Igihe nyacyo cya fluorescence PCR ni uburyo bwo gupima ibicuruzwa byose nyuma ya buri cyerekezo cya polymerase (PCR) cyizengurutsa ADN ikoresheje fluorophore.Uburyo bukoreshwa mukugereranya urutonde rwihariye rwa ADN murugero rwo kugeragezwa nuburyo bwimbere cyangwa hanze.Kuva yatangira, PCR igereranya PCR ivuga ko yarushijeho gukundwa nabarimu ba laboratoire.
Ihame rya Fluorescence PCR: Fluorescence PCR, yabanje kwitwa TaqManPCR nyuma ikaza no kwitwa Real-TimePCR, ni tekinike nshya yo kubara aside nucleic yakozwe na PE (PerkinElmer) muri Amerika muri 1995. Ubuhanga bushingiye ku kongeramo iperereza ryanditseho fluorescent cyangwa rihuye irangi rya fluorescent kuri PCR isanzwe kugirango igere kumikorere yayo.Ihame: uko reaction ya PCR igenda, ibicuruzwa bya PCR birundanya kandi ubukana bwikimenyetso cya fluorescent cyiyongera muburyo bungana.Hamwe na buri cyiciro, ibimenyetso bya fluorescence byegeranijwe kugirango dushobore gukurikirana ihinduka ryumubare wibicuruzwa nimpinduka zimbaraga za fluorescence bityo tubone igishushanyo mbonera cya fluorescence.
Muri rusange, umurongo wa florescence amplification umurongo urashobora kugabanywamo ibice bitatu: icyiciro cya signal ya fluorescence icyiciro, ibimenyetso bya fluorescence ibimenyetso byerekana amplification icyiciro hamwe nicyiciro cya plateau.Mugihe cyibimenyetso byerekana inyuma, ibimenyetso byongerewe imbaraga bya fluorescence bipfukiranwa nibimenyetso bya fluorescence kandi impinduka mubicuruzwa ntizishobora kugenwa.Mu cyiciro cya plateau, ibicuruzwa byongera imbaraga ntibikigenda byiyongera cyane, nta sano iri hagati yumubare wibicuruzwa byarangiye n’amafaranga yatangiriyeho, kandi nimero ya kopi ya ADN itangira ntishobora kubarwa hashingiwe ku mubare wa PCR wanyuma.Gusa mugice cya amplification amplification yicyapa cya fluorescente hariho umurongo ugaragara hagati ya logarithm yumubare wibicuruzwa bya PCR hamwe nintangiriro yicyitegererezo, kandi dushobora guhitamo kubara ibi muriki cyiciro.Kugirango byoroherezwe kugereranya no kugereranya, ibitekerezo bibiri byingenzi byinjijwe muburyo nyabwo bwa fluorescent quantitative PCR tekinike: igipimo cya fluorescence nagaciro ka CT.
Irembo ni igiciro cyakozwe muburyo bwa fluorescence amplification curve.icyiciro cyerekana icyerekezo cya PCR.
Ct agaciro: numubare wizunguruko ikimenyetso cya fluorescence muri buri reaction ya reaction cyakozwe kugirango igere kumurongo wagenwe.
Isano iri hagati yagaciro ka Ct nicyitegererezo cyo gutangira: ubushakashatsi bwerekanye ko agaciro ka Ct ya buri nyandikorugero gafite umurongo ugereranije na logarithm yo gutangira kopi yumubare wicyitegererezo, kopi nyinshi za numero yo gutangira kopi, ntoya Ct. agaciro.Indangagaciro za Ct zirahagaze neza.Igipimo gisanzwe gishobora gukorwa hifashishijwe igipimo kizwi cyo gutangira gukoporora nimero, aho umurongo uhuza utambitse ugereranya logarithm yumubare wintangiriro ya kopi naho vertical vertical igereranya agaciro ka Ct nkuko bigaragara mumashusho hepfo.
Kubwibyo, nukubona Ct agaciro ka sample itazwi, intangiriro yo gukoporora umubare wurwo rugero irashobora kubarwa uhereye kumurongo usanzwe.
Agaciro Ct ntigahoraho kandi gashobora guterwa ningero zitandukanye nibikoresho bitandukanye, nubwo icyitegererezo kimwe gisubirwamo inshuro 2 kurigikoresho kimwe, agaciro ka Ct karashobora gutandukana.
Umubare wa fluorescence urasobanura: Isuzuma ryinshi rya fluorescence rishobora kugabanywa mubice bya fluorescent hamwe n amarangi ya fluorescente bitewe nibimenyetso byakoreshejwe.Ubushakashatsi bwa Fluorescent burimo tekinoroji ya Beacon (tekinoroji ya beacon ya tekinoroji, ihagarariwe na Tagyi y'Abanyamerika), TaqMan probe (ihagarariwe na ABI) hamwe na tekinoroji ya FRET (ihagarariwe na Roche);Irangi rya fluorescent ririmo irangi rya fluorescent irangi hamwe n irangi rya fluorescent irangi, uhagarariye amarangi ya fluorescent atuzuye ni SYBRGreen I, isanzwe ikoreshwa ubu;kwiyuzuza Ubusanzwe uhagarariye amarangi ya fluorescent atuzuye ni SYBRGreenⅠ;irangi ryuzuye rya fluorescent ni EvaGreen, LCGreen, nibindi
SYBRGreenI ni ADN isanzwe ikoreshwa irangi irangi rya fluorescent PCR, ihuza bidasanzwe na ADN ebyiri.Muburyo bwubuntu, SYBRGreenI isohora fluorescence idakomeye, ariko iyo ihujwe na ADN ebyiri, fluorescence yayo yiyongera inshuro 1000.Kubwibyo, ibimenyetso byose bya fluorescence bitangwa nigisubizo biragereranywa nubunini bwa ADN ebyiri zihari kandi byiyongera uko ibicuruzwa byongera imbaraga byiyongera.
Ibyiza bya ADN ihuza imirongo ibiri irangi: igishushanyo cyoroshye cyubushakashatsi, primers 2 gusa zisabwa, nta mpamvu yo gukora iperereza, nta mpamvu yo gukora iperereza ryinshi kugirango ryipimishe byihuse genes nyinshi, ubushobozi bwo gukora isesengura ryumurongo wo gutondeka, gerageza umwihariko wa amplification reaction, igiciro cyambere cyambere, rusange muri rusange bityo rero ikoreshwa cyane mubushakashatsi murugo no mumahanga.
Uburyo bwa Fluorescent (tekinike ya Taqman): Iyo PCR ikozwe neza, hongeweho primers hamwe na probe yihariye ya fluorescent.Iyo iperereza ridahwitse, ikimenyetso cya fluorescence cyatanzwe nitsinda ryabanyamakuru cyakirwa nitsinda ryazimye kandi ntirigaragazwa nigikoresho cya PCR;mugihe cyo kongera PCR (mugice cyo kwagura), ibikorwa bya 5'-3 'clavage ya Taq enzyme itesha agaciro iperereza mu buryo bwimikorere, bigatuma umunyamakuru fluorescence itsinda kandi yazimye itsinda rya fluorescence
Porogaramu ya fluorescent yuzuye PCR.
Ubushakashatsi bwibinyabuzima bya molekuline:
1. Isesengura rya acide nucleic aside.Isesengura ryinshi kandi ryujuje ubuziranenge bw’indwara zandura, gutahura mikorobe ziterwa na virusi cyangwa virusi, nk'icyorezo cya grippe A (H1N1) giherutse, kumenya umubare wa kopi y’imiterere y’ibimera n’inyamaswa zanduye, kumenya igipimo cya RNAi idakora, n'ibindi.
2. Isesengura rya gene itandukanye.Kugereranya imiterere ya gene itandukanya ingero zavuwe (urugero: kuvura ibiyobyabwenge, kuvura umubiri, kuvura imiti, nibindi), itandukaniro ryerekana imiterere ya genes yihariye mubice bitandukanye no kwemeza microarray ya cDNA cyangwa ibisubizo byerekana imvugo itandukanye.
3. Kumenya SNP.Kumenya polymorphism nucleotide imwe ningirakamaro mu kwiga ubushakashatsi ku kwandura indwara ku giti cye cyangwa ku muntu ku giti cye ku miti yihariye, kandi kubera imiterere yubuhanga ya beacons ya molekile, iyo amakuru akurikirana ya SNP amaze kumenyekana, biroroshye kandi neza kuri koresha ubu buhanga kugirango bumenye byinshi SNP.
4. Kumenya Methylation.Methylation ifitanye isano n'indwara nyinshi zabantu, cyane cyane kanseri, kandi Laird yatangaje uburyo bwitwa Methylight, buvura ADN mbere yo kwongerwaho imbaraga kugirango cytosine idafite methyletike iba uracil kandi cytosine methylated itagira ingaruka, ikoresheje primers yihariye na Taqman kugirango itandukane na ADN methylated na methylated .birenze.
Ubushakashatsi mu by'ubuvuzi:
1. Kwipimisha mbere yo kubyara: abantu ntibashobora kuvura indwara zomurage ziterwa nibintu byahinduwe, kandi kugeza ubu, barashobora kugabanya gusa umubare wabana barwaye bavutse binyuze mugukurikirana mbere yo kubyara kugirango birinde ko habaho indwara zitandukanye.Ubu ni uburyo budahwitse bwemerwa byoroshye nabagore batwite.
2. Gutahura indwara ya virusi: Isuzuma rya fluorescent PCR ryemerera kumenya umubare wa virusi nka gonococcus, Chlamydia trachomatis, Mycoplasma solium, virusi ya papilloma, virusi ya herpes simplex, virusi ya hepatite, virusi ya grippe, Mycobacterium igituntu, EBV na cytomegalovirus.Ifite ibyiza byo kwiyumvisha ibintu byinshi, ingano ntoya y'icyitegererezo, kwihuta no koroshya ugereranije nuburyo gakondo bwo kwipimisha.
3. Isuzuma ryimiti yibiyobyabwenge: isesengura ryinshi rya virusi ya hepatite B (HBV) na virusi ya hepatite C (HCV) ryerekana ko isano iri hagati yumutwaro wa virusi ningaruka zibiyobyabwenge.Niba serumu ya HBV-ADN igabanutse mugihe cyo kuvura lamivudine hanyuma ikongera ikiyongera cyangwa ikarenga urwego rwabanje, byerekana ihinduka rya virusi.
4. Kwipimisha Oncogenetike: Nubwo uburyo bwo gukura kw'ibibyimba butarasobanuka neza, biremewe hose ko ihinduka ryimiterere ya gen ariryo ntandaro yo guhinduka kwa oncogeneque.Kongera imvugo no guhinduka kwa oncogène birashobora kugaragara mugihe cyambere cyibibyimba byinshi.Igihe nyacyo fluorescence PCR ntigikora gusa mugutahura ihinduka ryimiterere ya gen, ariko kandi irashobora kumenya neza imvugo ya oncogène.Ubu buryo bwakoreshejwe kugirango hamenyekane imiterere ya genes zitandukanye zirimo telomerase HTERT gene, karande granulocytic leukemia WT1, gene ya oncogenic ER, kanseri ya prostate PSM, hamwe na virusi ziterwa na kanseri.
Byahinduwe na www.DeepL.com/Translator (verisiyo yubuntu)
Igihe cyo kohereza: Jun-21-2022