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I-PCR yeyona nto isetyenziswa kakhulu itekhnoloji yokukhulisa i-nucleic acid kwaye isetyenziswa kakhulu ngenxa yobuntununtunu kunye nokucaciswa kwayo.Nangona kunjalo, i-PCR idinga ukuphindaphindwa kwe-thermal denaturation kwaye ayikwazi ukususa imida yokuxhomekeka kwizixhobo kunye nezixhobo, ezithintela ukusetyenziswa kwayo kuvavanyo lwecandelo leklinikhi.

Ukusukela ekuqaleni kweminyaka yoo-1990, iilabhoratri ezininzi ziye zaqalisa ukuphuhlisa itekhnoloji yokukhulisa ubushushu obungaguqukiyo obungadingi kuthotywa kwe-thermal.Ngoku baye baphuhlisa itekhnoloji yokukhulisa i-isothermal ye-loop-mediated, itekhnoloji yokukhulisa i-isothermal yokubuyisela i-isothermal, iteknoloji yokukhulisa isangqa se-isothermal, kunye nokuxhomekeka kokulandelelana kwe-nucleic acid.Itekhnoloji yokukhulisa i-Isothermal kunye nobunye ubuchwepheshe. 

Li-oop-mediated isothermal amplification

Umgaqo wokukhulisa usekelwe kwinto yokuba i-DNA ikwimo eguquguqukayo yokulingana malunga nama-65°C.Xa nayiphi na i-primer i-base-paired kwaye yandiswe kwi-complementary ye-DNA ephindwe kabini, enye i-strand iya kuhlukana kwaye ibe yinto enye.

Kulo bushushu, i-DNA isebenzisa ii-primers ezi-4 ezikhethekileyo ukuthembela kwi-strand-displacement DNA polymerase ukwenza i-synthesis ye-strand-displacement DNA self-circulate ngokuqhubekayo.

Okokuqala misela imimandla ethile ye-6 F3, F2, F1, B1, B2, B3 kwi-gene ekujoliswe kuyo, kwaye emva koko uyila iiprimers ezi-4 ngokusekelwe kule mimandla ethile ye-6 (njengoko kubonisiwe kumfanekiso ongezantsi):

I-front internal primer (FIP) yenziwe nge-F1c kunye ne-F2.

I-primer yangaphakathi yangasemva (i-BIP) yenziwe nge-B1c kunye ne-B2, kwaye i-TTTT isetyenziswe njenge-spacer phakathi.

I-primers yangaphandle i-F3 kunye ne-B3 ngokulandelanayo yenziwe yimimandla ye-F3 kunye ne-B3 kwi-gene ekujoliswe kuyo.

Itekhnoloji yokukhulisa i-nucleic acid isothermal

Kwinkqubo yokusabela kwe-LAMP, ukuxinwa kwe-primer yangaphakathi ngamaxesha amaninzi kune-primer yangaphandle.I-primer yangaphakathi idibaniswa okokuqala kunye ne-template strand ukudibanisa i-strand encedisayo ukwenza i-DNA double strand.Emva koko, i-primer yangaphandle idibaniswe ne-template strand ukwenza i-DNA double strand.Ngaphantsi kwesenzo se-polymerase ye-BstDNA, i-strand encedisayo eyenziwa yi-primer yangaphakathi iyakhululwa.Emva kothotho lokusabela, umsonto ohambelanayo ekugqibeleni wenza umsonto omnye we-DNA kunye nesakhiwo se-dumbbell.

Isakhiwo se-dumbbell DNA i-strand enye ngokwayo isetyenziswe njenge template ngokuqhubekayo ukwenza i-transitional stem-loop structure DNA enesiphelo esivulekileyo.I-primers yangaphakathi nangaphandle ikhokela i-stem-loop ye-stem-loop i-DNA ukuba iqhube ngokuqhubekayo i-strand displacement kunye nokuphendula okwandisiweyo, kwaye ekugqibeleni yenze izakhiwo ezininzi ze-stem-loop ezinobude obuhlukeneyo.DNA umxube.

Nucleic acid isothermal amplification technology2

Izinto eziluncedo kunye nokungalunganga kwe-loop-mediated isothermal amplification

Izinto ezilungileyo ze-LAMP:

(1) Ubuchule obuphezulu bokukhulisa, obunokwandisa ngokufanelekileyo iikopi ze-1-10 zejini elijoliswe kuyo ngaphakathi kwe-1h, kunye nokusebenza kakuhle kwe-amplification ngamaxesha angama-10-100 kwi-PCR eqhelekileyo.

(2) Ixesha lokuphendula lifutshane, ukuchaneka komelele, kwaye akukho sixhobo sikhethekileyo sifunekayo.

Ukusilela kweLAMP:

(1) Iimfuno zeeprimers ziphezulu kakhulu.

(2) Imveliso eyandisiweyo ayinakusetyenziselwa i-cloning kunye nokulandelelana, kodwa ingasetyenziselwa isigwebo kuphela.

(3) Ngenxa yobuthathaka obunamandla, kulula ukwenza i-aerosols, ebangela ukungahambi kakuhle kunye nokuchaphazela iziphumo zovavanyo.

Strand displacement ukukhulisa

I-Strand displacement amplification (SDA) bubuchule be-in vitro isothermal DNA yokukhulisa esekwe kwimpendulo ye-enzymatic eyacetywa kuqala ngumphengululi waseMelika u-Walker ngo-1992.

Inkqubo esisiseko ye-SDA ibandakanya i-endonuclease yokuthintela, i-polymerase ye-DNA ene-strand displacement activity, izibini ezimbini ze-primers, i-dNTPs, kunye ne-calcium kunye ne-magnesium ion kunye ne-buffer systems.

Umgaqo wokukhulisa i-strand displacement usekwe kulandelelwano lokuqaphela i-endonuclease ye-chemical modified restriction kuzo zombini iziphelo ze-DNA ekujoliswe kuyo.I-endonuclease ivula i-gap kwi-DNA ye-strand kwindawo yayo yokuqaphela, kwaye i-polymerase ye-DNA yandisa i-gap 3 'Ukuphela kwaye ithathe indawo ye-DNA strand elandelayo.

Iintambo ezitshintshiweyo ze-DNA zinokudityaniswa kunye ne-primers kwaye zandiswe zibe yimisonto ephindwe kabini nge-DNA polymerase.Le nkqubo iphindaphindwa ngokuqhubekayo, ukwenzela ukuba ulandelelwano lwethagethi lwandiswe ngokufanelekileyo.

Nucleic acid isothermal yokukhulisa ubuchwepheshe3

Izinto ezilungileyo kunye nokungalunganga kweteknoloji yokukhulisa i-strand displacement

Izinto eziluncedo zeSDA:

Ukuphumelela kokukhulisa kuphezulu, ixesha lokuphendula lifutshane, ukucaciswa komelele, kwaye akukho sixhobo sikhethekileyo sifunekayo.

Ukusilela kwe-SDA:

Iimveliso azifani, kwaye ezinye iimveliso ezidibeneyo kunye ne-double-stranded zihlala ziveliswa kumjikelezo we-SDA, kwaye umsila uya kwenzeka ngokuqinisekileyo xa ufunyenwe yi-electrophoresis.

Rukukhulisa isangqa olling

I-Rolling circle amplification (RCA) icetywayo ngokuzoba kwindlela yokukhuphela i-DNA kwizinto eziphilayo ze-pathogenic ngokujikeleza isangqa.Ibhekisela ekusetyenzisweni kwe-DNA ye-setyhula ene-single-stranded njenge-template kwiqondo lokushisa elingaguqukiyo, kunye ne-polymerase ekhethekileyo ye-DNA (efana ne-Phi29) ) Ngaphantsi kwesenzo sokujikeleza isangqa se-DNA synthesis ukufezekisa ukukhulisa i-gene ekujoliswe kuyo.

I-RCA inokwahlulwa ibe yi-linear amplification kunye ne-exponential amplification.Ukusebenza kwe-RCA yomgca kunokufikelela kwi-105amaxesha, kunye nokusebenza kwe-exponential RCA kunokufikelela kwi-109amaxesha.

Umahluko olula, njengoko kubonisiwe kumzobo ongezantsi, ulwandiso lomgca a lusebenzisa i-primer e-1 kuphela, i-exponential amplification b ine-primers ezi-2.

Nucleic acid isothermal amplification technology4

Linear RCA ikwabizwa ngokuba single primer RCA.I-primer ibophelela kwi-DNA yesetyhula kwaye yongezwa ngesenzo se-DNA polymerase.Imveliso yilayini yomgca omnye kunye nenani elikhulu lokuphindaphinda ulandelelwano ngamawaka amaxesha ubude belophu enye.

Ekubeni imveliso ye-RCA yomgca isoloko ixhunywe kwi-primer yokuqala, ukulungiswa lula kwesignali kuyinzuzo enkulu.

I-RCA ye-Exponential, eyaziwa ngokuba yi-Hyper branched amplification HRCA (Hyper branched RCA), kwi-RCA ye-exponential, enye i-primer ikhulisa imveliso ye-RCA, i-primer yesibini idibanisa kunye nemveliso ye-RCA kwaye yandisa, kwaye ukutshintshwa sele kuboshwe kwimveliso ye-RCA I-primers esezantsi yandisa i-strand, kwaye iphinda isandiso kunye nokutshintshwa kwe-RCA ye-amplification yokuvelisa imveliso ye-RCA.

Nucleic acid isothermal amplification technology5

Izinto eziluncedo kunye nezingeloncedo zokukhulisa isangqa se-nucleic acid

Izinto eziluncedo zeRCA:

Uvakalelo oluphezulu, ukuchaneka okulungileyo kunye nokusebenza okulula.

Ukusilela kwe-RCA:

Iingxaki ezingasemva ngexesha lokubonwa komqondiso.Ngexesha lokuphendula kwe-RCA, iprobe ye-padlock engajikelezikanga kunye ne-template ye-DNA okanye i-RNA ye-probe engapheliyo inokuvelisa imiqondiso ethile yangasemva. 

Nucleicacid ulandelelwano-based amplification

I-Nucleic acid sequence-based amplification (NASBA) bubuchwepheshe obutsha obuphuhliswe kwisiseko se-PCR.Yinto eqhubekayo kunye ne-isothermal nucleic acid amplification ekhokelwa ngamabini e-primers kunye nolandelelwano lomgqugquzeli we-T7.Ithekhnoloji inokukhulisa i-template ye-RNA malunga namaxesha angama-109 malunga neeyure ze-2, amaxesha angama-1000 aphezulu kunendlela ye-PCR eqhelekileyo kwaye ayifuni izixhobo ezizodwa.

Le teknoloji isetyenziselwe ukuxilongwa ngokukhawuleza kwezifo ngokukhawuleza njengoko kubonakala, kwaye iinkampani ezininzi ngoku zisebenzisa le ndlela kwiikiti zokufumanisa i-RNA.

Nangona ulwandiso lwe-RNA lunokusebenzisa itekhnoloji ye-PCR yokukhuphela umva, i-NASBA ineengenelo zayo: inokuqhutywa phantsi kweemeko zobushushu obungatshintshiyo, kwaye izinzile kwaye ichanekile kunetekhnoloji ye-PCR yemveli.

Ukusabela kuku 41 degrees celcius kwaye kufuna AMV (avian myeloblastosis virus) reverse transcriptase, RNase H, T7 RNA polymerase kunye nepere yeprimers ukugqiba.

Inkqubo ikakhulu ibandakanya:

I-primer yangaphambili iqulethe ukulandelelana okuhambelanayo komgqugquzeli we-T7.Ngethuba lokuphendula, i-primer yangaphambili ibophelela kwi-RNA strand kwaye i-catalyzed yi-enzyme ye-AMV ukwenza i-DNA-RNA i-strand kabini.

I-RNase H yetyisa i-RNA kwi-hybrid-strand ephindwe kabini kwaye igcina i-DNA enomsonto omnye.

Ngaphantsi kwesenzo se-reverse primer kunye ne-enzyme ye-AMV, i-DNA kabini i-strand ene-T7 umgqugquzeli wokulandelelana yenziwa.

Ngaphantsi kwesenzo se-T7 RNA polymerase, inkqubo yokubhala igqityiwe kwaye inani elikhulu le-RNA ekujoliswe kuyo liveliswa.

Nucleic acid isothermal amplification technology6

Izinto eziluncedo zeNASBA:

(1) I-primer yayo inolandelelwano lomgqugquzeli we-T7, kodwa i-DNA yangaphandle ephindwe kabini ayinayo i-T7 ulandelelwano lomgqugquzeli kwaye ayikwazi ukunyuswa, ngoko ke le teknoloji inobuchwephesha obuphezulu kunye novelwano.

(2) I-NASBA idibanisa ngokuthe ngqo inkqubo yokubhalwa kwe-reverse kwi-amplification reaction, inciphisa ixesha lokuphendula.

Izinto ezingalunganga zeNASBA:

(1) Amacandelo okusabela anzima ngakumbi.

(2) Iintlobo ezintathu zee-enzymes ziyafuneka ukwenza ukusabela kubize kakhulu.


Ixesha lokuposa: Aug-06-2021