Real Time PCR, nke a makwaara dị ka PCR quantitative ma ọ bụ qPCR, bụ usoro maka nleba anya na nyocha ngwa ngwa PCR.
N'ihi na quantitative PCR nwere uru nke dị mfe ọrụ, ngwa ngwa na adaba, elu uche, ezi repeatability, na ala mmetọ ọnụego, ọ na-ọtụtụ ebe na-eji na ọgwụ ule, ọgwụ irè ntule, mkpụrụ ndụ okwu nnyocha, transgenic nnyocha, mkpụrụ ndụ ihe nketa nchọpụta, pathogen nchọpụta, anụmanụ na osisi nchọpụta., Nnwale nri na ubi ndị ọzọ.
Ya mere, ma ị na-etinye aka na nyocha bụ isi na sayensị ndụ, ma ọ bụ ndị ọrụ nke ụlọ ọrụ ọgwụ, ụlọ ọrụ na-azụ anụ anụmanụ, ụlọ ọrụ nri, ma ọ bụ ọbụna ndị ọrụ nyocha ọpụpụ na ụlọ ọrụ kwarantaini, ngalaba nlekota gburugburu ebe obibi, ụlọ ọgwụ na ngalaba ndị ọzọ, ị ga-abụ karịa ma ọ bụ obere ekpughere ma ọ bụ ịkwesịrị ịma ihe ọmụma nke ịmụta PCR quantitative.

Ụkpụrụ nke Real Time PCR

PCR Real Time bụ usoro eji agbakwunye ihe fluorescent na sistemu mmeghachi omume PCR, a na-enyocha ike mgbaàmà fluorescence na usoro mmeghachi omume PCR ozugbo site na ngwa PCR ọnụ, na n'ikpeazụ, a na-enyocha ma hazie data nnwale ahụ.

【Usoro nkwalite】bụ usoro nke na-akọwa usoro dị ike nke PCR.Usoro nkwuwapụta nke PCR abụghị n'ezie usoro mgbawa ọkọlọtọ, kama ọ bụ usoro sigmoid.

[Nhazi nke ikpo okwu nkwuwa okwu]Site na mmụba nke ọnụ ọgụgụ cycles nke PCR, nkwụsị nke DNA polymerase, nkwụsị nke dNTPs na primers, na mgbochi nke mmeghachi omume site na mmeghachi omume nke ngwaahịa pyrophosphate, wdg, PCR anaghị agbasawanye ngwa ngwa., ma emechaa banye n'ala dị larịị.

[Mpaghara Uto Mgbapụta nke Mgbapụta Amụba]Ọ bụ ezie na oge dị larịị dịgasị iche iche nke ukwuu, na mpaghara ụfọdụ nke mpaghara mmụba nke mmụba, nkwughachi dị mma nke ukwuu, nke dị oke mkpa maka nyocha ọnụọgụ nke PCR.

[ọnụ ahịa ọnụ na uru Ct]Anyị na-edobe uru njedebe nke nchọpụta fluorescence n'ọnọdụ kwesịrị ekwesị na mpaghara uto nke nkwuputa ụda, ya bụ uru ọnụ ụzọ (Tthreshold).Nkwụsị nke ọnụ ụzọ ọnụ ụzọ na nkwunye nkwalite bụ uru Ct, ya bụ, uru Ct na-ezo aka na ọnụ ọgụgụ nke okirikiri (Threshold Cycle) mgbe uru ọnụ ụzọ ruru.

Eserese dị n'okpuru na-egosi n'ụzọ doro anya njikọ dị n'etiti ahịrị ọnụ ụzọ na akụkụ mmụba, ọnụ ụzọ na uru Ct.

【Kedu ka esi agụta ọnụ ọgụgụ?】

Egosiputara ya site na tiori mgbakọ na mwepụ na uru Ct nwere mmekọrịta kwụ ọtọ na logarithm nke ọnụọgụ ndebiri mbụ.Real Time PCR na-enyocha ngwaahịa PCR n'ezie ma na-agụpụta ha n'oge oge mmụba.

Maka usoro PCR nke ọ bụla, DNA mụbara nke ukwuu site na ugboro abụọ, n'oge na-adịghịkwa anya ruru ala dị larịị.

Na-eche na ọnụọgụ mmalite DNA bụ A₀ , ka n cycles gachara, enwere ike igosipụta ọnụọgụ ngwaahịa DNA dị ka:

A _n =A ₀ ×2ⁿ

Mgbe ahụ, ka ọnụọgụ DNA mbụ A 0 na-abawanye, ngwa ngwa ọnụ ọgụgụ ngwaahịa a na-abawanye na-erute uru nchọpụta An , na ọnụ ọgụgụ nke cycles mgbe ị rutere An bụ uru Ct.Ya bụ, ka ọnụọgụ DNA nke mbụ A 0 na-abawanye, na mbụ n'usoro nkwuwa okwu na-arị elu, yana ọnụ ọgụgụ cycles chọrọ n'otu aka ahụ dị ntakịrị.

Anyị na-eme gradient dilution nke ọkọlọtọ nke ama ama ma na-eji ya dị ka a template maka Real Time PCR, na a usoro nke amplification curves ga-enwetara n'otu n'otu etiti oge n'usoro nke malite DNA ego si ọzọ ka obere.Dị ka usoro njikọ dị n'etiti uru Ct na logarithm nke ọnụọgụ mmalite nke ndebiri, aEnwere ike ịmepụta [ụkpụrụ ọkọlọtọ].

Site n'ịgbanwe uru Ct nke ihe nlele ahụ na ntinye uche a na-amaghị ama n'ime usoro ọkọlọtọ, enwere ike nweta ọnụọgụ mbụ nke ihe nlele ahụ na ntinye uche a na-amaghị, nke bụ ụkpụrụ quantitative nke Real Time PCR.

Usoro nchọpụta nke Real Time PCR

Real Time PCR na-achọpụta ngwaahịa nkwalite PCR site n'ịchọpụta ike ọkụ na sistemu mmeghachi omume.

Ụkpụrụ nke Ụzọ Mgbakwunye Fluorescent Dye】

Agba agba fluorescent, dị ka TB Green ® , nwere ike nonspecifically ejikọta na abụọ stranded DNA na PCR usoro na fluoresce na-ekekọta.

Mmetụta fluorescence dị na sistemu mmeghachi omume mụbara nke ukwuu site na mmụba nke okirikiri PCR.Site n'ịchọpụta ike nke fluorescence, ọnụ ọgụgụ nke DNA amplification na usoro mmeghachi omume nwere ike nyochaa ozugbo, na mgbe ahụ, ọnụ ọgụgụ nke mmalite template na sample nwere ike gbanwee.

【Ụkpụrụ nke usoro nyocha fluorescent】

nyocha fluorescentbụ usoro nucleic acid nwere otu fluorescent na njedebe 5 na otu quenching na njedebe 3, nke nwere ike jikọta ya na template.Mgbe nyocha ahụ adịghị emebi emebi, ndị otu na-emenyụ ọkụ na-emenyụ ọkụ fluorescence nke fluorophore na-apụta na enweghị ike ịcha ọkụ.Mgbe nyocha ahụ mebiri emebi, ihe fluorescent ga-ekewa ma wepụta fluorescence.

A na-agbakwunye nyocha fluorescent na ngwọta mmeghachi omume PCR.N'oge annealing usoro, fluorescent nyocha ga-ejikọta na kpọmkwem ọnọdụ nke template.N'oge usoro ndọtị, 5′→3′ exonuclease ọrụ nke PCR enzyme nwere ike decompose fluorescent nyocha hybridized na template, na fluorescent ihe na-dissociated ka emit fluorescence.Site n'ịchọpụta ike nke fluorescence nke nyocha na sistemu mmeghachi omume, enwere ike nweta ebumnuche nke nyochaa ọnụọgụ mmụba nke ngwaahịa PCR.

【Nhọrọ nke usoro nchọpụta fluorescence】

Ọ bụrụ na a na-eji ya ịmata ọdịiche dị n'usoro na homology dị elu ma mee nchọpụta multiplex PCR dị ka nyocha SNP, usoro nyocha nke fluorescent agaghị edochi anya.
Maka nnwale PCR Real Time ndị ọzọ, enwere ike iji usoro chimera fluorescent dị mfe, dị mfe ma dị ọnụ ala.

probesStrong nkọwapụta, ike nke multiplex PCR

adịghị mkpirisi

Nhọrọ dị elu maka mmụba;

multiplex PCR enweghị ike ịrụ ya Mkpa chepụta kpọmkwem nyocha, ọnụ ahịa dị elu;

mgbe ụfọdụ imepụta nyocha siri ike

Ngwaahịa ndị emetụtara: