Direct PCR yog cov tshuaj tiv thaiv uas ncaj qha siv tsiaj los yog cov ntaub so ntswg rau amplification yam tsis muaj nucleic acid extraction.Hauv ntau txoj hauv kev, ncaj qha PCR ua haujlwm zoo li PCR li niaj zaus

Qhov sib txawv tseem ceeb yog cov kev cai tsis siv rau hauv PCR ncaj qha, cov qauv tuaj yeem raug ncaj qha rau PCR cov tshuaj tiv thaiv yam tsis muaj nucleic acid extraction, tab sis muaj cov kev cai sib xws rau kev ua siab ntev ntawm cov enzymes thiab kev sib raug zoo ntawm qhov tsis cuam tshuam nrog cov tshuaj tiv thaiv PCR ncaj qha.

Txawm hais tias muaj ntau dua lossis tsawg dua PCR inhibitors hauv cov qauv piv txwv, ncaj qha PCR tseem tuaj yeem ua tiav kev txhim kho txhim khu kev qha raws li kev ua ntawm enzymes thiab buffers.Cov tshuaj tiv thaiv PCR ib txwm xav tau cov tshuaj zoo nucleic acid ua tus qauv, uas tuaj yeem cuam tshuam qhov kev ua tiav ntawm PCR cov tshuaj tiv thaiv yog tias tus qauv muaj cov proteins thiab lwm yam impurities.Direct PCR yog tam sim no ib qho ntawm cov thev naus laus zis nrov tshaj plaws hauv kev kuaj mob molecular.

01 Direct PCR yog thawj zaug siv rau tsiaj thiab nroj tsuag

Daim ntawv thov thawj zaug ntawm PCR ncaj qha yog nyob rau hauv thaj tsam ntawm cov tsiaj thiab cov nroj tsuag, xws li cov ntshav, cov ntaub so ntswg thiab cov plaub hau ntawm nas, miv, qaib, luav, yaj, nyuj, thiab lwm yam., cov nplooj thiab cov noob, thiab lwm yam., siv los kawm txog genotyping, transgenic, plasmid detection, Gene knockout analysis, DNA source identification, species identifications, SNP.

Cov teb no muaj qee qhov zoo sib xws, uas yog, lub hom phiaj cov ntsiab lus tseem ceeb heev thiab nucleic acid extraction yog teeb meem, yog li PCR ncaj qha tsis tuaj yeem txuag lub sijhawm thiab muaj kev cuam tshuam me me rau cov txiaj ntsig, tab sis kuj txuag nqi.

Cov PCR ncaj qha siv rau kev kuaj kab mob yog qhov teeb meem ntawm xyoo tas los no, qee cov tuam txhab PCR reagent tau siv zog ntau hauv cov lus qhia no thaum ua kev tsim kho tshiab.Tshwj xeeb tshaj yog nyob rau hauv qhov kev kis tus kab mob COVID-19 no, ntau yam khoom kuaj pom tau tshwm sim hauv khw, xws li SARS-CoV-2 Nucleic Acid Detection Kit (Multiplex PCR Fluorescent Probe Method) tshawb fawb thiab tsim los ntawm Foregene, uas siv Real-time RT PCR thev naus laus zis (rRT-PCR) rau kev kuaj pom zoo ntawm SARS-CoV nucleus lossis SARS-CoV-2. .

Foregene yog ib qho ntawm cov tuam txhab uas siv Direct PCR thev naus laus zis, txhawm rau kuaj pom qhov qub ORF1ab, N, E, thiabtxawv lineages nucleic acids in human nasopharyngeal or oropharyngeal swab samples xws li SARS-CoV-2 B.1.1.7 lineage (UK), B.1.351 lineage (ZA), B.1.617 lineage (IND) and P.1 lineage (BR).

02  Reagents xav tau rau PCR ncaj qha

Qauv Lysate

Cov qauv lysate tuaj yeem teeb tsa los ntawm koj tus kheej lossis yuav.Qhov sib txawv ntawm cov khoom sib txawv ntawm lysate yuav ua rau lub peev xwm lysing txawv, thiab tom qab ntawd lub sij hawm lysing yuav txawv me ntsis.Piv txwv li, rau kev npaj cov ntaub so ntswg tsiaj, 30 feeb los yog hmo ntuj lysis feem ntau pom zoo, thiab cov tshuaj lysis rau cov kab mob nyob ntawm 3-10 feeb.

PCR master mix

Nws raug nquahu kom siv kub-pib DNA polymerase los txhim kho qhov tshwj xeeb amplification thiab ua kom muaj peev xwm nthuav dav.Lub hauv paus ntawm PCR ncaj qha yog ib qho kev ua siab ntev polymerase.

Tshem tawm lossis inhibit cov khoom hauv cov qauv uas cuam tshuam rau DNA amplification

Tom qab cov qauv ua tiav nrog cov lysate, cov protein, lipids thiab lwm cov khib nyiab ntawm tes yuav raug tso tawm, cov tshuaj no yuav inhibit PCR cov tshuaj tiv thaiv.Yog li ntawd, ncaj qha PCR yuav tsum tau ntxiv cov kev tshem tawm los yog inhibitors kom txo tau qhov cuam tshuam ntawm cov yam ntxwv no.

03  Ib qho kev sau ntawm tsib lub ntsiab lus kev paub ntawm PCR ncaj qha

Ua ntej, Direct PCR thev naus laus zis yog kev siv tshuab PCR ncaj qha rau ntau yam kev kuaj lom.Nyob rau hauv cov kev mob no, tsis tas yuav tsum tau cais thiab rho tawm cov nucleic acid, ncaj qha siv cov ntaub so ntswg qauv raws li cov khoom, thiab ntxiv lub hom phiaj gene primers yog ua cov tshuaj tiv thaiv PCR.

Thib ob, Direct PCR thev naus laus zis tsis yog tsuas yog ib txwm siv DNA template amplification technology, tab sis kuj suav nrog RNA template thim rov qab PCR.

Thib peb, Direct PCR thev naus laus zis tsis yog tsuas yog ua haujlwm ncaj qha rau PCR cov txiaj ntsig zoo ntawm cov qauv ntaub so ntswg, tab sis kuj suav nrog Real-Time qPCR cov tshuaj tiv thaiv, uas yuav tsum muaj cov tshuaj tiv thaiv kom muaj zog tiv thaiv keeb kwm yav dhau los cuam tshuam muaj peev xwm thiab endogenous fluorescence quenchers lub peev xwm antagonistic.

Plaub, cov qauv tsom los ntawm Direct PCR thev naus laus zis tsuas yog xav tau kev tso tawm nucleic acid templates, thiab tsis txhob tshem tawm cov proteins, polysaccharides, ntsev ions, thiab lwm yam uas cuam tshuam rau PCR cov tshuaj tiv thaiv.Uas yuav tsum tau cov nucleic acid polymerase thiab PCR Mix nyob rau hauv cov tshuaj tiv thaiv system kom muaj zoo heev kuj thiab adaptability kom paub meej enzyme kev ua si thiab replication raug nyob rau hauv complex tej yam kev mob.

Thib tsib, cov qauv ntawm cov ntaub so ntswg tsom los ntawm Direct PCR thev naus laus zis yam tsis muaj kev kho mob nucleic acid ntxiv thiab tus nqi ntawm cov qauv me me, uas yuav tsum muaj cov txheej txheem tshuaj tiv thaiv kom muaj qhov ua tau zoo heev thiab ua kom muaj txiaj ntsig zoo.