwePCR.

[Threshold value uye Ct value]Isu tinoisa kukosha kwemuganho wekuonekwa kwefluorescence panzvimbo yakakodzera munzvimbo yekukura kweiyo amplification curve, kureva kukosha kwechikumbaridzo (Threshold).Kupindirana kwechikumbaridzo kukosha uye kukwidziridzwa curve ndiyo Ct kukosha, kureva kuti, kukosha kweCt kunoreva nhamba yezvikamu (Threshold Cycle) apo kukosha kwechikumbaridzo kwasvika.

Girafu pazasi inoratidza zvakajeka hukama pakati pechikumbaridzo mutsara uye amplification curve, chikumbaridzo uye Ct kukosha.

【Nzira yekuenzanisa sei?】

Izvo zvakaratidzwa nedzidziso yemasvomhu kuti kukosha kweCt kune hukama hwemutsara hwakasiyana nelogarithm yenhamba yekutanga matemplate.Real Time PCR inocherekedza PCR amplification zvigadzirwa munguva chaiyo uye inozvienzanisa panguva yeexponential amplification chikamu.

Kune yega yega kutenderera kwePCR, iyo DNA yakawedzera exponentially ne2 nguva, uye nekukurumidza yakasvika pabani.

Tichifunga kuti huwandu hwekutanga DNA ndiA₀ , mushure mekutenderera kwen, iyo theoretical huwandu hwechigadzirwa cheDNA inogona kuratidzwa se:

A _n =A ₀ ×2ⁿ

Zvadaro, iyo yekutanga DNA yakawanda A 0 ndeye, nokukurumidza kuwanda kwechigadzirwa chakasimudzirwa chinosvika pakuonekwa kukosha An , uye nhamba yezvikamu paunosvika kuAn ndiyo Ct kukosha.Ndokunge, iyo yakawanda yekutanga DNA huwandu A 0, iyo yekutanga iyo amplification curve inokwira, uye inoenderana iyo inodiwa nhamba yema cycle n idiki.

Isu tinoita gradient dilution yeyakajairwa yekumisikidzwa uye tinoishandisa setemplate yeChaiyo Nguva PCR, uye nhevedzano yeamplification curves inozowanikwa panguva dzakaenzana mukurongeka kwekutanga DNA huwandu kubva pakuwedzera kusvika kushoma.Zvinoenderana nehukama hwemutsara pakati pe Ct kukosha uye logarithm yenhamba yekutanga matemplate, a[standard curve] inogona kugadzirwa .

Nekutsiva kukosha kweCt yesample ine kusazivikanwa kusingazivikanwe muyero curve, iyo yekutanga template huwandu hwesampuresi ine isingazivikanwe yekumisikidza inogona kuwanikwa, inova iyo yehuwandu nheyo yeReal Time PCR.

Kuona nzira yeChaiyo Nguva PCR

Real Time PCR inoona PCR amplification zvigadzirwa nekuona iyo fluorescence kusimba mukuita system.

Nheyo yeFluorescent Dye Embedding Method】

Fluorescent dhayi, yakadai seTB Green ®, inogona kusungirirwa zvisina kufanira kuDNA ine tambo mbiri muPCR systems uye fluoresce pakusunga.

Iyo fluorescence intensity mune reaction system yakawedzera exponentially nekuwedzera kwePCR kutenderera.Nekuona iyo fluorescence intensity, huwandu hweDNA amplification muhurongwa hwekuita hunogona kutariswa munguva chaiyo, uye ipapo huwandu hwekutanga template mumuenzaniso hunogona kudzokororwa kufungidzirwa.

【Nheyo yefluorescent probe nzira】

fluorescent probeis nucleic acid sequence neboka refluorescent pamagumo e5 uye boka rekudzima pamagumo e3, iyo inogona kunyatsosunga kune template.Kana iyo probe yakasimba, iyo fluorescence inobudiswa nefluorophore inodzimwa neboka rekudzima uye haigoni fluoresce.Kana iyo probe yakaora, chinhu chefluorescent chinoparadzanisa uye chinoburitsa fluorescence.

A fluorescent probe inowedzerwa kune PCR reaction solution.Munguva yeannealing process, iyo fluorescent probe inosunga kune chaiyo chinzvimbo chetemplate.Munguva yekuwedzera, iyo 5′ 3 ′ exonuclease chiitiko chePCR enzyme inogona kukanganisa fluorescent probe yakasanganiswa netemplate, uye chinhu chefluorescent chinoparadzaniswa kuti chibudise fluorescence.Nekuona iyo fluorescence kusimba kweiyo probe mune maitiro sisitimu, chinangwa chekutarisa huwandu hwekuwedzera kwePCR chigadzirwa chinogona kuwanikwa.

【Kusarudzwa kweFluorescence Detection Method】

Kana ikashandiswa kusiyanisa kutevedzana nepamusoro homology uye kuita multiplex PCR yekuona seSNP typing analysis, iyo fluorescent probe nzira haidzoreki.
Kune dzimwe Chaiyo Nguva PCR kuyedza, yakapusa, nyore uye yakaderera-inodhura fluorescent chimera nzira inogona kushandiswa.

probesSimba chaiyo, inokwanisa multiplex PCR

Kuperevedza

High specificity zvinodiwa kuti amplification;

multiplex PCR haigone kuitwaInodiwa kugadzira maprobes chaiwo, mutengo wakakwira;

dzimwe nguva probe dhizaini yakaoma

Related Products: