Real-time fluorescence quantitative PCR bụ usoro maka ịlele mkpokọta ngwaahịa mgbe ọ bụla polymerase chain reaction (PCR) na mmeghachi omume mmụba DNA site na iji fluorophore.A na-eji usoro a wee tụọ usoro DNA dị iche iche n'ime ihe nlele nke a ga-anwale site na ụzọ ntụaka ime ma ọ bụ nke mpụga.Kemgbe mmalite ya, nyocha nke fluorescent quantitative PCR abụrụla nke ndị nkuzi ụlọ nyocha na-ewu ewu.
Ụkpụrụ PCR Fluorescence: Fluorescence PCR, nke mbụ a na-akpọ TaqManPCR na emesịa bụkwa Real-TimePCR, bụ usoro ọnụọgụ ọnụọgụ nucleic acid ọhụrụ nke PE (PerkinElmer) mepụtara na USA na 1995. Usoro ahụ dabere na mgbakwunye nke nyocha nke a na-akpọ fluorescently ma ọ bụ kwekọrọ na ya. fluorescent agba na PCR nkịtị iji nweta ọrụ ọnụọgụ ya.Ụkpụrụ: ka mmeghachi omume PCR na-aga n'ihu, ngwaahịa mmeghachi omume PCR na-agbakọta na ike nke mgbaàmà fluorescent na-abawanye na nha nha.Na okirikiri nke ọ bụla, a na-anakọta mgbaama ike ọkụ fluorescence ka anyị wee nwee ike nyochaa mgbanwe na ọnụọgụ ngwaahịa site na ngbanwe nke ike fluorescence wee si otú ahụ nweta eserese mgbaba fluorescence.
N'izugbe, enwere ike kewaa usoro mgbama ọkụ fluorescence ụzọ atọ: oge mgbama mgbama n'azụ ọkụ, mgbama mgbama ọkụ ọkụ na oge mgbago ugwu.N'oge mgbama n'azụ, mgbama fluorescence emelitere na-ekpuchi ya site na mgbama ndabere fluorescence na enweghị ike ikpebi ọnụọgụ ngwaahịa.N'ime oge ala dị larịị, ngwaahịa mmụba anaghịzi abawanye nke ukwuu, enweghị njikọ kwụ ọtọ n'etiti ọnụọgụ ngwaahịa ikpeazụ na ọnụọgụ ndebiri mmalite, enweghị ike ịgbakọ ọnụọgụ mbipụta DNA mbido dabere na ọnụọgụ PCR ikpeazụ.Naanị n'ime oge mmụba nke mgbama fluorescent ka enwere mmekọrịta dị n'etiti logarithm nke ego ngwaahịa PCR na ọnụọgụ ndebiri mmalite, anyị nwere ike họrọ ịkọwapụta nke a n'oge a.Maka ịdị mma nke nha na ntụnyere, ewebatala echiche abụọ dị oke mkpa n'ime ezigbo oge fluorescent quantitative teknụzụ PCR: ọnụ ụzọ ọkụ na uru CT.
Ọnụ ụzọ bụ uru ahaziri ahazi n'usoro mgbago fluorescence.akụkụ nke exponential nke PCR amplification.
uru Ct: bụ ọnụọgụ okirikiri nke mgbama fluorescence na tube mmeghachi omume ọ bụla megoro ka ọ rute uru ngalaba ahaziri.
Mmekọrịta dị n'etiti uru Ct na ndebiri mmalite: ọmụmụ egosila na uru Ct nke template ọ bụla nwere mmekọrịta dị n'ahịrị na logarithm nke nọmba mmalite nke ndebiri ahụ, ka ọtụtụ mbipụta nke nọmba nbipụta mmalite, na-ebelata Ct. uru.Ụkpụrụ Ct kwụsiri ike.Enwere ike iji ọkọlọtọ nwere nọmba mbido ama ama, ebe nhazi kwụ ọtọ na-anọchi anya logarithm nke nọmba mmalite na nhazi kwụ ọtọ na-anọchi anya uru Ct dị ka egosiri na foto dị n'okpuru.
Ya mere, site n'inweta uru Ct nke ihe nlele amaghị, ọnụọgụ mmalite nke ihe nlele ahụ nwere ike gbakọọ site na ọkọlọtọ ọkọlọtọ.
Ọnụ ego Ct adịghị adịgide adịgide ma nwee ike imetụta ụdị dị iche iche na ngwá ọrụ dị iche iche, ọ bụrụgodị na a na-emegharị otu ihe ahụ ugboro 2 n'otu ngwá ọrụ ahụ, uru Ct nwere ike ịdị iche.
Ọnụ ọgụgụ nke fluorescence assays: Enwere ike kewaa nyocha nke fluorescence n'ime nyocha fluorescent na ihe eji esiji fluorescent dabere na akara ejiri.Ihe nyocha nke fluorescent gụnyere teknụzụ Beacon (teknụzụ beacon molecular, nke American Tagyi nọchiri anya ya), nyocha TaqMan (nke ABI nọchiri anya ya) na teknụzụ FRET (nke Roche nọchiri anya ya);ihe na-acha ọkụ na-acha ọkụ na-agụnye ihe na-esi ísì ụtọ nke na-egbuke egbuke na ihe na-acha ọkụ ọkụ na-adịghị edozi, onye nnọchiteanya nke ihe eji eme ihe na-adịghị edozi bụ SYBRGreen I, nke a na-ejikarị eme ihe ugbu a;juputara Ihe nnochite anya nke ihe eji esiji fluorescent na-ejujughi bu SYBRGreenⅠ;eju mmiri fluorescent juputara bụ EvaGreen, LCGreen, wdg.
SYBRGreenI bụ ihe a na-ejikarị DNA na-ejikọta agba maka PCR fluorescent, nke na-ejikọta na-abụghị kpọmkwem na DNA nwere eriri abụọ.Na steeti nweere onwe ya, SYBRGreenI na-ebunye ọkụ ọkụ na-adịghị ike, mana ozugbo ejiri ya na DNA nwere eriri abụọ, fluorescence ya na-abawanye ugboro 1000.Ya mere, mkpokọta fluorescence mgbama na-ewepụta site na mmeghachi omume dabara na ọnụọgụ DNA nwere eriri abụọ ma na-abawanye ka ngwaahịa mmụba na-abawanye.
Uru nke eriri DNA na-ekekọta eriri abụọ: nhazi nnwale dị mfe, naanị 2 primers chọrọ, enweghị mkpa imepụta nyocha, ọ nweghị mkpa imepụta ọtụtụ nyocha maka nnwale ngwa ngwa nke ọtụtụ mkpụrụ ndụ ihe nketa, ikike ịme nyocha mgbaze ebe mgbaze, nwalee nkọwapụta nke mmeghachi omume mmụba, ọnụ ala mmalite dị ala, ezigbo izugbe ya mere a na-ejikarị eme nchọpụta n'ụlọ na mba ọzọ.
Usoro nyocha fluorescent (Teknụ Taqman): Mgbe a na-eme nkwalite PCR, a na-agbakwunye otu ụzọ primers yana otu nyocha fluorescent.Mgbe nyocha ahụ adịghị emebi emebi, mgbaàmà fluorescence nke ndị otu ndị nta akụkọ na-ewepụta na-abanye n'ime otu ahụ kwụsịrị ma ọ bụghị ngwa PCR achọpụtara ya;n'oge nkwalite PCR (na oge ndọtị), ọrụ 5'-3' nke nbibi nke enzyme Taq na-emebi nyocha ahụ nke ọma, na-eme ka ndị nta akụkọ fluorescence otu na menyụọ fluorescence otu.
Ngwa nke fluorescent quantitative PCR.
Nchoputa ihe omumu ihe omimi:
1. Ọnụ ọgụgụ nke nucleic acid analysis.Nyocha ọnụọgụ na qualitative nke ọrịa na-efe efe, nchọpụta nke microorganisms pathogenic ma ọ bụ nje, dị ka ọrịa influenza A (H1N1) na-adịbeghị anya, nchọpụta nke mkpụrụ ndụ mkpụrụ ndụ nke osisi transgenic na anụmanụ, nchọpụta nke RNAi gene inactivation rate, wdg.
2. Nyocha okwu mkpụrụ ndụ dị iche iche.Ntụnyere ọdịiche nke mkpụrụ ndụ ihe nketa n'etiti ihe nlele emere (dịka ọgwụgwọ ọgwụ, ọgwụgwọ anụ ahụ, ọgwụgwọ kemịkalụ, wdg), nkwupụta okwu dị iche iche nke mkpụrụ ndụ ihe nketa akọwapụtara n'ụdị dị iche iche yana nkwenye nke microarray cDNA ma ọ bụ nsonaazụ ngosipụta dị iche.
3. Nchọpụta SNP.Nchọpụta nke otu nucleotide polymorphisms dị mkpa maka ọmụmụ ihe nke onye ọ bụla nwere ike ịnweta ọrịa dị iche iche ma ọ bụ nzaghachi nke onye ọ bụla na ọgwụ ndị dị iche iche, na n'ihi usoro mgbagwoju anya nke mgbama molecular, ozugbo a mara usoro nke SNP, ọ dị mfe ma dị mma. jiri usoro a maka nchọpụta SNP dị elu.
4. Nchọpụta methylation.Methylation na-ejikọta ya na ọtụtụ ọrịa mmadụ, karịsịa ọrịa cancer, na Laird kọrọ usoro a na-akpọ Methylight, nke na-agwọ DNA tupu mmụba nke mere na cytosine unmethylated na-aghọ uracil na methylated cytosine adịghị emetụta ya, na-eji primers kpọmkwem na Taqman nyocha iji mata ọdịiche dị n'etiti DNA methylated na unmethylated. .ọzọ mmetụta.
Nnyocha ahụike:
1. Nchọpụta tupu ime nwa: ndị mmadụ enweghị ike ịgwọ ọrịa ihe nketa sitere na mkpụrụ ndụ ihe nketa gbanwere, na ka ọ dị ugbu a, naanị ha nwere ike ibelata ọnụ ọgụgụ ụmụaka na-arịa ọrịa nke a mụrụ site n'ịleba anya tupu ọmụmụ nwa iji gbochie ọrịa dị iche iche nke eketa.Nke a bụ usoro na-adịghị emerụ ahụ nke ụmụ nwanyị dị ime na-anabata ngwa ngwa.
2. Nchọpụta ihe na-akpata ọrịa: The fluorescent quantitative PCR assay na-enye ohere ikpebi ọnụọgụ nke nje ndị dị ka gonococcus, Chlamydia trachomatis, Mycoplasma solium, virus papilloma mmadụ, nje virus herpes simplex, nje immunodeficiency mmadụ, nje ịba ọcha n'anya, nje influenza, Mycobacterium ụkwara nta na cytome , EB.Ọ nwere uru nke ịdị elu dị elu, nha nlele dị ala, ngwa ngwa na ịdị mfe ma e jiri ya tụnyere usoro nyocha ọdịnala.
3. Ntụle nrụpụta ọgwụ: ọnụọgụ ọnụọgụ nke nje ịba ọcha n'anya B (HBV) na nje ịba ọcha n'anya C (HCV) na-egosi na mmekọrịta dị n'etiti ibu nje na ịdị irè nke ọgwụ ụfọdụ.Ọ bụrụ na ọkwa ọbara nke HBV-DNA na-ebelata n'oge ọgwụgwọ lamivudine wee na-abawanye ọzọ ma ọ bụ gafere ọkwa gara aga, ọ na-egosi mmụgharị nje.
4. Nnwale oncogenetic: Ọ bụ ezie na usoro nke mmepe tumor amabeghị nke ọma, a na-anabatakarị na mmụgharị na mkpụrụ ndụ ihe nketa dị mkpa bụ isi ihe kpatara mgbanwe oncogenic.Enwere ike ịhụ mmụba na ngbanwe nke oncogenes na mmalite nke ọtụtụ etuto ahụ.Real-time fluorescence quantitative PCR abụghị nanị dị irè n'ịchọpụta mmụgharị na mkpụrụ ndụ ihe nketa, kama ọ pụkwara ịchọpụta n'ụzọ ziri ezi okwu nke oncogenes.A na-eji usoro a chọpụta okwu nke mkpụrụ ndụ ihe nketa dị iche iche gụnyere telomerase hTERT gene, granulocytic leukemia WT1 na-adịghị ala ala, mkpụrụ ndụ oncogenic ER, mkpụrụ ndụ cancer prostate PSM, na mkpụrụ ndụ ihe nketa metụtara tumor.
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Oge nzipu: Jun-21-2022