ʻO ka PCR fluorescence quantitative manawa maoli he ala no ke ana ʻana i ka huina o ka huahana ma hope o kēlā me kēia pōʻaiapili kaulahao polymerase (PCR) i loko o kahi pane hoʻonui DNA me ka fluorophore.Hoʻohana ʻia ke ʻano no ka helu ʻana i nā kaʻina DNA kikoʻī i ka laʻana e hoʻāʻo ʻia e nā ʻano kuhikuhi i loko a i waho paha.Mai kona hoʻomaka ʻana, ua lilo ka fluorescent quantitative PCR assays i mea kaulana loa me nā kumu hoʻokolohua.
ʻO ke kumukānāwai PCR Fluorescence: ʻO Fluorescence PCR, i kapa mua ʻia ʻo TaqManPCR a ma hope aku ʻo Real-TimePCR, he ʻenehana helu nucleic acid hou i hoʻomohala ʻia e PE (PerkinElmer) ma USA i ka makahiki 1995. Hoʻokumu ʻia ka ʻenehana ma ka hoʻohui ʻana i kahi ʻimi fluorescently labeled a i ʻole like. ka waiʻona fluorescent i ka PCR maʻamau e hoʻokō i kāna hana quantitative.ʻO ke kumu: i ka hoʻomaka ʻana o ka hopena PCR, hōʻiliʻili nā huahana hopena PCR a piʻi ka ikaika o ka hōʻailona fluorescent i ka like like.Me kēlā me kēia pōʻai, hōʻiliʻili ʻia kahi hōʻailona fluorescence i hiki iā mākou ke nānā i ka hoʻololi ʻana i ka nui o ka huahana ma o ka hoʻololi ʻana i ka ikaika fluorescence a no laila e loaʻa ai kahi kiʻi kiʻi fluorescence amplification curve.
Ma keʻano laulā, hiki ke hoʻokaʻawale i ka pihi hoʻonui fluorescence i ʻekolu mau ʻāpana: ka pae hōʻailona o ka fluorescence background, ka pae hoʻonui hoʻonui ʻana i ka hōʻailona fluorescence a me ka pae plateau.I ka wā o ka hōʻailona hōʻailona hope, ua uhi ʻia ka hōʻailona fluorescence i hoʻonui ʻia e ka hōʻailona hope fluorescence a ʻaʻole hiki ke hoʻoholo i ka nui o ka huahana.Ma ka pae pāpū, ʻaʻole e piʻi nui ka hua hoʻonui, ʻaʻohe pilina laina ma waena o ka nui o ka huahana hope a me ka nui template hoʻomaka, a ʻaʻole hiki ke helu ʻia ka helu kope DNA hoʻomaka ma muli o ka nui o ka huahana PCR hope.Aia wale ma ka māhele hoʻonui exponential o ka hōʻailona fluorescent kahi pilina laina ma waena o ka logarithm o ka nui o ka huahana PCR a me ka nui o ka hoʻomaka ʻana, a hiki iā mākou ke koho e helu i kēia ma kēia pae.No ka maʻalahi o ka helu ʻana a me ka hoʻohālikelike ʻana, ua hoʻokomo ʻia ʻelua manaʻo koʻikoʻi i loko o ka ʻenehana PCR fluorescent quantitative: ka paepae fluorescence a me ka waiwai CT.
ʻO ka paepae kahi waiwai i hoʻonohonoho ʻia ma ka pihi hoʻonui fluorescence.ka māhele exponential o ka hoʻonui PCR.
Waiwai Ct: ʻo ia ka helu o nā pōʻaiapuni i loaʻa i ka hōʻailona fluorescence i kēlā me kēia paipu hopena a hiki i ka waiwai domain i hoʻonohonoho ʻia.
ʻO ka pilina ma waena o ka waiwai Ct a me ke kumu hoʻomaka: ua hōʻike nā haʻawina he pilina laina ko ka waiwai Ct o kēlā me kēia maʻamau me ka logarithm o ka helu kope hoʻomaka o kēlā maʻamau, ʻoi aku ka nui o nā kope o ka helu kope hoʻomaka, ʻoi aku ka liʻiliʻi o ka Ct. waiwai.Paʻa ka waiwai Ct.Hiki ke hana ʻia kahi ʻōkuhi maʻamau me ka helu kope hoʻomaka i ʻike ʻia, kahi i hōʻike ʻia ai ka logarithm o ka helu kope hoʻomaka a ʻo ka hoʻonohonoho kū i ka waiwai Ct e like me ke kiʻi ma lalo nei.
No laila, ma ka loaʻa ʻana o ka waiwai Ct o kahi laʻana i ʻike ʻole ʻia, hiki ke helu ʻia ka helu kope hoʻomaka o kēlā hāpana mai ka pihi maʻamau.
ʻAʻole paʻa ka waiwai Ct a hiki ke hoʻololi ʻia e nā laʻana like ʻole a me nā mea kani like ʻole, ʻoiai inā e hana hou ʻia ka hāpana like i 2 mau manawa ma ka mea kani like, hiki ke ʻokoʻa ka waiwai Ct.
Nā ho'āʻo ʻana o ka fluorescence quantitative: Hiki ke hoʻokaʻawale ʻia nā hōʻike fluorescence quantitative i loko o nā probes fluorescent a me nā mea ʻulaʻula ma muli o nā māka i hoʻohana ʻia.Loaʻa i nā ʻenehana Fluorescent ka ʻenehana Beacon (ʻenehana molecular beacon, i hōʻike ʻia e American Tagyi), nā Probes TaqMan (i hōʻike ʻia e ABI) a me ka ʻenehana FRET (i hōʻike ʻia e Roche);ʻO nā mea hoʻoluʻu fluorescent ʻo ia ka SYBRGreen I, ka mea maʻamau i kēia manawa;saturated ʻO SYBRGreenⅠ ka ʻelele maʻamau o nā mea hoʻonaninani ʻole-saturated;ʻO nā mea hoʻonani fluorescent saturated ʻo EvaGreen, LCGreen, etc.
ʻO SYBRGreenI kahi mea hoʻopaʻa paʻa DNA maʻamau i hoʻohana ʻia no ka PCR fluorescent, kahi e hoʻopaʻa ʻole ai i ka DNA kaula-lua.Ma kona kūlana kūʻokoʻa, hoʻopuka ʻo SYBRGreenI i kahi fluorescence nāwaliwali, akā hoʻokahi i hoʻopaʻa ʻia i ka DNA stranded pālua, piʻi ka fluorescence i 1000-pa.No laila, ʻo ka hōʻailona fluorescence holoʻokoʻa i hoʻokuʻu ʻia e kahi pane e like me ka nui o ka DNA stranded pālua i kēia manawa a piʻi aʻe i ka piʻi ʻana o ka huahana amplification.
ʻO nā mea maikaʻi o nā mea hoʻopaʻa paʻa DNA kaulua: hoʻolālā hoʻokolohua maʻalahi, 2 mau kumu mua e pono ai, ʻaʻohe pono e hoʻolālā i nā ʻimi noiʻi, ʻaʻohe pono e hoʻolālā i nā ʻimi he nui no ka hoʻāʻo wikiwiki ʻana i nā genes he nui, hiki ke hana i ka hoʻoheheʻe ʻana o ka helu heheʻe, hoʻāʻo i ka kikoʻī o ka ka hoʻonui ʻana, ke kumu kūʻai haʻahaʻa, ka laulā maikaʻi a no laila ʻoi aku ka maʻamau i ka noiʻi ma ka home a ma waho.
ʻO ke ʻano o ka ʻimi Fluorescent (Taqman technique): Ke hoʻokō ʻia ka PCR amplification, hoʻohui ʻia kahi pālua o nā primers me kahi ʻimi fluorescent kikoʻī.Ke hoʻopaʻa paʻa ka ʻimi, ʻo ka hōʻailona fluorescence i hoʻokuʻu ʻia e ka hui mea hoʻolaha e hoʻopili ʻia e ka hui kinai ʻia a ʻaʻole ʻike ʻia e ka mea hana PCR;i ka wā o ka hoʻonui ʻana o PCR (i ka manawa hoʻonui), ʻo ka hana cleavage 5'-3' o ka enzyme Taq e hoʻohaʻahaʻa i ka probe enzymatically, e hana ana i ka hui fluorescence reporter a me ka hui fluorescence kinai.
Nā noi o ka PCR quantitative fluorescent.
Ka noiʻi molecular biology:
1. Ka helu helu nucleic acid.Ka helu helu a me ka qualitative o nā maʻi infectious, ka ʻike ʻana i nā microorganism pathogenic a i ʻole nā viruses, e like me ka maʻi maʻi influenza A (H1N1) hou, ka ʻike ʻana i nā helu kope o nā mea kanu transgenic a me nā holoholona, ka ʻike ʻana i nā helu RNAi gene inactivation, etc.
2. Ka nānā 'ana i ka hō'ike gene.Ka hoʻohālikelike ʻana i nā ʻokoʻa hōʻike genes ma waena o nā laʻana i mālama ʻia (e laʻa me ka mālama ʻana i ka lāʻau lapaʻau, mālama kino, mālama kemika, a me nā mea ʻē aʻe), nā ʻokoʻa hōʻike o nā genes kikoʻī i nā pae like ʻole a me ka hōʻoia ʻana o ka microarray cDNA a i ʻole nā hopena hōʻike ʻokoʻa.
3. ʻIke SNP.He mea nui ka ʻike ʻana i nā polymorphisms nucleotide hoʻokahi no ke aʻo ʻana i ka maʻalahi o kēlā me kēia kanaka i nā maʻi like ʻole a i ʻole ka pane ʻana o kēlā me kēia kanaka i nā lāʻau lapaʻau kūikawā, a ma muli o ke ʻano akamai o nā beacons molecular, ke ʻike ʻia ka ʻike kaʻina o kahi SNP, maʻalahi a pololei. e hoʻohana i kēia ʻenehana no ka ʻike SNP kiʻekiʻe.
4. ʻIke ʻana i ka methylation.Hoʻopili ʻia ka methylation me nā maʻi he nui o ke kanaka, ʻoi aku ka maʻi kanesa, a ua hōʻike ʻo Laird i kahi ʻenehana i kapa ʻia ʻo Methylight, nāna e mālama i ka DNA ma mua o ka hoʻonui ʻana i lilo ai ka cytosine unmethylated i uracil a ʻaʻole i hoʻopilikia ʻia ka cytosine methylated, me ka hoʻohana ʻana i nā primers kikoʻī a me Taqman probes e hoʻokaʻawale i waena o ka methylated a me ka unmethylated DNA. .ʻoi aku ka maʻalahi.
Ka noiʻi lāʻau lapaʻau:
1. Ka maʻi ma mua o ka hānau ʻana: ʻaʻole hiki i nā kānaka ke mālama i nā maʻi hoʻoilina i hoʻololi ʻia e nā mea genetic i hoʻololi ʻia, a hiki i kēia manawa, hiki iā lākou ke hōʻemi wale i ka helu o nā pēpē maʻi i hānau ʻia ma o ka nānā ʻana i ka prenatal no ka pale ʻana i ke ʻano o nā maʻi hoʻoilina.He ʻano hana ʻole kēia i ʻae ʻia e nā wahine hāpai.
2. ʻO ka ʻike ʻana i ka maʻi: ʻO ka hoʻomaʻamaʻa PCR fluorescent quantitative hiki ke hoʻoholo i ka nui o nā pathogens e like me gonococcus, Chlamydia trachomatis, Mycoplasma solium, maʻi papilloma kanaka, maʻi herpes simplex, maʻi immunodeficiency kanaka, maʻi hepatitis, maʻi influenza, Mycobacterium tuberculosis, EBV a me cytomegalovirus.Loaʻa iā ia nā pōmaikaʻi o ka naʻau kiʻekiʻe, ka liʻiliʻi liʻiliʻi, ka wikiwiki a me ka maʻalahi i hoʻohālikelike ʻia i nā ʻano hoʻokolohua kuʻuna.
3. Hoʻoholo i ka pono o ka lāʻau lapaʻau: hōʻike ʻia ka helu ʻana o ka maʻi hepa B (HBV) a me ka maʻi hepatitis C (HCV) i ka pilina ma waena o ka hoʻouka viral a me ka pono o kekahi mau lāʻau.Inā emi ke kiʻekiʻe serum o HBV-DNA i ka wā o ka mālama ʻana i ka lamivudine a laila piʻi hou a ʻoi aku paha ma mua o ka pae mua, he hōʻailona ia o ka hoʻololi ʻana i ka maʻi virus.
4. ʻO ka hoʻāʻo oncogenetic: ʻOiai ʻaʻole i maopopo ke ʻano o ka ulu ʻana o ka maʻi tumora, ua ʻae nui ʻia ʻo ka hoʻololi ʻana i nā genes kūpono ke kumu kumu o ka hoʻololi oncogenic.Hiki ke ʻike ʻia ka hoʻonui ʻana i ka ʻōlelo a me ka hoʻololi ʻana o nā oncogenes i ka wā mua o nā maʻi maʻi he nui.ʻAʻole maikaʻi wale ka PCR fluorescence quantitative i ka ʻike ʻana i nā mutation i nā genes, akā hiki ke ʻike pololei i ka ʻōlelo o ka oncogenes.Ua hoʻohana ʻia kēia ʻano hana no ka ʻike ʻana i ka hōʻike ʻana o nā ʻano genes like ʻole me ka telomerase hTERT gene, ka maʻi maʻi granulocytic leukemia WT1 gene, ka oncogenic ER gene, ka prostate cancer PSM gene, a me nā genes viral pili i ka tumo.
Unuhi ʻia me www.DeepL.com/Translator (mana manuahi)
Ka manawa hoʻouna: Iune-21-2022