Hauv kev tshuaj ntsuam xyuas molecular, polymerase saw cov tshuaj tiv thaiv (PCR) tau ntev tau suav tias yog tus qauv kub rau kev kuaj pom nucleic acid. Nyob rau hauv xyoo tas los no, isothermal amplification technologies, sawv cev los ntawm recombinase polymerase amplification (RPA), tau txais kev xav ntau ntxiv vim lawv ceev, yooj yim, thiab haum rau ntawm - qhov chaw kuaj.
Txawm hais tias ob qho tib si PCR thiab RPA yog nucleic acid amplification txoj hauv kev, lawv sib txawv hauv cov qauv kev ua haujlwm, cov khoom siv, thiab cov xwm txheej ntawm daim ntawv thov, ua rau txhua lub tshuab tsim nyog rau kev kuaj mob sib txawv.
1. Lub hauv paus ntsiab lus tseem ceeb: Thermal Cycling vs. Constant-Temperature Amplification
PCR yog cov txheej txheem biochemical muaj peev xwm nthuav tawm ib qho DNA molecule rau ntau lab luam nyob rau lub sijhawm luv luv. Cov tshuaj tiv thaiv yog nyob ntawm kev rov ua dua thermal cycling thiab muaj peb kauj ruam tseem ceeb:
1.Denaturation (90–95 degree) – Muab ob npaug rau- DNA stranded tau muab cais ua ib leeg.
2.Annealing (55–65 degree) – Primers khi rau lub hom phiaj sib lawv liag.
3.Extension (72 degree) – DNA polymerase synthesizes DNA strands tshiab.
Los ntawm cov ntsuas kub no - tswj cov voj voog, lub hom phiaj DNA tau nthuav dav nthuav dav nrog qhov tseeb. Txawm li cas los xij, PCR yuav tsum tau siv cov cuab yeej thermal cycling thiab feem ntau siv sijhawm 1-2 teev kom tiav.
Hauv qhov sib piv, RPA ua haujlwm ntawm qhov ntsuas kub tsis tu ncua ntawm 37-42 degree, ua kom nrawm nucleic acid amplification yam tsis muaj thermal cycling. Cov thev naus laus zis tso siab rau kev sib koom tes ntawm cov proteins recombinase, ib leeg - tso tseg DNA- khi cov proteins, thiab strand- tshem tawm DNA polymerase kom ua tiav lub hom phiaj tshwj xeeb loj hauv isothermal tej yam kev mob.
DNA polymerase siv nyob rau hauv RPA muaj strand displacement kev ua si tab sis tsis muaj 5'→ 3' exonuclease kev ua. Thaum lub sij hawm amplification, cov primers sab nraud txuas nrog lub template strand. Thaum lawv ntsib ob - cov cheeb tsam tso tseg uas tau tsim los ntawm cov primers sab hauv, cov polymerase tsis tuaj yeem degrade downstream strand thiab hloov pauv nws, tsim cov khoom lag luam amplification tshiab.
Tag nrho cov tshuaj tiv thaiv tau ua tiav nyob rau hauv qhov kub thiab txias, tshem tawm qhov xav tau ntawm cov cuab yeej siv nyuaj thiab ua kom muaj tseeb qhov tseeb - ntawm - kev saib xyuas thiab teb - raws li kev sim. Los ntawm kev koom ua ke rov qab transcriptase, RPA kuj tuaj yeem siv rau RNA lub hom phiaj nrhiav pom.
Amplification cov txiaj ntsig tuaj yeem txheeb xyuas tau siv ntau hom kev nyeem ntawv, suav nrog kev tshawb pom qhov kawg, tiag - lub sijhawm fluorescence saib xyuas, lateral flow assays, thiab tuaj yeem txuas ntxiv nrog CRISPR-Cas systems, microfluidics, lossis tom ntej- tiam tom ntej (NGS) rau kev txhim kho qib siab.
Daim Ntawv Thov Scenarios: Laboratory Precision vs. On -Site Rapid Testing
PCR tau dhau los ua ib qho tseem ceeb tshaj plaws kev tshuaj ntsuam xyuas technologies thiab tseem yog tus qauv kub rau kev kuaj nucleic acid.
Ua tsaug rau nws qhov raug siab, tus nqi -kev ua tau zoo, thiab kev ua kom muaj zog, PCR tau siv dav hauv kev kuaj mob, cov chaw kuaj mob hauv nruab nrab, cov chaw tshawb fawb, thiab cov phiaj xwm loj - ntsuas ntsuas, qhov twg cov txheej txheem thiab cov neeg ua haujlwm tau txais kev kawm tau yooj yim.
RPA, ntawm qhov tod tes, muaj qhov zoo meej hauv kev nrawm, ntawm - qhov chaw kuaj mob. Nrog kev nthuav dav ua tiav hauv tsawg li 20 feeb, cov khoom siv tsawg tsawg, thiab kev ua haujlwm yooj yim, RPA yog qhov tsim nyog rau.
- Kev kho mob thawj zaug thiab kev teeb tsa xwm txheej ceev
- Kev lis kev cai thiab kev soj ntsuam ciam teb
- Kev tshuaj ntsuam xyuas tsiaj thiab nroj tsuag kab mob
- Kev kuaj ib puag ncig thiab zaub mov kev nyab xeeb
Thaum ua ke nrog cov khoom siv nqa tau yooj yim thiab cov hlab ntsha ntws sab nraud, RPA ua kom ceev thiab txhim khu kev qha cov tshuaj tiv thaiv kab mob sab nraud cov chaw sim tshuaj.
MIRA (Multienzyme Isothermal Rapid Amplification)yog Amp Future tus tswv isothermal amplification technology. Nws belongs rau tib tsev neeg kev txawj ntse xws li RPA thiab tau txais kev tshoov siab los ntawm ntuj DNA recombination thiab kho cov txheej txheem, siv ntau lub zog enzymes kom ua tiav cov nucleic acid amplification ceev nyob rau hauv qhov kub thiab txias.
MIRA ntiav cov txheej txheem sib koom ua ke ntawm tsib lub enzymes tseem ceeb, suav nrog helicase, recombinase, ib leeg -stranded DNA-binding protein, thiab DNA polymerase, los pab:
- Kev khiav hauj lwm kub ntau yam: 25℃-45 degree
- Kev nthuav dav nrawm: tau tshwm sim hauv 20 feeb
- Siab rhiab heev thiab tshwj xeeb
- Txhim khu kev nyab xeeb thiab txo qhov kev pheej hmoo kis kab mob
MIRA yog ib lub tshuab nucleic acid amplification technology tshwj xeeb uas tsim los rau qhov tseeb ntawm -site thiab taw tes- ntawm - kev kuaj mob.
Lub tsev ntawm MIRA platform, Amp Yav Tom Ntej tau tsim ib qho kev nthuav dav ntawm cov khoom lag luam rau POCT sai molecular, suav nrog:
Rapid Nucleic Acid Release Agent:
Amplifiable nucleic acid yog tau nyob rau hauv 5 feeb ntawm chav tsev kub; tsawg inhibition, tso cai rau tag nrho cov qauv thauj khoom txog li 50 μL; chav thauj kub thiab khaws cia, tsis muaj kev tiv thaiv lub teeb.
Isothermal Nucleic Acid Amplification Reagent:
Amplification ua tiav hauv 5-20 feeb, nrawm thiab ua haujlwm tau zoo; ntau hom reagent (lyophilized hmoov, lyophilized microspheres, thiab lwm yam); Ntau yam kev nthuav qhia tshwm sim (electrophoresis, fluorescence, xeem strips).
Miniaturized Portable Devices:
Digital isothermal detectors, hnab tshos{0}}cov cuab yeej ntsuas qhov loj me, yooj yim tuav hauv ib txhais tes, nruab nrog lub txhab nyiaj fais fab, tso koj tawm ntawm qhov kev txwv ntawm chav kuaj thiab nqa qhov tseeb nucleic acid kuaj rau thaj chaw.
Ib leeg/ntau yam xeem strips:
Optimized raws li txoj kev MIRA rau kev sib raug zoo dua; ua ke nrog MIRA thev naus laus zis, ua kom pom kev yooj yim.
Fully Automated Nucleic Acid Amplification Analyzer:
Yuav tsum tsuas yog ib qho qauv thauj khoom ua haujlwm rau kev kuaj pom zoo tag nrho; nrhiav kom tau txwv mus rau ib daim ntawv; isothermal amplification thiab tsis siv neeg capping txo qhov kev pheej hmoo ntawm kev sib kis.
Ntawm -Site Rapid Testing package:
Ntawm - qhov - kuaj qhov chaw, txhua lub sijhawm, nyob qhov twg, tag nrho hauv ib pob; ua tiav kev sim hauv 30 feeb, yog thiab rhiab heev.
Yog xav paub ntxiv txog cov khoom lag luam lossis cov kev daws teeb meem, thov koj xav tiv tauj peb.