COVID-19 yog ib yam kab mob sib kis los ntawm tus mob hnyav ua pa nyuaj rau tus mob Coronavirus Hom 2. Thaum tus neeg kis tus kab mob, cov tsos mob tshwm sim feem ntau suav nrog kub taub hau, hnoos, thiab ua tsis taus pa.
Cov qauv siv los kuaj tau tuaj yeem sau los ntawm nasopharyngeal swabs lossis oropharyngeal swabs.
Tus txheej txheem ntawm kev kuaj pom tus mob coronavirus yog polymerase saw cov tshuaj tiv thaiv, PCR.Qhov no yog ib txoj kev siv dav hauv molecular biology.Nws tuaj yeem luam tawm ntau lab mus rau ntau lab ntawm cov DNA tshwj xeeb.
Tus kab mob coronavirus tshiab muaj ib lub genome RNA ntev heev.Txhawm rau txhawm rau txheeb xyuas cov kab mob no los ntawm PCR, RNA molecules yuav tsum tau hloov mus rau hauv lawv cov DNA txuas ntxiv los ntawm kev thim rov qab transcriptase, thiab tom qab ntawd cov DNA uas tau tsim tshiab tuaj yeem nthuav dav los ntawm cov txheej txheem PCR, uas feem ntau hu ua RT-PCR.
RT-PCR txheej txheem
RNA rho tawm
Txhawm rau ua qhov no, tus kab mob RNA yuav tsum tau muab rho tawm.Ntau yam khoom siv RNA purification tuaj yeem siv rau kev sib cais yooj yim, ceev thiab zoo.
Txhawm rau rho tawm cov kab mob RNA siv cov khoom siv coj mus muag, ua ntej ntxiv cov qauv mus rau lub raj microcentrifuge thiab tom qab ntawd sib tov nrog cov lysis tsis ua haujlwm.Qhov no tsis yog denatured heev thiab feem ntau muaj phenol thiab guanidine isothiocyanate.Tsis tas li ntawd, RNase inhibitors feem ntau muaj nyob rau hauv lysis buffer kom ntseeg tau tias kev sib cais ntawm cov kab mob tsis zoo RNA.
Tom qab ntxiv cov lysis tsis, vortex lub raj sib xyaw los ntawm pulse thiab incubate ntawm chav tsev kub.Tus kab mob no yog lysed nyob rau hauv cov mob denaturing heev muab los ntawm lysis tsis.
Tom qab cov qauv yog lysed, ib lub raj centrifuge yog siv rau cov txheej txheem purification.Cov qauv yog loaded rau hauv lub centrifuge raj thiab ces centrifuged.
Cov txheej txheem no yog cov txheej txheem rho tawm cov txheej txheem uas cov theem nyob ruaj khov muaj cov silica gel matrix.
Nyob rau hauv kev pom zoo ntsev thiab pH, RNA molecules khi rau silica membrane.
Nyob rau tib lub sijhawm, cov protein thiab lwm yam kab mob raug tshem tawm.
Tom qab centrifugation, muab lub raj centrifuge tso rau hauv ib lub raj ntim huv, pov tseg cov lim dej, thiab tom qab ntawd ntxiv cov ntxhua khaub ncaws.
Muab lub raj tso rau hauv lub centrifuge dua kom quab yuam cov ntxhua khaub ncaws los ntawm daim nyias nyias.Qhov no yuav tshem tawm tag nrho cov seem impurities ntawm daim nyias nyias, tsuas yog RNA khi rau silica gel.
Tom qab cov qauv raug ntxuav, muab lub raj tso rau hauv ib lub raj microcentrifuge huv thiab ntxiv cov elution tsis.
Tom qab ntawd nws yog centrifuged kom yuam cov elution tsis los ntawm daim nyias nyias.Lub elution tsis tshem tawm cov kab mob RNA los ntawm kab rov tav thiab tau txais RNA purified tsis muaj cov proteins, inhibitors, thiab lwm yam kab mob.
Mixed concentrate
Tom qab rho tawm tus kab mob RNA, cov kauj ruam tom ntej yog npaj cov tshuaj tiv thaiv sib xyaw rau PCR amplification.Hauv cov kauj ruam no, siv cov concentrate.Cov tshuaj concentrated no yog cov tshuaj premixed concentrated uas muaj premix, thim rov qab transcriptase, nucleotides, pem hauv ntej primer, thim rov qab primer, TaqMan sojntsuam thiab DNA polymerase.
Thaum kawg, txhawm rau ua kom tiav cov tshuaj tiv thaiv no, RNA template ntxiv.Cov hlab yog tov los ntawm pulse vortexing, thiab tom qab ntawd cov tshuaj tiv thaiv sib xyaw yog thauj mus rau hauv PCR phaj.PCR phaj feem ntau muaj 96 qhov dej thiab tuaj yeem txheeb xyuas ntau yam qauv tib lub sijhawm.
PCR amplification
Tom ntej no, muab lub phaj tso rau hauv lub tshuab PCR, uas yog qhov tseem ceeb ntawm thermal cycler.
Lub sijhawm tiag tiag RT-PCR yog siv los kuaj pom tus kabmob tshiab 2019 los ntawm kev nthuav dav lub hom phiaj ua ntu zus hauv RdrRP noob, E noob thiab N noob.Qhov kev xaiv ntawm cov hom phiaj yog nyob ntawm cov primer thiab sojntsuam ib ntus.
Thawj kauj ruam ntawm RT-PCR yog thim rov qab.Thawj strand ntawm complementary DNA yog synthesized, uas yog pib los ntawm PCR rov qab primer, uas khi rau cov complementary ib feem ntawm tus kab mob RNA genome.Tom qab ntawd thim rov qab transcriptase ntxiv DNA nucleotides rau 3′ kawg ntawm cov primer los ua ke DNA ntxiv rau cov kab mob RNA.Qhov kub thiab lub sijhawm ntawm cov kauj ruam no yog nyob ntawm cov primers, lub hom phiaj RNA, thiab rov qab transcriptase siv.
Tom ntej no, thawj kauj ruam denaturation yog siv, uas ua rau lub denaturation ntawm RNA-DNA hybrid.Cov kauj ruam no yog tsim nyog los qhib DNA polymerase.Nyob rau tib lub sijhawm, thim rov qab transcriptase yog inactivated.
PCR muaj cov txheej txheem thermal.Txhua lub voj voog muaj denaturation, annealing thiab txuas ntxiv cov kauj ruam.
Cov kauj ruam denaturation muaj cua sov cov tshuaj tiv thaiv chamber rau 95 degrees Celsius thiab siv nws rau denaturation ntawm ob-stranded DNA template.
Nyob rau hauv cov kauj ruam tom ntej, cov tshuaj tiv thaiv kub yog txo mus rau 58 degrees Celsius, tso cai rau pem hauv ntej primer rau anneal rau lub complementary ib feem ntawm nws ib leeg-stranded DNA template.Lub annealing kub ncaj qha nyob ntawm qhov ntev thiab muaj pes tsawg leeg ntawm primer.
Hauv cov kauj ruam txuas ntxiv, DNA polymerase synthesizes DNA tshiab strand uas yog ntxiv rau DNA template strand.Los ntawm kev ntxiv cov nuclei dawb ntxiv rau cov qauv hauv 5′ txog 3′ kev taw qhia los ntawm cov tshuaj tiv thaiv sib tov.Qhov kub ntawm cov kauj ruam no yog nyob ntawm DNA polymerase siv.
Tom qab thawj lub voj voog, tau txais ob lub hom phiaj DNA.
Tom qab ntawd, nkag mus rau lub voj voog thib ob.Qhov ob-stranded DNA yog denatured los tsim ob leeg-stranded DNA molecules.
Nyob rau hauv cov kauj ruam tom ntej, cov tshuaj tiv thaiv kub yog txo, cov primers yog annealed rau txhua ib leeg-stranded DNA template, thiab lub Taq-txiv neej soj ntsuam yog annealed rau lub complementary ib feem ntawm lub hom phiaj DNA.
Lub TaqMan sojntsuam muaj cov fluorophore covalently txuas mus rau 5'kawg ntawm oligonucleotide sojntsuam.Thaum zoo siab los ntawm lub teeb ci ntawm lub cycler, fluorophore emits fluorescence.Tsis tas li ntawd, qhov kev sojntsuam yog tsim los ntawm quencher ntawm 3′ kawg.Qhov sib thooj ntawm cov neeg sau xov xwm gene mus rau lub quencher tiv thaiv kom pom cov fluorescence.
Hauv cov kauj ruam txuas ntxiv, DNA polymerase synthesizes ib txoj hlua tshiab.Thaum cov polymerase ncav cuag TaqMan sojntsuam, nws cov haujlwm endogenous 5′nuclease cleaves qhov kev sojntsuam, cais cov dye los ntawm lub quencher.
Nrog rau txhua lub voj voog ntawm PCR, ntau cov dye molecules raug tso tawm, ua rau muaj kev nce hauv fluorescence siv proportional rau cov amplicons synthesized.
Txoj kev no tso cai rau kwv yees tus naj npawb ntawm ib ntu uas muaj nyob hauv cov qauv.Tus naj npawb ntawm ob-stranded DNA fragments doubles nyob rau hauv txhua lub voj voog.Yog li, PCR tuaj yeem siv los txheeb xyuas cov qauv me me.
Kev ntsuas fluorescent teeb liab, tungsten halogen teeb, lim excitation, reflector, lens, emission filter thiab charged device-siv CCD lub koob yees duab.
Kauj Ruam 4 Tshawb xyuas
Kev ntsuas fluorescent teeb liab, tungsten halogen teeb, lim excitation, reflector, lens, emission filter thiab charged device-siv CCD lub koob yees duab.
Lub teeb lim los ntawm lub teeb yog cuam tshuam los ntawm lub reflector, hla los ntawm condenser lens, thiab tsom mus rau qhov chaw ntawm txhua lub qhov.Tom qab ntawd cov fluorescence emissions los ntawm lub qhov yog reflected los ntawm daim iav, hla los ntawm lub emission lim, thiab raug kuaj los ntawm lub koob yees duab CCD.Hauv txhua lub voj voog PCR, lub teeb fluorophore tus kheej tuaj yeem kuaj pom los ntawm CCD.
Nws converts lub teeb ntes rau hauv cov ntaub ntawv digital.Txoj kev no yog hu ua PCR real-time, thiab nws tso cai rau lub sijhawm saib xyuas kev ua tiav ntawm PCR cov tshuaj tiv thaiv.
Post lub sij hawm: Lub Xya hli ntuj-19-2021