Fluorescence quantitative PCR (nke a makwaara dị ka TaqMan PCR, nke a na-akpọ FQ-PCR) bụ teknụzụ ọnụọgụ ọnụọgụ ọhụrụ nke PE (Perkin Elmer) mepụtara na United States na 1995. Nkà na ụzụ a dabere na PCR nkịtị site n'ịgbakwunye fluorescent labeled probes.Tụnyere PCR na-agbanwe agbanwe, FQ-PCR nwere ọtụtụ uru iji ghọta ọrụ ọnụọgụ ya.Edemede a bu n'obi ịkọwa nkenke nke njirimara, ụkpụrụ, ụzọ na ngwa nke teknụzụ.
1 atụmatụ
FQ-PCR ọ bụghị naanị nwere uche dị elu nke PCR nkịtị, kamakwa n'ihi ngwa nke nyocha fluorescent, ọ nwere ike ịchọpụta ozugbo mgbanwe nke mgbaàmà fluorescent n'oge mmụba PCR site na sistemu njikwa fotoelectric iji nweta nsonaazụ quantitative, nke na-emeri ọtụtụ adịghị ike nke PCR nkịtị, yabụ na ọ nwekwara nkọwa dị elu nke hybridization DNA na teknụzụ nyocha dị elu.
Dịka ọmụmaatụ, ọ dị mkpa ka a hụ ngwaahịa PCR n'ozuzu site na agarose gel electrophoresis na ethidium bromide na-eji ọkụ ultraviolet ma ọ bụ polyacrylamide gel electrophoresis na ncha ọlaọcha.Nke a ọ bụghị nanị na-achọ ọtụtụ ngwá ọrụ, kamakwa ọ na-ewe oge na mgbalị.Unyi eji Ethidium bromide na-emerụ ahụ mmadụ, usoro nnwale ndị a gbagwojuru anya na-enye ohere maka mmetọ na ihe na-ezighị ezi.Otú ọ dị, FQ-PCR kwesịrị imeghe mkpuchi otu ugboro n'oge nbudata ihe nlele, na usoro na-esote bụ ọrụ mechiri emechi-tube, nke na-adịghị achọ PCR post-processing, na-ezere ọtụtụ ihe ndọghachi azụ na PCR arụ ọrụ.Nnwale a na-ejikarị ABI7100 PCR thermal cycler nke ụlọ ọrụ PE mepụtara.
Ngwa ahụ nwere njirimara ndị a: ① Ngwa ngwa ngwa: Enwere ike iji ya maka nyocha ngwaahịa DNA na RNA PCR, nchọpụta mkpụrụ ndụ ihe nketa, nchọpụta pathogen, na njikarịcha ọnọdụ PCR.② Ụkpụrụ ọnụọgụ pụrụ iche: N'iji nyocha ndị a na-akpọ fluorescently, ọnụ ọgụgụ nke fluorescence ga-agbakọ na okirikiri PCR mgbe mkpali laser gasịrị, iji nweta nzube nke quantification.③ Ịrụ ọrụ dị elu: Igwe ọkụ ọkụ PCR 9600 arụnyere, kọmpụta na-achịkwa 1 ruo 2 awa iji mezue mmụba na nha nke sample 96 na-akpaghị aka na synchronously.④ Ọ dịghị mkpa maka gel electrophoresis: Ọ dịghị mkpa itughari na electrophoresis sample, dị nnọọ na-eji a pụrụ iche nyocha chọpụta kpọmkwem na mmeghachi omume tube.⑤ Enweghị mmetọ na pipeline: A na-anakwere tube mmeghachi omume pụrụ iche zuru oke na usoro nchịkwa fotoelectric, yabụ na ọ dịghị mkpa ichegbu onwe gị maka mmetọ.⑥ Nsonaazụ bụ mmeghari: ọnụọgụ ike ike dị elu ruru iwu ise nke ịdị ukwuu.Ya mere, ebe ọ bụ na e mepụtara nkà na ụzụ a nke ọma, ọtụtụ ndị nchọpụta sayensị ji ya kpọrọ ihe ma tinye ya n'ọrụ n'ọtụtụ ebe.
2 Ụkpụrụ na ụzọ
Ụkpụrụ na-arụ ọrụ nke FQ-PCR bụ iji 5′→3′ exonuclease ọrụ nke Taq enzyme iji gbakwunye nyocha nke akpọrọ fluorescently na sistemụ mmeghachi omume PCR.Nchọpụta ahụ nwere ike iji ụdị DNA dị n'ime usoro mmalite mee ngwakọ kpọmkwem.A na-akpọ njedebe 5 nke nyocha ahụ na fluorescence emission gene FAM (6-carboxyfluorescein, fluorescence emission peak na 518nm), na 3'end na-akpọrọ The fluorescence quenching group TAMRA (6-carboxytetramethylrhodamine, fluorescence nke 3). phosphorylated iji gbochie ịgbatị nyocha ahụ n'oge mmụba PCR.Mgbe nyocha ahụ ka dịgidere, otu ndị na-ekpo ọkụ na-egbochi ikuku ikuku nke otu na-ekupụta.Ozugbo e kewapụrụ ìgwè ndị na-emepụta ihe na ndị na-emenyụ ọkụ ahụ, a na-ebuli ihe mgbochi ahụ, na njupụta anya na 518nm na-abawanye ma na-achọpụta ya site na usoro nchọpụta fluorescence. N'ime usoro nhazigharị, nyocha ahụ na-ejikọta ya na DNA template, na Taq enzyme na mgbatị mgbatị na-aga n'akụkụ DNA template na mgbatị nke primer.Mgbe ebipụrụ nyocha ahụ, a na-ahapụ mmetụta nkwụsị ahụ wee hapụ mgbama fluorescent.Mgbe ọ bụla edepụtaghachiri template ahụ, a na-ebipụ ihe nyocha, yana ntọhapụ nke mgbama fluorescent.Ebe ọ bụ na enwere mmekọrịta otu na otu n'etiti ọnụ ọgụgụ fluorophores a tọhapụrụ na ọnụ ọgụgụ nke ngwaahịa PCR, enwere ike iji usoro a kọwaa template ahụ n'ụzọ ziri ezi.Ngwa nnwale a na-ejikarị ABI7100 PCR thermal cycler nke ụlọ ọrụ PE mepụtara, enwere ike iji igwe igwe ọkụ ndị ọzọ.Ọ bụrụ na ejiri usoro mmeghachi omume ABI7700 mee ihe maka nnwale ahụ, mgbe emechara mmeghachi omume ahụ, enwere ike ịnye nsonaazụ ọnụọgụ ozugbo site na nyocha kọmputa.Ọ bụrụ na ị na-eji ndị ọzọ thermal cyclers, ị kwesịrị iji a fluorescence detector tụọ fluorescence mgbaàmà na mmeghachi omume tube n'otu oge gbakọọ RQ +, RQ-, △RQ.RQ + na-anọchi anya ruru nke luminescence ike nke fluorescent emission otu nke sample tube ka luminescence ike nke quenching otu, RQ- na-anọchi anya ruru nke abụọ na oghere tube, △ RQ (△ RQ = RQ + -RQ-) na-anọchi anya ego nke fluorescence esi eme mgbanwe n'oge PC nwere ike nweta mgbanwe mgbaàmà.N'ihi iwebata nyocha nke fluorescent, a na-emeziwanye nkọwa nke nnwale ahụ nke ọma.Nchọpụta nyocha kwesịrị n'ozuzu izute ọnọdụ ndị a: ① Ogologo nyocha kwesịrị ịdị ihe dịka 20-40 bases iji hụ na nkọwapụta nke njide.②Ọdịnaya nke ntọala GC dị n'etiti 40% na 60% iji zere mbịgharị nke otu usoro nucleotide.③ Zenarị ngwakọ ma ọ bụ mechie na primers.④ Nkwụsi ike nke njide n'etiti nyocha na template dị ukwuu karịa nkwụsi ike nke njide n'etiti primer na template, ya mere uru Tm nke nyocha kwesịrị ịdịkarịa ala 5 Celsius C karịa uru Tm nke primer.Na mgbakwunye, ntinye nke nyocha, homology n'etiti nyocha na usoro template, na anya n'etiti nyocha na primer niile nwere mmetụta na nsonaazụ nnwale.
Ngwaahịa ndị emetụtara:
China Real Time PCR Easyᵀᴹ-Taqman emeputa na Supplier |Foregene (foreivd.com)
Oge nzipu: Ọktoba 15-2021
