Direct PCR ke karabelo e sebelisang ka kotloloho lisele tsa liphoofolo kapa tsa semela bakeng sa ho holisa ntle le ho ntšoa ha nucleic acid.Ka litsela tse ngata, PCR e tobileng e sebetsa joalo ka PCR e tloaelehileng

Phapang e kholo ke "buffer" ea tloaelo e sebelisoang ho PCR e tobileng, sampole e ka beoa ka kotloloho ho karabelo ea PCR ntle le ho ntšoa ha nucleic acid, empa ho na le litlhoko tse tsamaellanang tsa mamello ea li-enzyme le ho lumellana ha buffer e amehang karabelong e tobileng ea PCR.

Leha ho na le li-inhibitors tsa PCR tse ngata kapa tse nyane lisampoleng tse tloaelehileng, PCR e tobileng e ntse e ka fihlela matlafatso e tšepahalang tlasa ts'ebetso ea li-enzyme le li-buffer.Mokhoa oa setso oa PCR o hloka boleng ba nucleic acid ea boleng bo holimo joalo ka template, e ka thibelang tsoelo-pele e ntle ea karabelo ea PCR haeba template e na le liprotheine le litšila tse ling.Direct PCR hajoale ke e 'ngoe ea mahlale a tsebahalang haholo lefapheng la tlhahlobo ea limolek'hule.

01 Direct PCR qalong e ne e sebelisetsoa liphoofolo le semela

Ts'ebeliso ea pele ea PCR e tobileng e sebakeng sa liphoofolo le limela, joalo ka mali, lisele le moriri oa rat, katse, khoho, mmutla, linku, likhomo, joalo-joalo, makhasi a limela le lipeo, joalo-joalo, tse sebelisetsoang ho ithuta genotyping, transgenic, plasmid analysis, Gene knockout analysis, DNA source ID.

Masimo ana a na le likarolo tse ling tse tloaelehileng, ke hore, sepheo sa liphatsa tsa lefutso li batla li phahame 'me ho ntšoa ha nucleic acid ho thata, kahoo PCR e tobileng e ke ke ea boloka nako feela le ho ba le tšusumetso e nyenyane liphellong, empa hape e boloka litšenyehelo.

PCR e tobileng e sebelisetsoang ho lemoha pathogen ke taba ea lilemong tsa morao tjena, bahlahisi ba bang ba PCR ba entse boiteko bo bongata ntlheng ena ha ba etsa mekhoa e mecha.Haholo-holo seoa sena sa COVID-19, lihlahisoa tse joalo tse ngata tsa ho sibolla li hlahile 'marakeng, joalo ka SARS-CoV-2 Nucleic Acid Detection Kit (Multiplex PCR Fluorescent Probe Method) e entsoeng lipatlisiso le ho ntlafatsoa ke Foregene, e sebelisang theknoloji ea Real-time RT PCR (rRT-PCR) bakeng sa tlhahlobo ea boleng ba SARS kapa SARS-Cophary2 lisampole tsa swab.

Foregene ke e 'ngoe ea lik'hamphani tse sebelisang theknoloji ea Direct PCR, bakeng sa ho lemoha lintho tse tloaelehileng tsa ORF1ab, N, E, lephapang lineages nucleic acids ho disampole tsa swab tsa nasopharyngeal kapa oropharyngeal tse kang SARS-CoV-2 B.1.1.7 lineage (UK), B.1.351 leloko (ZA), B.1.617 leloko (IND) le P.1 leloko (BR).

02  Lisebelisoa tse hlokahalang bakeng sa PCR e tobileng

Mohlala oa Lysate

Mohlala oa lysate o ka hlophisoa ke uena kapa oa rekoa.Phapang ea ho hlophisoa ha lihlahisoa tse fapaneng tsa lysate e tla etsa hore bokhoni ba leshano bo fapane, ebe nako ea ho bua leshano e tla fapana hanyane.Mohlala, bakeng sa ho lokisoa ha lisampole tsa linama tsa liphoofolo, hangata ho khothaletsoa metsotso e 30 kapa lysis ea bosiu bo le bong, 'me tharollo ea lysis bakeng sa livaerase e tloha metsotso e 3-10.

PCR master mix

Ho khothaletsoa ho sebelisa DNA polymerase e chesang ho qala ho matlafatsa matlafatso e khethehileng le ho eketsa bokhoni ba ho holisa.Koko ea PCR e tobileng ke polymerase e mamellang haholo.

Tlosa kapa thibela likarolo tsa sampuli tse amang DNA amplification

Kamora hore sampole e sebetsoe ka lysate, liprotheine, lipids le litšila tse ling tsa lisele li tla lokolloa, lintho tsena li tla thibela karabelo ea PCR.Ka hona, PCR e tobileng e hloka ho eketsoa ha ho tlosoa kapa li-inhibitors tse lumellanang ho fokotsa tšusumetso ea lintlha tsena.

03  Pokello ea lintlha tse hlano tsa tsebo ea PCR e tobileng

Taba ea pele, theknoloji ea Direct PCR ke theknoloji e tobileng ea PCR bakeng sa disampole tse fapaneng tsa baeloji.Tlas'a boemo bona ba tekheniki, ha ho na tlhoko ea ho arola le ho ntša nucleic acid, ho sebelisa sampole ea linama ka kotloloho joalo ka ntho, 'me u kenyelle liphatlalatso tsa liphatsa tsa lefutso ke ho etsa karabelo ea PCR.

Taba ea bobeli, thekenoloji ea Direct PCR ha se feela mokhoa oa khale oa ho hōlisa template ea DNA, empa hape e kenyelletsa RNA template reverse transcription PCR.

Taba ea boraro, thekenoloji ea Direct PCR ha e etse feela karabelo ea boleng ba PCR lisampoleng tsa linama, empa hape e kenyelletsa karabelo ea nako ea 'nete ea qPCR, e hlokang hore sistimi ea karabelo e be le tšitiso e matla ea anti-background fluorescence le endogenous fluorescence e tima bokhoni ba bohanyetsi.

Ea bone, mehlala e lebisitsoeng ke theknoloji ea Direct PCR e hloka feela ho lokolloa ha li-template tsa nucleic acid, 'me u se ke ua tlosa liprotheine, li-polysaccharides, li-ion tsa letsoai, joalo-joalo tse sitisang karabelo ea PCR.E hlokang nucleic acid polymerase le PCR Mix tsamaisong ea karabelo ho ba le khanyetso e ntle haholo le ho ikamahanya le maemo ho netefatsa ts'ebetso ea enzyme le ho nepahala ha ho ikatisa tlasa maemo a thata.

Ea bohlano, sampole ea linama tse lebisitsoeng ho thekenoloji ea Direct PCR ntle le kalafo ea ntlafatso ea nucleic acid mme palo ea template e nyane haholo, e hlokang hore sistimi ea karabelo e be le kutlo e phahameng haholo le bokhoni ba ho holisa.