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Ibintu byinshi byavumbuwe mu mateka yubuhanga bwo gutahura mubitekerezo byanjye ni tekinoroji ikingira indwara ishingiye ku ihame rya antigen-antibody yihariye ihuza, ikoranabuhanga rya PCR hamwe n’ikoranabuhanga rikurikirana.Uyu munsi tuzavuga kubijyanye n'ikoranabuhanga rya PCR.Ukurikije ubwihindurize bwa tekinoroji ya PCR, abantu basanzwe bagabanya ikoranabuhanga rya PCR mubisekuru bitatu: tekinoroji isanzwe ya PCR, igihe nyacyo cya fluorescent numero ya PCR nubuhanga bwa PCR.

Ctekinike ya PCR

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KARY MULLIS (1944.12.28-2019.8.7)

Kary Mullis yahimbye urunigi rwa polymerase (reaction ya polymerase reaction, PCR) mu 1983. Bavuga ko igihe yari atwaye umukunzi we, yahise agira igitekerezo cyo guhumeka maze atekereza ku ihame rya PCR (ku nyungu zo gutwara).Kary Mullis yahawe igihembo cyitiriwe Nobel muri Chimie mu 1993. Ikinyamakuru New York Times cyagize kiti: "Umwimerere kandi ukomeye, hafi yo kugabanya ibinyabuzima mu bihe byabanjirije PCR na nyuma ya PCR.

Ihame rya PCR: Munsi ya catalizike ya polymerase ya ADN, ADN yumugore wa ADN ikoreshwa nkicyitegererezo, naho primer yihariye ikoreshwa nkintangiriro yo kwaguka, naho umukobwa wumugozi ADN yuzuzanya nicyitegererezo cyababyeyi ADN ikopororwa muri vitro ikoresheje denatration, annealing, kwagura nizindi ntambwe.Nubuhanga bwa ADN synthesis amplification tekinoroji muri vitro, ishobora kwihuta kandi byumwihariko kwagura ADN iyo ari yo yose muri vitro.

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Ibyiza bya PCR isanzwe
1.Uburyo bwa kera, bwuzuye amahame mpuzamahanga no murugo
2.Igiciro gito cyibikoresho reagent
3.Ibicuruzwa bya PCR birashobora kugarurwa kubindi bigeragezo bya biologiya
Basabwe Foregene imashini ya PCR:
Ibicuruzwa bifitanye isano: https://www.foreivd.com/pcr-herotm-kuri-dye-ibicuruzwa/
Ibibi bya PCR isanzwe
1.byoroshye kwanduza
2.imikorere itoroshye
3.isesengura ryujuje ubuziranenge
4.Gucisha make
5.Hano hari amplification idasanzwe, kandi mugihe umurongo udasanzwe ufite ubunini bungana na bande yintego, ntibishobora gutandukanywa
 
Capillary electrophoresis ishingiye kuri PCR
Mu gusubiza amakosa ya PCR isanzwe, abayikora bamwe bazanye ibikoresho bishingiye ku ihame rya capillary electrophoresis.Intambwe ya electrophoreis nyuma ya amplification ya PCR irangiye muri capillary.Ibyiyumvo biri hejuru, kandi itandukaniro ryibanze rishobora gutandukana kandi amplification irashobora kubarwa na MAERKER.ibicuruzwa.Ikibi nuko ibicuruzwa bya PCR bigikenewe gukingurwa no gushyirwa mubikoresho, kandi haracyari ibyago byinshi byo kwanduza.

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CapillaryEYamazaki

 

2. Ikirangantego nyacyo cya fluorescent PCR (Quantitative Real-time PCR, qPCR) tekinorojiFluorescent quantitative PCR, nanone yitwa Real-Time PCR, ni tekinoroji nshya ya acide nucleic acide yakozwe na PE (Perkin Elmer) mumwaka wa 1995. Amateka yiterambere rya fluorescent quantitative PCR ni amateka yintambara zitera imbaraga ibihangange nka ABI, Roche, na Bio-Rad.Niba ubishaka, urashobora kubigenzura.Ubu buhanga nubuhanga bukuze kandi bukoreshwa cyane igice cya kabiri cya PCR.

Imashini isabwa qPCR Imashini: https: //www.foreivd.com/mini-yukuri-igihe-pcr-imikorere-impamvu-sf2sf4-ibicuruzwa/

Uburyo bwo gusiga irangi rya Fluorescent (SYBR Icyatsi I):SYBR Icyatsi cya I ni cyo gikoreshwa cyane muri ADN ihuza irangi rya PCR igereranya, ihuza bidasanzwe na ADN ebyiri.Muri leta yubuntu, SYBR Green isohora fluorescence nkeya, ariko iyo ihujwe na ADN ebyiri, fluorescence yayo yiyongera inshuro 1000.Kubwibyo, ibimenyetso byose bya fluorescente bitangwa nigisubizo bigereranwa nubunini bwa ADN ebyiri zihari kandi biziyongera hamwe no kongera ibicuruzwa byongerewe.Kubera ko irangi rihuza bidasanzwe na ADN ikubye kabiri, ibisubizo byiza bitari byo bishobora kubyara.

Ibicuruzwa bifitanye isano: https://www.foreivd.com/ukuri-igihe-pcr-easytm-sybr-icyatsi--kit-product/

Uburyo bwa Fluorescent probe (tekinoroji ya Taqman): MugiheKwiyongera kwa PCR, iperereza ryihariye rya fluorescent ryongewe mugihe kimwe na primers.Iperereza ni umurongo wa oligonucleotide, hamwe nitsinda ryabanyamakuru ba fluorescent hamwe nitsinda rya florescent quencher ryanditse kumpande zombi.Iyo iperereza ridahwitse, ikimenyetso cya fluorescent cyatanzwe nitsinda ryabanyamakuru cyakirwa nitsinda rizimya, kandi gutahura Nta kimenyetso cya fluorescent;mugihe cyo kongera PCR (murwego rwo kwaguka), ibikorwa bya 5'-3 'Dicer ya Taq enzyme izajya igogora kandi itesha agaciro iperereza, kugirango itsinda ryumunyamakuru fluorescent hamwe nitsinda rya quencher fluorescent ritandukane, kugirango sisitemu yo gukurikirana fluorescence Ikimenyetso cya fluorescente gishobora kwakirwa, ni ukuvuga ko burigihe burigihe urunigi rwa ADN rwongerewe imbaraga, rukerekana ibimenyetso bya fluorescent ya PCR.Uburyo bwa Taqman probe nuburyo bukoreshwa cyane mugushakisha ivuriro.

Ibicuruzwa bifitanye isano: https://www.foreivd.com/quickeasy%e1%b5%80%e1%b4%b9-igihe- gihe-pcr-kit-taqman-product/

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Ibyiza bya qPCR
1.Uburyo burakuze kandi ibikoresho bifasha na reagent biruzuye
2.Igiciro giciriritse cya reagent
3.byoroshye gukoresha
4.Kumenyekanisha cyane kandi byihariye
 
Ibibi bya qPCR

Guhinduranya kwa gene iganisha ku kubura gutahura.
Gutahura ibisubizo byubushakashatsi bwibanze ntibishobora kugenwa.
Hariho ikosa rinini mugihe ukoresheje umurongo usanzwe wo gutahura umubare.
 
3. Ikoranabuhanga rya PCR (Digital PCR, dPCR) tekinoroji
Digital PCR nubuhanga bwo kugereranya byimazeyo molekile ya nucleic.Ugereranije na qPCR, PCR ya digitale irashobora gusoma mu buryo butaziguye umubare wa molekile ya ADN / RNA, niwo mubare wuzuye wa molekile ya acide nucleique muburyo bwo gutangira.Mu 1999, Bert Vogelstein na Kenneth W. Kin-zler basabye ku mugaragaro igitekerezo cya dPCR.
 
Muri 2006, Fluidigm niyambere mu gukora chip yubucuruzi ishingiye kuri dPCR.Muri 2009, Life Technologies yatangije sisitemu ya OpenArray na QuantStudio 12K Flex dPCR.Muri 2013, Life Technologies yatangije sisitemu ya QuantStudio 3DdPCR, ikoresha tekinoroji ya nanoscale microfluidic chip ikora cyane kugirango ikwirakwize ingero kuri selile 20.000.in reaction.

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Mu mwaka wa 2011, Bio-Rad yashyize ahagaragara igikoresho gishingiye ku gitonyanga cya QX100 dPCR, ikoresha ikoranabuhanga ry’amazi mu mavuta kugira ngo igabanye icyitegererezo ku mazi 20.000 y’amazi-y’amavuta, kandi ikoresha isesengura ry’ibitonyanga mu gusesengura ibitonyanga.Mu mwaka wa 2012, RainDance yashyize ahagaragara igikoresho cya RainDrop dPCR, itwarwa na gaze y’umuvuduko ukabije, kugira ngo igabanye buri sisitemu isanzwe y’imyitozo ngororamubiri irimo miriyoni 1 kugeza kuri miliyoni 10 za picoliter yo mu rwego rwa mikoro.

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Kugeza ubu, Digital PCR yashizeho ibice bibiri byingenzi, ubwoko bwa chip nubwoko butonyanga.Ntakibazo cyubwoko bwa PCR, amahame yibanze aragabanya kugabanuka, amaherezo ya PCR no gukwirakwiza Poisson.Sisitemu isanzwe ya PCR irimo aside nucleic templates igabanijwemo ibice ibihumbi icumi bya PCR reaction, bigabanywa kuri chip cyangwa microdroplets, kuburyo buri reaction irimo ibishoboka byose byerekana molekile yicyitegererezo, kandi reaction ya molekile imwe ya PCR irakorwa.Mugusoma fluorescence Kuboneka cyangwa kutaboneka kw'ikimenyetso birabaze, kandi umubare wuzuye ukorwa nyuma ya kalibrasi yo gukwirakwiza imibare ya Poisson.

Ibikurikira nibiranga urubuga rwa PCR rwinshi nakoresheje:

1. Bio-Rad QX200 igitonyanga cya digitale PCR Bio-RadQ.Igikorwa kiragoye, kandi ibyago byo guhumana ni bito.

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Xinyi TD1 micro-igitonyanga digitale PCRXinyi TD1 ni porogaramu ya PCR yo murugo, inzira yibanze yo gutahura: kubyara 30.000-50.000 byamazi-y-amavuta muri peteroli binyuze mumashanyarazi, byongerera imbaraga igikoresho rusange cya PCR, hanyuma ugahita unyura Umusomyi wigitonyanga asoma ibimenyetso bya fluorescent ya buri gitonyanga.Byombi ibisekuruza hamwe no gusoma mururu rubuga bikorerwa muri chip yabugenewe ifite ibyago bike byo kwanduza.

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 STILLA Naica micro-ibitonyanga chip digitale PCRSTILLA Naica ni urubuga rushya rwa PCR.Uburyo bwibanze bwo gutahura ni: ongeraho igisubizo cyibisubizo kuri chip, shyira chip muri micro-droplet generation na amplification sisitemu, hanyuma ubyare 30.000 mikoro.Gukwirakwiza kuri chip, kandi PCR amplification irangiye kuri chip.Hanyuma chip yongerewe imbaraga yimurirwa muri sisitemu yo gusesengura micro-igitonyanga, kandi ibimenyetso bya fluorescent bisomwa no gufata amashusho.Kubera ko inzira zose zibera muri chip ifunze, ibyago byo kwandura ni bike.

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4. ThermoFisher QuantStudio 3D chip digitale PCR

ThermoFisher QuantStudio 3D nubundi buryo bwa chip bushingiye kuri sisitemu ya PCR.Uburyo bwibanze bwo gutahura ni: ongeramo igisubizo cyibisubizo mubikwirakwiza, hanyuma ukwirakwize igisubizo kiboneye kuri chip hamwe na microwell 20.000 binyuze mukwirakwiza., shyira chip kumashini ya PCR kugirango wongere, hanyuma ushyire chip mubasomyi hanyuma ufate ifoto kugirango usome ibimenyetso bya fluorescent.Igikorwa kiragoye, kandi inzira yose ikorerwa muri chip ifunze, kandi ibyago byo kwandura ni bike.

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5. JN MEDSYS Ibisobanuro bya chip digitale PCR

JN MEDSYS Ubusobanuro nuburyo bushya bwa chip-bwoko bwa sisitemu ya PCR.Uburyo bwibanze bwo gutahura ni: ongeraho igisubizo cyibisubizo mubisaba, hanyuma ukwirakwize igisubizo cya reaction ku miyoboro 10,000 PCR yashyizwe mumiyoboro ya PCR binyuze mubisaba.Kuri chip ya microporome, igisubizo cyibisubizo byinjira muri chip binyuze mubikorwa bya capillary, hanyuma umuyoboro wa PCR hamwe na chip ushyirwa kumashini ya PCR kugirango wongere imbaraga, hanyuma amaherezo chip ashyirwa mubasomyi kugirango basome ibimenyetso bya fluorescent bafata ifoto.Igikorwa kiragoye.Ibyago byo kwandura ni bike.

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Ibipimo bya buri platform ya PCR igizwe nincamake kuburyo bukurikira:

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Ibipimo byerekana isuzuma rya sisitemu ya PCR ni: umubare wibice byacitsemo ibice, umubare wimiyoboro ya fluorescente, ibikorwa bigoye hamwe ningaruka zo kwanduza.Ariko icy'ingenzi ni ukumenya neza.Uburyo bumwe bwo gusuzuma urubuga rwa PCR ni ugukoresha sisitemu nyinshi ya PCR kugirango igenzure, kandi ubundi buryo ni ugukoresha ibintu bisanzwe bifite agaciro nyako.

Ibyiza bya dPCR
1.Kugera ku mubare wuzuye
2.Ibyiyumvo bihanitse kandi byihariye
3.Urashobora gutahura kopi ntoya
Ibibi bya dPCR1. Ibikoresho bihenze na reagent 2. Igikorwa kitoroshye nigihe kirekire cyo gutahura 3. Urwego ruto rwo kumenya

Kugeza ubu, ibisekuru bitatu byikoranabuhanga rya PCR bifite inyungu zabyo nibibi, kandi buriwese afite aho akorera, kandi ntabwo ari isano igisekuru gisimbuza ikindi.Iterambere rihoraho ryikoranabuhanga ryinjije imbaraga nshya mu ikoranabuhanga rya PCR, bituma rishobora gufungura icyerekezo kimwe gikoreshwa, bigatuma acide nucleic acide yoroha kandi neza.
Inkomoko: Dr. Yuan aragutwara kwipimisha
 
Ibicuruzwa bisabwa:


Igihe cyo kohereza: Ugushyingo-18-2022