Iyo genomic kutevedzana kweakawanda mavhairasi ave achizivikanwa.Nucleic acid probes izvo zvikamu zvipfupi zveDNA zvakagadzirirwa kusanganiswa neanokwana evhairasi DNA kana RNA zvikamu.Iyo polymerase chain reaction (PCR) inzira inoshanda zvakanyanya yekuona hutachiona.High throughput diagnostic nzira dzakagadzirwa munguva pfupi yapfuura.
A. Nucleic acid hybridization technique
Nucleic acid hybridization, kunyanya kusanganisira Maodzanyemba blotting (Maodzanyemba) uye Maodzanyemba blotting (Maodzanyemba), inokurumidza kuvandudza nzira nyowani mundima yekuongorora hutachiona.Chikonzero cheiyo hybridization assay ndeye kushandisa zvikamu zvipfupi zveDNA (zvinodaidzwa kuti "probe") zvakagadzirirwa kusanganiswa nekuwedzera kwehutachiona hweDNA kana RNA zvikamu.Nekupisa kana kurapa kwealkaline, DNA kana RNA yakaganhurwa yakapetwa kaviri inopatsanurwa kuita tambo imwe chete uye yobva yamiswa parutsigiro rwakasimba.Mushure meizvozvo, probe inowedzerwa uye yakasanganiswa neyakanangwa DNA kana RNA.Sezvo probe yakanyorwa neisotope kana isiri-radioactive nuclide, chinangwa cheDNA kana RNA chinogona kuonekwa kuburikidza ne autoradiography kana ne biotin-avidin system.Sezvo mazhinji ehutachiona genomes akaumbwa uye akateedzana, anogona kuwonekwa pachishandiswa hutachiona-chaiyo kutevedzana se probes mumuenzaniso.Parizvino, nzira dzekusanganisa dzinosanganisira: dot blot, in situ hybridization mumaseru, DNA blotting(DNA) (Southern blot) uye RNA blotting(RNA) (Northern blot).
B.PCR Technology
Mumakore achangopfuura, nhevedzano ye in vitro nucleic acid amplification maitiro akagadzirwa zvichibva paPCR, kuyedza hutachiona husina hanya kana husingarimike.PCR inzira inogona kusanganisa chaiyo DNA kutevedzana ne in vitro polymerase reaction.Maitiro ePCR anosanganisira kutenderera kwekupisa kwematanho matatu: denaturation, annealing, uye kuwedzera Pakupisa kwakanyanya (93 ℃ ~ 95 ℃), iyo DNA yakapetwa kaviri inopatsanurwa kuita maviri single strands DNA;ipapo pakudziya kwakadzika (37 ℃ ~ 60 ℃), maviri akagadzirwa nucleotide primers anneal kune anowirirana DNA zvikamu;nepo pakudziya kwakakodzera kweTaq enzyme (72 ℃), kuumbwa kwemacheni matsva eDNA kunotanga kubva kuprimer 3'kumagumo vachishandisa DNA inopindirana sematemplate uye nucleotides imwechete semidziyo.Saka mushure mekutenderera kwega kwega, cheni imwe yeDNA inogona kukwidziridzwa kuita macheni maviri.Kudzokorora maitiro aya, cheni yega yega yeDNA yakagadzirwa mune imwe denderedzwa inogona kushandiswa setemplate mune inotevera kutenderera, uye nhamba yemaketani eDNA inopetwa kaviri muchikamu chega chega, zvinoreva kuti kugadzirwa kwePCR kunowedzerwa mu 2n log kumhanya.Mushure memakumi maviri neshanu kusvika makumi matatu kutenderera, kugadzirwa kwePCR kunoonekwa kuburikidza ne electrophoresis, uye chaiyo DNA zvigadzirwa zvinogona kucherechedzwa pasi peUV mwenje (254nm).Nekuda kwekunaka kwayo, kunzwa, uye nyore, PCR yakagamuchirwa mukuongororwa kwekiriniki kwehutachiona hwakawanda hwehutachiona hwakadai seHCV, HIV, CMV, uye HPV.Sezvo PCR inonyatsonzwa, inogona kuona hutachiona DNA pafg level, kuvhiya kunofanirwa kuitwa nekuchenjerera kudzivirira manyepo.Pamusoro pezvo, mhedzisiro yakanaka mukuyedzwa kwenucleic acid hazvireve kuti kune hutachiona hwehutachiona mumuenzaniso.
Nekushandiswa kwakakura kwePCR tekinoroji, matekiniki matsva uye nzira dzinogadzirwa zvichibva paPCR tekinoroji kune akasiyana bvunzo chinangwa.Semuenzaniso, iyo chaiyo nguva yehuwandu PCR inogona kuona hutachiona huwandu;in situ PCR inoshandiswa kuona hutachiona hwehutachiona munyama kana masero;Iyo nested PCR inogona kuwedzera iyo chaiyo yePCR.Pakati pavo, nguva chaiyo yehuwandu PCR yakagadziridzwa nekukurumidza.Mazhinji matekiniki matsva, akadai seTaqMan hydrolysis probe, hybridization probe, uye molecular beacon probe, akasanganiswa kuita chaiyo nguva yehuwandu hwePCR maitiro, ayo anoshandiswa zvakanyanya mukutsvagisa kwekiriniki.Kunze kwekuziva huwandu hwehutachiwana huri mumvura yemurwere nemazvo, nzira iyi inogona kushandiswawo kuona inoshivirira zvinodhaka.Naizvozvo, chaiyo nguva yehuwandu PCR inonyanya kushandiswa mukurapa maitiro ekuongorora uye kushivirira zvinodhaka kuongororwa.
C. High-throughput kuonekwa kweviral nucleic acids
Kuti isangane nezvinodiwa zvekuongororwa nekukurumidza kwezvirwere zvitsva zvinotapukira, nzira dzakasiyana-siyana dzekuona dzepamusoro, senge DNA chips(DNA), dzakavambwa.Kune machipisi eDNA, maprobe chaiwo anogadzirwa uye anonamirwa kune madiki silicon machipi mune yakanyanya density kugadzira DNA probe microarray (DNA) inogona kusanganiswa nemuenzaniso.Chiratidzo chehybridization chinogona kufananidzwa neconfocal microscope kana laser scanner uye inogona kugadziridzwazve nekombuta uye data hombe rine chekuita nemajini akasiyana rinogona kuwanikwa.Kune marudzi maviri eDNA chip.Iyo "synthesis chip" yakafanana neinotevera: iyo chaiyo oligonucleotides inogadzirwa zvakananga pamachipi.Imwe DNA dziva chip.Iwo akaumbwa majini kana PCR zvigadzirwa zvakadhindwa zvakarongeka pachisiraidhi.Chakanakira cheDNA chip tekinoroji ndeyekuona panguva imwe chete yehuwandu hukuru hwekutevedzana kweDNA.Iyo yazvino vhezheni ye pathogen yekuona chip inogona kuona anopfuura zana nemazana manomwe mavhairasi emunhu kamwechete.DNA chip tekinoroji yakagadzirisa matambudziko echinyakare nucleic acid hybridization nzira uye ine yakakura kwazvo mashandisirwo muhutachiona hwehutachiona uye epidemiological kudzidza.
Nguva yekutumira: Zvita-23-2020