I-real-time fluorescence quantitative PCR yindlela yokulinganisa isixa esipheleleyo semveliso emva komjikelo ngamnye we-polymerase chain reaction (PCR) kwi-DNA amplification reaction usebenzisa i-fluorophore.Indlela isetyenziselwa ukulinganisa ulandelelwano oluthile lwe-DNA kwisampuli ukuba ivavanywe ngeendlela zokubhekisela zangaphakathi okanye zangaphandle.Ukusukela oko yaqalayo, iimvavanyo ze-PCR zobungakanani befluorescent ziye zanda zithandwa ngootitshala baselabhoratri.
Fluorescence PCR umgaqo: Fluorescence PCR, okokuqala ebizwa TaqManPCR kwaye kamva kwakhona Real-TimePCR, yindlela entsha nucleic acid quantification ubuchule PE (PerkinElmer) e-USA ngo 1995. Ubuchule isekwe ekongezweni kweprobe ebhalwe ngefluorescently okanye ehambelanayo. Idayi ye-fluorescent kwi-PCR eqhelekileyo ukufezekisa umsebenzi wayo wobuninzi.Umgaqo: njengoko i-PCR reaction iqhubeka, iimveliso ze-PCR reaction ziyaqokelelana kwaye ubunzulu besignali ye-fluorescent bunyuka ngokulinganayo.Ngomjikelezo ngamnye, isignali ye-fluorescence intensity iqokelelwa ukuze sikwazi ukubeka iliso utshintsho kwixabiso lemveliso ngokutshintsha kwe-fluorescence intensity kwaye ngaloo ndlela sifumane igrafu ye-fluorescence amplification curve.
Ngokubanzi, ijika lokukhulisa i-fluorescence linokwahlulwa libe ngamanqanaba amathathu: inqanaba lophawu lwangasemva lwe-fluorescence, isigaba sokukhulisa uphawu lwe-fluorescence kunye nesigaba seplateau.Ngexesha lenqanaba lophawu lwangasemva, isignali ye-fluorescence eyandisiweyo igqunywa ngumqondiso ongasemva we-fluorescence kwaye utshintsho kwixabiso lemveliso alunakuchazwa.Kwinqanaba le-plateau, imveliso yokukhulisa imveliso ayisayi kunyuka ngokukhawuleza, akukho budlelwane bomgca phakathi kwexabiso lemveliso yokugqibela kunye nenani le-template yokuqala, kwaye inombolo yokuqala yekopi ye-DNA ayinakubalwa ngokusekelwe kwixabiso lokugqibela lemveliso ye-PCR.Kuphela kwisigaba sokukhulisa i-exponential yesignali ye-fluorescent kukho ubudlelwane bomgca phakathi kwe-logarithm yexabiso lemveliso ye-PCR kunye nesixa setemplate sokuqala, kwaye sinokukhetha ukulinganisa oku kweli nqanaba.Ukuze kube lula ukulinganisa kunye nokuthelekisa, iikhonsepthi ezimbini ezibaluleke kakhulu ziye zaziswa kwi-real-time fluorescent quantitative technology PCR: i-fluorescence threshold kunye nexabiso le-CT.
Umda lixabiso elimiselwe ngokwendalo kwigophe lokukhulisa i-fluorescence.inqanaba lokwandisa iPCR.
Ixabiso le-Ct: linani lemijikelo apho umqondiso we-fluorescence kwi-tube nganye yokusabela ufikelele kwixabiso le-domain ebekiwe.
Ubudlelwane phakathi kwexabiso le-Ct kunye ne-template yokuqala: izifundo zibonise ukuba ixabiso le-Ct le-template nganye linobudlelwane bomgca kunye ne-logarithm yenombolo yekopi yokuqala yaloo template, iikopi ezininzi zenombolo yokuqala yekopi, i-Ct encinci. ixabiso.Amaxabiso e-Ct azinzile.Ijika eliqhelekileyo linokwenziwa kusetyenziswa umgangatho kunye nenombolo yekopi yokuqala eyaziwayo, apho i-horizontal coordinate imele i-logarithm yenombolo yokuqala yekopi kwaye i-vertical coordinate imele ixabiso le-Ct njengoko kubonisiwe kumfanekiso ongezantsi.
Ngoko ke, ngokufumana ixabiso le-Ct yesampuli engaziwayo, inombolo yokuqala yekopi yaloo sampuli ingabalwa kwi-curve eqhelekileyo.
Ixabiso le-Ct alikho rhoqo kwaye lingaphenjelelwa ngamasampuli ahlukeneyo kunye nezixhobo ezahlukeneyo, nokuba isampuli efanayo iphinda iphindwe ngamaxesha e-2 kwisixhobo esifanayo, ixabiso le-Ct lingahluka.
Ubungakanani bovavanyo lwe-fluorescence: Ubungakanani bovavanyo lwe-fluorescence lunokwahlulwa lube ziiprobe ze-fluorescent kunye nedayi ye-fluorescent ngokuxhomekeke kwiziphawuli ezisetyenzisiweyo.Iiprobes zeFluorescent ziquka iteknoloji yeBeacon (iteknoloji ye-molecular beacon, emelwe yi-American Tagyi), i-TaqMan probes (emelwe yi-ABI) kunye neteknoloji ye-FRET (emelwe nguRoche);Idayi zefluorescent ziquka iidayi zefluorescent ezigcweleyo kunye nedayi ye-fluorescent engahluthiyo, ummeli oqhelekileyo wedayi ye-fluorescent engahluthisiyo yi-SYBRGreen I, esetyenziswa ngokuqhelekileyo ngoku;saturated Ummeli oqhelekileyo weedayi zefluorescent ezingahluthisiyo yi-SYBRGreenⅠ;iidayi zefluorescent ezigcweleyo zi-EvaGreen, LCGreen, njl.
I-SYBRGreenI yidayi ebophelela i-DNA exhaphakileyo ye-fluorescent PCR, ebophelela ngokungangqalanga kwi-DNA ephindwe kabini.Kwimeko yayo ekhululekileyo, i-SYBRGreenI ikhupha i-fluorescence ebuthathaka, kodwa xa ibotshelelwe kwi-DNA ephindwe kabini, i-fluorescence yayo yonyuka ka-1000.Ke ngoko, isiginali ye-fluorescence iyonke ekhutshwe yi-reaction ilingana nobungakanani be-DNA ephindwe kabini ekhoyo kwaye iyanda njengoko imveliso yokukhulisa inyuka.
Izinto eziluncedo kwiidayi ezibophelelayo ze-DNA ezinemisonto ephindwe kabini: uyilo olulula lokulinga, kuphela ii-primers ezi-2 ezifunekayo, akukho mfuneko yokuyila iiprobe, akukho mfuneko yokuyila iiprobes ezininzi zovavanyo olukhawulezayo lweejini ezininzi, ukukwazi ukwenza uhlalutyo lwegophe lokunyibilika, ukuvavanya ubume bendlela. ukusabela kokukhulisa, ixabiso lokuqala eliphantsi, isiqhelo esilungileyo kwaye ke ngoko isetyenziswa kakhulu kuphando ekhaya nakwamanye amazwe.
Indlela ye-Fluorescent probe (ubuchule be-Taqman): Xa ulwandiso lwePCR lusenziwa, iperi yeprimers iyongezwa kunye neprobe ethile yefluorescent.Xa i-probe intact, isibonakaliso se-fluorescence esikhutshwe liqela lentatheli lixutywe liqela elicinyiweyo kwaye alibonwa sisixhobo sePCR;ngexesha lokwandiswa kwe-PCR (kwisigaba sokwandiswa), umsebenzi wokucoca we-5'-3' we-enzyme ye-Taq wehlisa i-probe nge-enzymatic, okwenza iqela lentatheli ye-fluorescence kunye neqela elicinyiweyo le-fluorescence.
Ukusetyenziswa kwe-fluorescent quantitative PCR.
Uphando lwebhayoloji yeemolekyuli:
1. Uhlalutyo lwe-nucleic acid yobungakanani.Uhlalutyo lobungakanani kunye nomgangatho wezifo ezithathelwanayo, ukufumanisa i-pathogenic microorganisms okanye iintsholongwane, ezifana nomkhuhlane we-influenza A (H1N1) yakutshanje, ukufumanisa amanani ekopi yofuzo yezityalo kunye nezilwanyana eziguquguqukayo, ukufumanisa i-RNAi gene inactivation rates, njl.
2. Uhlalutyo lwentsingiselo yemfuza eyahlukileyo.Ukuthelekiswa komehluko wokubonakaliswa kofuzo phakathi kweesampulu ezinyangweyo (umzekelo, unyango lwechiza, unyango ngokwasemzimbeni, unyango lwemichiza, njl. njl.), ukubonakaliswa kokwahluka kofuzo oluthile kwizigaba ezahlukeneyo kunye nokuqinisekiswa kwe-cDNA microarray okanye iziphumo zokubonisa umahluko.
3. Ukufunyanwa kwe-SNP.Ukufumanisa i-polymorphisms ye-nucleotide eyodwa kubalulekile ekufundweni komntu ngamnye kwizifo ezahlukeneyo okanye impendulo yomntu kumachiza athile, kwaye ngenxa yesakhiwo esinobuchule se-molecular beacon, emva kokuba ulwazi olulandelelanayo lwe-SNP luyaziwa, kulula kwaye luchanekile. sebenzisa obu buchule kubhaqo lwe-SNP ephezulu.
4. Ukufumanisa i-methylation.I-Methylation inxulunyaniswa nezifo ezininzi zabantu, ngakumbi umhlaza, kwaye uLaird uchaze ubuchule obubizwa ngokuba yiMethylight, ephatha i-DNA ngaphambi kokukhulisa ukuze i-cytosine i-unmethylated ibe yi-uracil kwaye i-methylated cytosine ayichaphazeleki, isebenzisa iiprimers ezithile kunye ne-Taqman probes ukwahlula phakathi kwe-methylated kunye ne-unmethylated DNA. .ubuthathaka ngakumbi.
Uphando lwezonyango:
1. Ukuxilongwa kwangaphambi kokubeletha: abantu abanakunyanga izifo ezibangelwa yimfuzo etshintshileyo, kwaye ukuza kuthi ga ngoku, banokunciphisa kuphela inani labantwana abagulayo abazalwa ngokujongwa kwangaphambili ukukhusela ukwenzeka kwezifo ezahlukeneyo zefa.Le yindlela engabonakaliyo eyamkelwa lula ngabasetyhini abakhulelweyo.
2. Ubhaqo Pathogen: The fluorescent quantitative PCR assay ivumela ukuzimisela quantitative of pathogens ezifana gonococcus, Chlamydia trachomatis, Mycoplasma solium, papilloma virus, herpes simplex virus, human immunodeficiency virus, virus hepatitis, virus influenza, Mycocycyculosis, Tuber EB TB.Ineenzuzo zobuntununtunu obuphezulu, ubungakanani besampulu esezantsi, ukukhawuleza kunye nokulula xa kuthelekiswa neendlela zovavanyo lwemveli.
3. Uvavanyo lokuphumelela kweziyobisi: uhlalutyo lobungakanani bentsholongwane ye-hepatitis B (HBV) kunye nentsholongwane ye-hepatitis C (HCV) ibonisa ukuba ubudlelwane phakathi komthamo wentsholongwane kunye nokusebenza kwamachiza athile.Ukuba inqanaba le-serum ye-HBV-DNA iyancipha ngexesha lonyango lwe-lamivudine kwaye iphinda inyuke okanye idlule inqanaba langaphambili, libonisa ukuguqulwa kwentsholongwane.
4. Uvavanyo lwe-Oncogenetic: Nangona indlela yokuphuhliswa kwe-tumor ingakacaci, iyamkelwa ngokubanzi ukuba ukuguqulwa kwezakhi zofuzo ezichaphazelekayo yimbangela esisiseko yokuguqulwa kwe-oncogenic.Ukwandiswa kwentetho kunye nokuguqulwa kwe-oncogenes kunokubonwa kwizigaba zokuqala zamathumba amaninzi.Ixesha lokwenyani le-fluorescence quantitative PCR ayisebenzi nje kuphela ekuboneni utshintsho kwijene, kodwa inokubona ngokuchanekileyo intetho ye-oncogenes.Le ndlela isetyenziselwe ukufumanisa ukubonakaliswa kweentlobo ezahlukeneyo zofuzo ezibandakanya i-telomerase hTERT gene, i-general granulocytic leukemia WT1 gene, i-oncogenic ER gene, i-prostate cancer ye-PSM gene, kunye ne-tumor-associated viral genes.
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Ixesha lokuposa: Jun-21-2022