PCR nubuhanga bukoreshwa cyane muri nucleic aside amplification kandi ikoreshwa cyane kubera sensibilité yayo kandi yihariye.Nyamara, PCR isaba gutandukanya ubushyuhe bwumuriro kandi ntishobora gukuraho imbogamizi zo kwishingikiriza kubikoresho nibikoresho, bigabanya imikoreshereze yabyo mugupima kwa muganga.

Kuva mu ntangiriro ya za 90, laboratoire nyinshi zatangiye guteza imbere tekinoroji yo kongera ubushyuhe budakenera guhindagurika.Noneho bateje imbere tekinoroji ya isothermal amplification tekinoroji, gusimbuza umugozi isothermal amplification tekinoroji, kuzenguruka uruziga isothermal amplification tekinoroji, hamwe na nucleic aside ikurikirana.Ikoreshwa rya Isothermal amplification tekinoroji nubundi buryo bwikoranabuhanga. 

Loop-medrated isothermal amplification

Ihame rya amplification rishingiye ku kuba ADN iri mu buryo bungana na 65 ° C.Iyo primer iyo ari yo yose ifatanije kandi ikagurwa kugeza ku gice cyuzuzanya cya ADN ebyiri, undi murongo uzatandukana ugahinduka umurongo umwe.

Kuri ubu bushyuhe, ADN ikoresha primers 4 yihariye kugirango yishingikirize kumurongo wa ADN polymerase kugirango ikore synthèse ya ADN yimuka ikomeza.

Banza umenye uturere 6 twihariye F3, F2, F1, B1, B2, B3 kuri gene yagenewe, hanyuma ushushanye primers 4 ukurikije uturere 6 twihariye (nkuko bigaragara ku gishushanyo gikurikira):

Imbere yimbere (FIP) igizwe na F1c na F2.

Imbere yimbere yimbere (BIP) igizwe na B1c na B2, naho TTTT ikoreshwa nka space hagati.

Imbere ya F3 na B3 igizwe nibice bya F3 na B3 kuri gen igenewe.

Muri sisitemu ya reaction ya LAMP, kwibanda kuri primer y'imbere ni inshuro nyinshi ugereranije na primer yo hanze.Imbere yimbere yabanje guhuzwa nicyitegererezo cyumugozi kugirango ushushanye umurongo wuzuzanya kugirango ugire ADN ya kabiri.Ibikurikira, primer yo hanze ihujwe nicyitegererezo cyumugozi kugirango ikore ADN ya kabiri.Mubikorwa bya BstDNA polymerase, umurongo wuzuzanya ukomatanya na primer y'imbere urekurwa.Nyuma yuruhererekane rwibisubizo, umurongo wuzuzanya amaherezo ukora umurongo umwe wa ADN ufite imiterere itavuga.

Imiterere ya dumbbell ADN umurongo umwe ubwayo ikoreshwa nkicyitegererezo kugirango uhore ukora imiterere yinzibacyuho ya ADN ifite impera ifunguye.Imbere ninyuma yambere iyobora imiterere yinzibacyuho ya stem-loop imiterere ya ADN kugirango ikomeze guhindagurika kwimuka no kwaguka, hanyuma amaherezo ikore stem-loop yubatswe ifite uburebure butandukanye.Uruvange rwa ADN.

Ibyiza nibibi bya loop-medrated isothermal amplification

Ibyiza bya LAMP:

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(2) Igihe cyo kubyitwaramo ni kigufi, umwihariko urakomeye, kandi nta bikoresho byihariye bisabwa.

Inenge za LAMP:

(1) Ibisabwa kuri primers ni byinshi cyane.

(2) Ibicuruzwa byongerewe imbaraga ntibishobora gukoreshwa mugukwirakwiza no gukurikiranya, ariko birashobora gukoreshwa gusa murubanza.

(3) Bitewe nubukangurambaga bukomeye, biroroshye gukora aerosole, bitera ibyiza bibi kandi bigira ingaruka kubisubizo.

SKwimura ibicuruzwa

Kwiyongera kwimurwa (SDA) ni muri vitro isothermal ADN tekinike yo kongera imbaraga zishingiye ku myitwarire ya enzymatique yatanzwe bwa mbere n’intiti y’umunyamerika Walker mu 1992.

Sisitemu shingiro ya SDA ikubiyemo kubuza endonuclease, ADN polymerase hamwe nigikorwa cyo kwimura imirongo, ibice bibiri bya primers, dNTPs, na calcium na magnesium ion na sisitemu ya buffer.

Ihame ryo kwimura imirongo ihindagurika rishingiye ku miti yahinduwe mu buryo bwa shimi endonuc Please kumenyekanisha ku mpande zombi za ADN.Endonuclease ifungura icyuho muri ADN umurongo aho yamenyekanye, kandi polymerase ya ADN yongerera icyuho 3 ′ Kurangiza no gusimbuza umurongo ukurikira wa ADN.

Imirongo imwe yasimbuwe ya ADN irashobora guhuzwa na primers hanyuma ikagurwa mumirongo ibiri na ADN polymerase.Iyi nzira isubirwamo ubudahwema, kugirango intego ikurikiranye yongerewe neza.

Ibyiza nibibi bya tekinoroji yo kwimura umurongo

Ibyiza bya SDA:

Gukora neza ni byinshi, igihe cyo kubyitwaramo ni kigufi, umwihariko urakomeye, kandi nta bikoresho byihariye bisabwa.

Inenge za SDA:

Ibicuruzwa ntabwo ari bimwe, kandi bimwe mubicuruzwa bimwe kandi byikubye kabiri bikorerwa muburyo bwa SDA, kandi umurizo byanze bikunze bizabaho mugihe byamenyekanye na electrophorei.

Rolling umuzenguruko

Kuzunguruka uruziga (RCA) bisabwa gushushanya uburyo bwo gukoporora ADN ibinyabuzima bitera indwara ukoresheje uruziga.Ryerekeza ku ikoreshwa rya ADN izunguruka imwe nk'icyitegererezo ku bushyuhe buhoraho, hamwe na polymerase idasanzwe ya ADN (nka Phi29)) Mu gikorwa cyo kuzunguruka uruziga ADN kugira ngo igere kuri gene igamije.

RCA irashobora kugabanywamo umurongo wo kwongera umurongo no kwongerwaho imbaraga.Imikorere yumurongo RCA irashobora kugera kuri 10⁵inshuro, hamwe nubushobozi bwa RCA yerekanwe bishobora kugera 10⁹ibihe.

Itandukaniro ryoroshye, nkuko bigaragara ku gishushanyo gikurikira, umurongo wo kwongera umurongo ukoresha gusa primer 1, amplification amplonction b ifite primers 2.

Umurongo RCA nanone witwa primer RCA imwe.Primer ihuza ADN izenguruka kandi ikongerwa nigikorwa cya ADN polymerase.Igicuruzwa ni umurongo umwe umurongo hamwe numubare munini wisubiramo inshuro ibihumbi nuburebure bwumuzingo umwe.

Kubera ko ibicuruzwa byumurongo RCA bihora bihujwe no gutangira primer, gukosora byoroshye ibimenyetso nibyiza byingenzi.

Exponential RCA, izwi kandi nka Hyper branched amplification HRCA (Hyper branched RCA), muri exponential RCA, primer imwe yongerera ibicuruzwa RCA, primer ya kabiri ivanga nibicuruzwa bya RCA ikaguka, kandi gusimburwa bimaze guhuzwa nibicuruzwa bya RCA Intangiriro zo hasi zongerera umurongo, kandi zigasubiramo kwagura no gusimbuza ibicuruzwa bya RCA.

Ibyiza nibibi byo kuzunguruka uruziga nucleic aside amplification

Ibyiza bya RCA:

Ibyiyumvo bihanitse, umwihariko mwiza nibikorwa byoroshye.

Inenge za RCA:

Ibibazo byinyuma mugihe cyo kumenya ibimenyetso.Mugihe cyimyitwarire ya RCA, iperereza ridafite uruziga hamwe nicyitegererezo ADN cyangwa RNA ya probe idafunze irashobora kubyara ibimenyetso byinyuma. 

Nucleicacid ikurikiranye ishingiye kuri amplification

Nucleic aside ikurikirana ishingiye kuri amplification (NASBA) ni ikoranabuhanga rishya ryakozwe hashingiwe kuri PCR.Nibikomeza kandi bya isothermal nucleic aside amplification iyobowe na primers hamwe na T7 ikurikirana.Ikoranabuhanga rirashobora kongera inyandikorugero RNA inshuro zigera kuri 109 mu masaha agera kuri 2, iruta inshuro 1000 kurenza uburyo busanzwe bwa PCR kandi ntibisaba ibikoresho byihariye.

Iri koranabuhanga ryakoreshejwe mugupima byihuse indwara zikimara kugaragara, kandi ibigo byinshi kuri ubu bikoresha ubu buryo mubikoresho bya RNA.

Nubwo amplification ya RNA ishobora kandi gukoresha ikoreshwa rya tekinoroji ya PCR, NASBA ifite ibyiza byayo: irashobora gukorwa mubihe byubushyuhe burigihe, kandi birahagaze neza kandi neza kuruta tekinoroji ya PCR.

Igisubizo kiri kuri dogere selisiyusi 41 kandi gisaba AMV (virusi ya avian myeloblastose) ihindura inyandiko mvugo, RNase H, T7 RNA polymerase hamwe na primers kugirango irangire.

Inzira ikubiyemo ahanini:

Imbere primer ikubiyemo urutonde rwuzuzanya rwa T7 promoter.Mugihe cyo kubyitwaramo, primer yimbere ihuza umurongo wa RNA kandi igaterwa na enzyme ya AMV kugirango ikore umurongo wa ADN-RNA.

RNase H igogora RNA muri Hybrid inshuro ebyiri kandi ikagumana ADN imwe.

Mubikorwa bya revers primer na enzyme ya AMV, hashyizweho umurongo wa ADN ebyiri urimo urutonde rwa T7.

Mubikorwa bya T7 RNA polymerase, inzira yo kwandukura yararangiye kandi hashyizweho umubare munini wintego RNA.

Ibyiza bya NASBA:

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Ibibi bya NASBA:

(1) Ibigize reaction biragoye.

(2) Ubwoko butatu bwa enzymes burasabwa kugirango igiciro cya reaction kibe kinini.