Molecular diagnosis technology siv molecular biology txoj kev los xyuas cov kev qhia thiab cov qauv ntawm cov khoom siv caj ces ntawm tib neeg lub cev thiab ntau yam kab mob, thiaj li ua tiav lub hom phiaj ntawm kev kwv yees thiab kuaj kab mob.

Nyob rau hauv xyoo tas los no, nrog rau kev kho dua tshiab thiab iteration ntawm molecular diagnostic technology, kev soj ntsuam daim ntawv thov ntawm molecular diagnostics tau ua ntau thiab ntau dua thiab nyob rau hauv-tob, thiab lub lag luam molecular diagnostics tau nkag mus rau ib lub sij hawm ntawm kev loj hlob sai.

Tus sau sau sau cov ntsiab lus ntawm kev tshuaj ntsuam xyuas cov thev naus laus zis hauv kev ua lag luam, thiab tau muab faib ua peb ntu: thawj ntu qhia txog PCR thev naus laus zis, ntu thib ob qhia txog nucleic acid isothermal amplification technology, thiab qhov thib ob qhia txog kev siv thev naus laus zis.

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Ntu I: PCR Technology

PCR technology

PCR (polymerase saw cov tshuaj tiv thaiv) yog ib qho hauv vitro DNA amplification technologies, nrog rau keeb kwm ntawm ntau tshaj 30 xyoo.

PCR thev naus laus zis tau tsim tawm hauv xyoo 1983 los ntawm Kary Mullis ntawm Cetus, Tebchaws Asmeskas.Mullis tau thov rau PCR patent hauv xyoo 1985 thiab luam tawm thawj daim ntawv PCR kev kawm ntawm Science hauv tib lub xyoo.Mullis yeej qhov Nobel nqi zog hauv Chemistry hauv 1993.

Basic Principles ntawm PCR

PCR tuaj yeem nthuav dav cov phiaj xwm DNA ntau dua li ib lab zaug.Lub hauv paus ntsiab lus yog tias nyob rau hauv catalysis ntawm DNA polymerase, niam txiv strand DNA yog siv los ua tus qauv, thiab cov primer tshwj xeeb yog siv los ua qhov pib ntawm kev txuas ntxiv.Nws yog replicated hauv vitro los ntawm cov kauj ruam xws li denaturation, annealing, thiab extension.Cov txheej txheem ntawm tus ntxhais strand DNA ntxiv rau niam txiv strand template DNA.

Tus txheej txheem PCR tau muab faib ua peb kauj ruam:

1. Denaturation: Siv qhov kub thiab txias los cais DNA ob txoj hlua.Cov hydrogen bonds ntawm DNA ob strands yog tawg ntawm qhov kub thiab txias (93-98 ° C).

2. Annealing: Tom qab ob-stranded DNA tau sib cais, qhov kub thiab txias yog txo kom cov primer khi rau ib leeg-stranded DNA.

3. Extension: Lub DNA polymerase pib tsim cov khoom sib txuas ntxiv raws li DNA strands los ntawm cov primers khi thaum qhov kub thiab txias.Thaum qhov txuas ntxiv ua tiav, lub voj voog tiav, thiab tus lej ntawm DNA tawg ob npaug.

Reciprocating peb kauj ruam 25-35 zaug, tus naj npawb ntawm DNA tawg yuav nce exponentially.

Lub ingenuity ntawm PCR yog tias cov primers sib txawv tuaj yeem tsim los rau cov hom phiaj sib txawv, yog li ntawd cov phiaj noob caj noob ces tuaj yeem nthuav dav hauv lub sijhawm luv luv.

Txog tam sim no, PCR tuaj yeem muab faib ua peb pawg, uas yog PCR zoo tib yam, fluorescent quantitative PCR thiab digital PCR.

Thawj tiam ntawm PCR zoo tib yam

Siv cov cuab yeej PCR zoo tib yam los nthuav tawm cov noob caj noob ces, thiab tom qab ntawd siv agarose gel electrophoresis txhawm rau txheeb xyuas cov khoom, tsuas yog kev soj ntsuam zoo tuaj yeem ua tiav.

Lub ntsiab tsis zoo ntawm thawj tiam PCR:

-Nyob rau qhov tsis muaj kev nthuav qhia tshwj xeeb thiab cov txiaj ntsig tsis zoo.

-Qhov kev tshawb pom yuav siv sij hawm ntev thiab kev ua haujlwm yog cumbersome.

-Tsuas yog qhov kev ntsuam xyuas zoo tuaj yeem ua tiav.

Qhov thib ob tiam fluorescence quantitative PCR

Fluorescence quantitative PCR (Real-Time PCR), tseem hu ua qPCR, yog siv los saib xyuas cov khoom sib txuam ntawm cov khoom lag luam los ntawm kev sib txuam ntawm cov teeb liab fluorescent los ntawm kev ntxiv cov fluorescent probes uas tuaj yeem qhia txog kev nce qib ntawm cov tshuaj tiv thaiv kab mob, thiab txiav txim cov txiaj ntsig los ntawm fluorescence nkhaus, thiab nws tuaj yeem ua kom muaj nuj nqis ntawm cov qauv kev sib tw.

Vim tias qPCR thev naus laus zis tau ua tiav hauv lub kaw lus kaw, qhov tshwm sim ntawm kev sib kis tau raug txo, thiab cov teeb liab fluorescence tuaj yeem raug saib xyuas kom muaj nuj nqis, yog li nws yog qhov siv dav tshaj plaws hauv kev kho mob thiab tau dhau los ua cov thev naus laus zis hauv PCR.

Cov tshuaj fluorescent siv nyob rau hauv real-time fluorescent quantitative PCR tuaj yeem muab faib ua: TaqMan fluorescent probes, molecular beacons thiab fluorescent dyes.

1) TaqMan fluorescent sojntsuam:

Thaum lub sij hawm PCR amplification, ib tug tshwj xeeb fluorescent sojntsuam yog ntxiv thaum ntxiv ib khub ntawm primers.Kev sojntsuam yog ib qho oligonucleotide, thiab ob qhov kawg tau sau npe nrog cov neeg sau xov xwm fluorescent pawg thiab pawg quencher fluorescent.

Thaum qhov kev sojntsuam tsis zoo, lub teeb liab fluorescent emitted los ntawm pawg neeg sau xov xwm yog absorbed los ntawm pawg quenching;Thaum lub sij hawm PCR amplification, lub 5′-3′ exonuclease kev ua ntawm Taq enzyme cleaves thiab degrades lub sojntsuam, ua tus reporter fluorescent pab pawg neeg thiab quencher Cov fluorescent pawg yog sib cais, kom lub fluorescence xyuas system tau txais lub fluorescence teeb liab, uas yog, txhua txhua lub sij hawm ib tug DNA strand fluorescent amplified, lub teeb liab yog ib tug amplified. tag nrho synchronized nrog tsim ntawm cov khoom PCR.

2) SYBR fluorescent dyes:

Hauv PCR cov tshuaj tiv thaiv, qhov ntau tshaj ntawm SYBR fluorescent dye yog ntxiv.Tom qab SYBR fluorescent dye tsis yog tshwj xeeb koom ua ke rau hauv DNA ob txoj hlua, nws tawm lub teeb liab fluorescent.Lub SYBR dye molecule uas tsis muab tso rau hauv cov saw yuav tsis tawm ib qho teeb meem fluorescent, yog li ua kom lub teeb liab fluorescent Qhov nce hauv PCR cov khoom lag luam yog synchronized tag nrho nrog kev nce hauv PCR cov khoom.SYBR tsuas yog khi rau ob-stranded DNA, yog li ntawd lub melting nkhaus yuav siv tau los txiav txim seb cov tshuaj tiv thaiv PCR tshwj xeeb.

3) Molecular beacons

Nws yog qia-voj ob-labeled oligonucleotide sojntsuam uas tsim cov qauv plaub hau ntawm txog 8 lub hauv paus ntawm 5 thiab 3 kawg.Cov nucleic acid sequences ntawm ob qho kawg yog ua ke ua ke, ua rau pawg fluorescent thiab pawg quenching nruj.Kaw, nws yuav tsis tsim fluorescence.

Tom qab cov khoom PCR raug tsim, thaum lub sij hawm annealing txheej txheem, nruab nrab ib feem ntawm lub molecular beacon yog ua ke nrog ib tug tshwj xeeb DNA ib theem zuj zus, thiab cov fluorescent noob yog cais los ntawm lub quencher noob los tsim fluorescence.

Lub ntsiab tsis zoo ntawm ob tiam PCR:

Qhov kev xav tau tseem tsis muaj, thiab kev tshawb pom ntawm cov qauv luam ntawv qis tsis raug.

Muaj keeb kwm muaj nuj nqis cuam tshuam, thiab qhov tshwm sim yog cuam tshuam rau kev cuam tshuam.

Peb tiam digital PCR

Cov PCR (DigitalPCR, dPCR, Dig-PCR) suav cov lej luam ntawm lub hom phiaj sib lawv liag los ntawm qhov kawg-point detection, thiab tuaj yeem ua tiav qhov kev tshawb pom tseeb tsis tas siv cov kev tswj sab hauv thiab cov qauv nkhaus.

Digital PCR siv qhov kawg ntawm kev tshawb nrhiav thiab tsis nyob ntawm tus nqi Ct (lub voj voog pib), yog li cov tshuaj PCR cov tshuaj tiv thaiv tsis tshua muaj kev cuam tshuam los ntawm kev ua kom muaj zog, thiab kev ua siab ntev rau PCR cov tshuaj tiv thaiv cov tshuaj tiv thaiv tau txhim kho, muaj qhov tseeb thiab kev rov ua dua tshiab.

Vim yog cov yam ntxwv ntawm kev nkag siab zoo thiab qhov tseeb siab, nws tsis yooj yim cuam tshuam los ntawm PCR cov tshuaj tiv thaiv cov tshuaj tiv thaiv, thiab nws tuaj yeem ua tiav qhov tseeb tsis muaj cov khoom lag luam, uas tau dhau los ua kev tshawb fawb thiab thov hotspot.

Raws li qhov sib txawv ntawm cov tshuaj tiv thaiv chav tsev, nws tuaj yeem muab faib ua peb hom: microfluidic, nti thiab droplet systems.