Siyabonga ngokuvakashela i-Nature.com.Inguqulo yesiphequluli oyisebenzisayo inokwesekwa okulinganiselwe kwe-CSS.Ukuze uthole ulwazi olungcono kakhulu, sincoma ukuthi usebenzise isiphequluli esibuyekeziwe (noma uvale imodi yokuhambisana ku-Internet Explorer).Okwamanje, ukuze uqinisekise ukuqhubeka nokusekela, sizobonisa isayithi ngaphandle kwezitayela ne-JavaScript.
Ukubaluleka kokuqapha amasampula emvelo kuye kwathakazelelwa kakhulu kusukela ekuqaleni kobhadane lwe-COVID-19, futhi eminye imizamo yokuqapha yenziwa kusetshenziswa izinga legolide, nakuba amasu asekelwe ku-qPCR emba eqolo.Ama-biosensors e-electrochemical DNA angahlinzeka ngendlela engabizi kakhulu. Isixazululo sokuqapha amasampula amanzi emvelo emazweni anemali engenayo ephansi naphakathi. Kulo msebenzi, sibonisa ukutholwa kwe-electrochemical ama-amplicon atholwe ku-Phi6 phage ehlukanisiwe namasampula amanzi echibi eline-spiked (i-surrogate edumile ye-SARS-CoV-2), kusetshenziswa i-ENIG ukuqedela ama-electrode e-PCB ngaphandle kokuguqulwa kwendawo.ucansi.Impendulo yenzwa ye-electrochemical iphawulwe kahle ezingcezuni ezimbili ze-DNA ezinobude obuhlukene (\({117}\,\hbox {bp}\) kanye \({503}\,\hbox {bp}\)), kanye umphumela kasawoti ku-PCR master mixes on methylene blue (MB) -DNA interactions.Imiphumela yethu ibonisa ukuthi ubude bezingcezu ze-DNA bunquma ngokuphawulekayo ukuzwela kwe-electrochemical futhi kubonisa kulo msebenzi ukuthi ikhono lokubona ama-amplicon amade ngaphandle kokuhlanzwa kwejeli kwemikhiqizo ye-PCR. kubalulekile ekulinganisweni kwendawo yamasampula amanzi.Isixazululo esizenzakalelayo somthamo wegciwane egazini sisebenza kahle.
Ukudluliswa kwegciwane emanzini bekwaziwa njengengozi yezempilo yomphakathi kusukela ngeminyaka yawo-1940s, nobufakazi bokuqala bokusuleleka emanzini kovendle nesifo sokusha kwesibindi kohlobo E1.Inhlangano Yezempilo Yomhlaba (i-WHO) ihlukanise amagciwane amaningana aphuma emanzini abaluleke kakhulu empilweni2.Igciwane lendabuko Izindlela zokuhlonza zithembele kumasu asekelwe ku-qPCR asezingeni legolide, azwela kakhulu futhi aqondile, kodwa adinga izisebenzi ezinekhono ukuba zihlole elabhorethri zisebenzisa amathuluzi abizayo.Kodwa-ke, emazweni anemali engenayo ephansi naphakathi (ama-LMIC) anezinsiza ezilinganiselwe, abantu. ukuhlolwa kwesampula cishe kuzoza kuqala kunokuqapha isampula yamanzi emvelo.Ngakho-ke, ezinye izindlela ezingabizi kakhulu ziyadingeka ukuze kuqashwe amasampula amanzi kanye namanzi angcolile ngesikhathi sangempela emazweni anemali engenayo ephansi naphakathi njengezixwayiso zakuqala zokuqubuka kwezifo eziqhamukayo, ngaleyo ndlela zibavikele emiphumeleni enzima yenhlalo-mnotho yobhubhane lwegciwane.Ama-electrochemical biosensors abiza kancane ama-nucleic acid anganikeza ikhambi elithembisayo lalesi sidingo esingafezeki.Iningi lalezi zinzwa ze-DNA zisebenza ngokuthi imicu ehambisanayo ye-DNA ayinyakazi ku-electrode. ingaphezulu futhi ihlanganise uma ukulandelana okufanayo kukhona kusampula.Lokhu kungase kuguqulelwe kube isignali ngamasu ahlukahlukene kagesi we-electrochemical kusetshenziswa izixhumanisi ze-redox ezifana ne-potassium iron/ferrocyanide.I-Methylene blue (MB) ingenye ye-molecule esebenzayo ye-redox, kubikwe ukuthi ihlangana ibe i-DNA enemicu ephindwe kabili (dsDNA) ngaphezu kokubophezela kwayo okungaqondile ku-DNA enomucu owodwa5,6.Imvelo ehlanganayo yama-MB ukuze akhe ama-MB-DNA complexes iwenza abe yisinqumo esithandwayo njengabalamuli be-redox ku-electrochemical DNA eminingana. ukumiswa kwezinzwa5,6,7,8,9.Nakuba ukuxutshwa kwe-MB ku-DNA kungaqondile, futhi ukucaciswa kwale nzwa ye-electrochemical kuncike kakhulu ekuhlanzekeni kweziqalo ezisetshenziselwa i-PCR noma i-isothermal amplification, ifaneleka kahle ekusebenziseni okwangempela. -isikhathi se-electrochemical-based qPCR noma i-fluorescence isothermal amplification njengenye indlela yokukala ukugxila kwe-DNA 9 .Kokunye ukuqaliswa okunjalo, Won et al.Ingaphezulu lama-electrode egolide ashintshwe nge-6-mercapto-1-hexanol (MCH) ngesikhathi sangempela ukukalwa kwamaamplikhoni e-PCR nge-MB kusetshenziswa i-difaultial pulse voltammetry (DPV)9. Kwezinye izimo, u-Ramirez et al. Ukutholwa kwe-SARS-CoV-2 emanzini angcolile ngokusabela kwe-RT-LAMP kusetshenziswa i-MB ngama-electrode aphrintwe isikrini. Ama-electrode ePlatinum nawo asetshenzisiwe. esetshenziswa njengama-electrode e-situ endaweni yesikhulumi se-PCR ye-microfluidic eklanyelwe ukubona ama-amplicons e-electrochemically ngesikhathi sokusabela 8.Zonke lezi zifundo zidinga ukuguqulwa okungaphezulu kwama-electrode, okusho ukwanda kwezindleko zokukhiqiza kanye nokusebenza ngenxa yezidingo ezikhethekile zokugcina ukuzinza kwalawa ma-electrode asebenzayo.
Isikimu sokuhamba komsebenzi sokutholwa kwe-electrochemical yama-amplicon atholwe ezinhlayiyeni zegciwane ezigxilile kumasampula amanzi echibi.
Sisanda kukhombisa inzwa ye-electrochemical ye-SARS-CoV-2 amplicon enama-electrode ebhodi ephrintiwe ashibhile (PCB) asuselwa ku-DPV kanye ne-cyclic voltammetry (CV) edalwe ukufakwa kwe-MB-DNA complexes ebusweni bama-electrode angashintshiwe ) okwamanje11.Sibika ukuthi izingcezu ezinde ze-DNA (N1-N2, \({943}\, \hbox) ezakhiwe kusetshenziswa i-CDC-enconywe i-N1 eya phambili ne-N2 reverse primers uma kuqhathaniswa nezingcezu ezimfushane {bp}\)) zibonise umugqa ongcono ekuphenduleni kwenzwa ( I-N1, \(72\,\hbox {bp}\)) yakhiwe kusetshenziswa amasethi e-N1 phambili kanye ne-N1 reverse primer. Lezi zifundo zibikwa kusetshenziswa ukuhlanjululwa kwe-DNA okulungiselelwe emanzini angenayo i-nuclease.Inkundla yasetshenziswa futhi ukuthola i-SARS-CoV -2 ama-amplicon kumasampula amanzi angcolile alingisiwe (atholwe ngamasampula esamba se-RNA e-spiking nge-SARS-CoV-2 RNA). Njengoba i-RNA isengozini yokugunda ngesikhathi sokuhlukaniswa nokucubungula phansi komfula,12,13 kunzima ukukhulisa izingcezwana ezinde ngale sampuli ehlukahlukene. Ngakho-ke, ukuboniswa kwenzwa ye-electrochemical ye-SARS-CoV-2 amplicon emanzini angcolile kukhawulelwe kusiqeshana \(72\,\hbox {bp}\) N1 esifushane.
Kulo msebenzi, siphenye ukuthi kungenzeka yini inzwa ye-electrochemical ye-ENIG PCB ye-phage Phi6 egxilile futhi ehlukanisiwe namasampula amanzi echibi (Fig. 1) . futhi ibe nolwelwesi lwe-lipid kanye nephrotheni eyi-spike.Ngenxa yalezi zizathu, i-bacteriophage Phi6 iyi-surrogate edumile ye-SARS-CoV-2 kanye namanye amagciwane e-RNA aguqiwe14,15.RNA ahlukaniswe nezinhlayiya zephage yasetshenziswa njengesifanekiso se-cDNA synthesis elandelwa I-PCR ukuze ithole izingcezu ezimbili ze-DNA ka-117 kanye namapheya angu-503 ubude.Ngokunikezwa inselele yokukhulisa \(943\,\hbox {bp}\) N1-N2 izingcezu emsebenzini wethu wangaphambili, siqondise izingcezu zobude obumaphakathi (\(117) \,\hbox {bp}\) kanye \(503 \,\hbox {bp}\)), ngokusekelwe kuma-primer atholakalayo.Impendulo yenzwa ye-electrochemical yacwaningwa ngendlela ehlelekile ebangeni elibanzi lokugxilisa ingqondo (\({10}\,{ \hbox {pg}/{\upmu \hbox {l}}}\) kuya \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}}\)) Kuzo zombili izingcezu ku ukuba khona kwe-MB, umphumela kasawoti ekuphenduleni kwenzwa uye wabonakala futhi waqinisekiswa ngokuphambene nezilinganiso ze-spectrophotometric.Iminikelo eyinhloko yalo msebenzi yilena elandelayo:
Ubude besiqephu se-DNA kanye nokuba khona kukasawoti kusampula kuthinta kakhulu ukuzwela.Imiphumela yethu ikhombisa ukuthi umsebenzi we-electrochemical uncike ezindleleni ezihlukene zokusebenzisana kwe-MB, i-DNA, kanye nenzwa ekuphenduleni kwe-voltammetric, kuye ngokugxila kwe-DNA nobude, nezingcezu ezinde zibonisa ukuzwela okuphezulu, nakuba usawoti unomphumela omubi ekusebenzisaneni kwe-electrostatic phakathi MB kanye ne-DNA.
Ukugxiliswa kwe-DNA kunquma indlela yokusebenzisana kwe-MB-DNA kuma-electrode angashintshiwe Sibonisa ukuthi izindlela ezihlukene zokusebenzisana kwe-MB-DNA zincike ekugxilweni kwe-DNA. Ekugxilweni kwe-DNA ngaphansi kwenani elincane \({\hbox {ng}/{\upmu \hbox {l}}}\), siqaphele ukuthi impendulo yamanje ye-electrochemical yanqunywa ngokuyinhloko i-adsorption ye-MB-DNA ku-electrode, kuyilapho ekugxilweni okuphansi Ekugxilweni okuphezulu kwe-DNA, impendulo yamanje ye-electrochemical yanqunywa ukuvinjelwa kwe-steric kwe-redox. umsebenzi ngenxa yokufakwa kwe-MB phakathi kwamapheya esisekelo se-DNA.
I-ENIG PCB-Based Electrochemical Sensing of Viral Nucleic Acids in Lake Water Samples Okubonwayo kwaqinisekiswa ngokutholwa kwe-electrochemical ye-Phi6-added \(503\,\hbox {bp}\) izingcezu ze-DNA ezitholwe kumasampula amanzi e-Powai Lake, IIT Mumbai Campus. Umphumela phage.
Izindleko eziphansi zokuqalisa kanye namandla okuhlanganiswa ezinhlelweni zokuqapha ezizenzakalelayo ngokuphelele, ama-oligonucleotide noma ama-aptamers kuma-electrode anempilo yeshelufu ende.
I-Phage Phi6 igciwane elivalekile le-dsRNA lomndeni we-Cytoviridae elithelela i-Pseudomonas syringae.I-genome ye-Phi6 phage ikhona ngendlela yezingcezu ezi-3: S (\(2.95\,\hbox {Kb}\)), M (\(4.07) \,\hbox {Kb}\)) kanye no-L (\ (6.37\ ,\hbox{Kb}\))16,17.Njengoba i-Phi6 phage ithelela uhlobo lwe-Pseudomonas olungaguli lwe-BSL-1, kuphephile ukusebenzisa futhi ingatshalwa kalula elabhorethri.I-Phage Phi6 kanye nomsingathi wayo i-Pseudomonas syringae ithengwe e-Felix d'Herelle Reference Centre for Bacterial Viruss, Laval University, Canada (izinombolo zekhathalogi yesikhungo i-HER-102 kanye ne-HER-1102, ngokulandelana) .I-Phi6 phage kanye nomsingathi wayo bavuselelwe njengoba kuyalelwe isikhungo sokubhekisela.I-Phage Phi6 yahlanzwa nge-plate lysis kanye ne-elution ukuze ithole iziqu zokugcina ngokuthi \(\cishe 10^{12}\,{\hbox {PFU}/\hbox { ml}}\) (amayunithi okwenza uqwembe/amamililitha).I-RNA yahlukaniswa nezinhlayiya zephage ezihlanziwe kusetshenziswa i-GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) ngokwemiyalelo yomkhiqizi. 100}\,{{\upmu \hbox {l}}}\) yafakwa lysed futhi i-lysate yalayishwa kukholomu ejikelezayo ukuze kuvunyelwe i-RNA ukuthi ibophezele kukholamu ye-resin .I-RNA ibe isicaciswa kusixazululo se-elution \({ 50}\,{{\upmu \hbox {l}}}\) inikezwe ikhithi. Linganisela ukuhlangana kwe-RNA ngokumunca kokuthi \(260\,\hbox {nm}\).I-RNA igcinwe kuma-aliquots kokuthi \ ({-80}\,{^{\circ }\hbox {C}}\) kuze kusetshenziswe futhi.\({2}\,{\upmu \hbox {g}}\) I-iScript cDNA Synthesis Kit (Bio -Rad Laboratories) yasetshenziswa njengesifanekiso se-cDNA synthesis ngokulandela imiyalelo yomkhiqizi.Kafushane, ukusabela kokuhlanganiswa kwe-cDNA kuhlanganisa izinyathelo ezi-3: kuqala ku-\({25}\,{^{\circ }\hbox {C}}\ )\({5}\,{\hbox {min} }\) , ukuhlehla okulotshiweyo kokuthi \({20}\,{\hbox {min}}\) kokuthi \({46}\,{^{\circ }\hbox {C}}\), bese uhlehla Isirekhoda siku-\({95}\,{^{\circ }\hbox {C}}\) kokuthi \({1}\,{\hbox {min }}\).Lapho isetshenziswa ngejeli ye-agarose engu-1%, i-cDNA yabonisa amabhande amathathu ahambisana nezingcezu ezintathu ze-RNA ezilindelekile (idatha engabonisiwe). Lezi zingcezu ezilandelayo zisetshenziswe ukukhulisa izingcezu ezimbili ze-DNA ezingu-117 no-503 bp ubude, usebenzisa i-cDNA njengesifanekiso se-PCR ku-miniPCR® mini8 thermal cycler:
Iziqalo ze-\(117\,\hbox {bp}\) kanye \(503\,\hbox {bp}\) zihambelana nama-nucleotide angu-1476-1575 wengxenye ye-M kanye nama-nucleotide angu-458-943 wengxenye L, ngokulandelana i-asidi .Yonke imikhiqizo ye-PCR ekhulisiwe yenziwe nge-electrophoresed kumajeli e-agarose angu-1%, futhi i-DNA ehlosiwe ekhulisiwe yahlanzwa kusetshenziswa i-GeneJET Gel Extraction Kit (Thermo Fisher Scientific).
Ichibi ekhempasini ye-IIT Mumbai (i-Powai Lake, i-Powai, i-Mumbai) lasetshenziswa ukwengeza izinhlayiya ze-phage. Amanzi echibi ahlungwa ngolwelwesi \({5}\,{\upmu \hbox {m}}\) ukuze lukhishwe. izinhlayiya ezimisiwe, kwase kwengezwa i-Phi6 phage. Engeza \({1}\,{\hbox {ml}}\) ye-\(10^{6}\,{\hbox {PFU}/\hbox {ml}}} \) kuya \( {100}\ ,{\hbox {ml}}\) ehlungiwe amanzi echibi, kokuthi \({4}\,{^{\circle}\hbox {C}}\).I-aliquot encane ibi Sihlole izindlela ezimbili ezihlukene zokugxilisa izinhlayiya zegciwane le-Phi6 ezine-spiked: (1) indlela ye-aluminium hydroxide adsorption-precipitation,19 egunyazwe ukuthi ihlanganise amagciwane amaningi e-RNA amboziwe avela kumasampula emvelo, kanye (2) ) Indlela yokugxilisa igciwane esekelwe ku-polyethylene glycol (PEG) yashintshwa ku-Flood et al.20 .Njengoba ukubuyisela ukusebenza kahle kwendlela esekelwe ku-PEG kutholakale kungcono kunendlela ye-aluminium hydroxide, indlela ye-PEG-based yasetshenziselwa ukugxilisa izinhlayiya ze-Phi6 ezivela kumasampula amanzi echibi.
Indlela ye-PEG eyasetshenziswa yaba kanje: I-PEG 8000 kanye \(\hbox {NaCl}\) zengezwe kumasampula amanzi echibi eline-Phi6 ukuze kutholwe u-8 % PEG 8000 kanye \(0.2\,\hbox {M} \) \( \ hbox {NaCl}\).Amasampuli afakwe ku-shaker\({4}\,{^{\circ }\hbox {C}}\)\({4}\,{\hbox {h}}\ ), bese i-centrifuged kokuthi \(4700 \,\hbox {g}\) ithi \({45}\,{\hbox {min}}\).Lahla i-supernatant bese umisa kabusha i-pellet kokuthi \({1}\, {\hbox {ml}}\) ku-supernatant efanayo.Konke ukuhlola kwe-spiking nokugxilisa igciwane kwenziwa nge-triplicate.Ngemva kokugxilisa ingqondo, i-aliquot encane ibekelwe ukukalwa kokusebenza kahle kokutakula nge-plaque assay.I-RNA yahlukaniswa njengoba kuchazwe ngaphambilini futhi yacaciswa kubhafa ye-elution ehlinzekwe kit\({40}\,{\upmu \hbox {l}}\). Njengoba ukugxilisa kwe-RNA kuzohluka kusuka kusampula kuye kusampula kokuphindwe kathathu, \({2}\,{\upmu \\) I-hbox {l}}\) ye-RNA isetshenziswa kuzo zontathu kungakhathaliseki ukuthi i-cDNA synthesis yamasampula. yasetshenziswa njengesifanekiso se-\({20}\,{\upmu \hbox {l}}\) PCR emijikelezweni engu-35 yokukhulisa \ (117\,\hbox {bp}\) kanye \(503\,\hbox { bp}\) izingcezu.Lawa masampuli amelwe njengokuthi “1:1″, okungukuthi ngaphandle kokuhlanjululwa.I-no-template control (NTC) yamiswa njengokulawula okunegethivu, kuyilapho i-cDNA ehlanganiswe kusetshenziswa i-RNA ehlukanisiwe nephage ehlanzekile yamiswa. njengesifanekiso sokulawula okuhle (i-PC).I-Quantitative PCR (qPCR) yenziwe ensimbini ye-Stratagene Mx3000P RT-PCR kusetshenziswa i-Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies).Ukusabela kwahlelwa ngokuphindwe kathathu njengangaphambilini. kuchaziwe.Umkhawulo womjikelezo (Ct) urekhodwe kuwo wonke amasampuli.Ngaphezu kwalokho, amasampula ahlanjululiwe aye \({1}\,{\upmu \hbox {l}}\) esebenzisa i-cDNA ehlanjululwe 1:100 emanzini echibi ahlungiwe njenge \({20}\,{\upmu \hbox {l}}\) PCR yemijikelezo engu-35. Lawa masampuli amelwe njengokuthi “1:100″.
Ama-electrode e-PCB akhiwe kusetshenziswa inqubo etholakalayo ethengiswayo engabizi kakhulu ye-Electroless Nickel Immersion Gold (ENIG) ngaphandle kwesidingo segolide elengeziwe.Imininingwane ye-electrode ye-ENIG PCB inemininingwane emsebenzini wethu wangaphambili11.Ngama-electrode e-ENIG PCB, izindlela zokuhlanza ama-electrode ezivamile njenge isixazululo se-piranha noma i-sulfuric acid cyclic voltammetry ayinconywa ngoba ingabangela ukuxebuka kongqimba oluncane lwegolide (ukujiya \(\approx\) \(100\,\hbox {nm }\)) bese kuveza izendlalelo zethusi ezingaphansi ezijwayele kuya ku-corrosion 21, 22, 23, 24, 25.Ngakho-ke, hlanza ama-electrode ngendwangu engena-lint eswakanyiswe nge-IPA.Isampula okumele lihlolwe lifakwe \({50}\,{\upmu \hbox {M}} }\) MB kokuthi \({4}\,{^{\circ }\hbox {C}}\)\({ 1}\,{\hbox {h}}\) ukuze uyifake kalula.Emsebenzini wethu odlule , siqaphele ukuthi ukuzwela nokuhambisana kwenzwa kwathuthukiswa ngokukhuphula ukugxiliswa kwe-MB 11 .Ngokusekelwe ekuthuthukisweni okubikwe emsebenzini wethu wangaphambili, sisebenzise \({50}\,{\upmu \hbox {M}}\) MB ukugxilisa ingqondo ukuze kushumeke i-DNA kulolu cwaningo.Ukutholwa kwe-electrochemical ye-DNA enezintambo ezimbili (ds-DNA) kungafinyelelwa kusetshenziswa ama-anionic noma ama-cationic intercalators.Nakuba ama-anionic intercalators ethola i-DNA ngokukhetha okungcono, adinga ukufukamela ebusuku, okuholela ekutholeni izikhathi ezinde. ngakolunye uhlangothi, ama-cationic intercalators afana ne-MB adinga izikhathi ezimfushane zokufukamela, cishe \({1}\,{\hbox {h}}\) ukuze kutholwe i-electrochemical ye-ds-DNA6.Isilinganiso ngasinye sibandakanya ukukhipha isampula ukuze ihlolwe i-electrode\({5}\,{{\upmu \hbox {l}}}\), bese ihlanzwa ngeragi eswakanyiswe yi-IPA, ngaphambi kokuqhubeka nelinye isampula.isilinganiso esisodwa.Isampula ngayinye ihlolwe kuma-electrode angu-5 ahlukene ngaphandle uma kushiwo ngenye indlela.Izilinganiso ze-DPV ne-CV zenziwe kusetshenziswa i-PalmSens Sensit Smart potentiostat, futhi isofthiwe ye-PSTrace yasetshenziselwa ukumiswa kwe-potentiostat nokutholwa kwedatha, okuhlanganisa izibalo eziphezulu zamanje.Izilungiselelo ezilandelayo zisetshenzisiwe. ngezilinganiso ze-DPV ne-CV:
I-DPV: Isikhathi Sokulingana = \(8\,\hbox {s}\), Isinyathelo Sogesi = \(3\,\hbox {mV}\), Pulse Voltage = \(25\,\hbox {mV}\) , ubude be-pulse = \(50\,\hbox {ms}\), izinga lokuskena = \({20}\,\hbox {mV/s}\)
I-CV: Isikhathi Sokulingana = \(8\,\hbox {s}\), Isinyathelo Sogesi = \(3\,\hbox {mV}\), Isilinganiso Sokushanela = \({300}\,\hbox {mV/ s }\)
Amagagasi aphezulu atholwe kuma-voltammogram e-DNA ahlanganiswe ne-\({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV, (d) \(117\,\hbox {bp}\) CV.
I-DPV kanye ne-CV voltammograms atholwe kuma-electrode e-ENIG PCB \({50}\,{\upmu \hbox {M}}\) MB ahlanganiswe ne-DNA (ekugxiliseni okungu-10–\({20}\,{\ hbox {ng) }/{\upmu \hbox {l}}}\) okungukuthi 0.13–\({0.26}\,{\upmu \hbox {M}}\) ye-\(117\,\hbox {bp}\ ) kanye no-0.03 –\({0.06}\,{\upmu \hbox {M}}\) ye-\(503\,\hbox {bp}\)).Ama-voltammogram amele aboniswa kuMfanekiso S1 Olwazini Olwengeziwe.Umfanekiso 2 ubonisa imiphumela yezilinganiso ze-DPV ne-CV (i-peak current) kusetshenziswa imikhiqizo ye-PCR ehlanjululwe ijeli.Uma kuqhathaniswa nezilinganiso ze-CV, izilinganiso ze-DPV zibonisa ukuzwela okuphakeme (okwamanje njengomsebenzi we-DNA concentration) ngoba ingemuva le-capacitive currents ezilinganisweni ze-CV lifihla imisinga ye-Faradaic 26 .Idatha ebhokisini ngalinye ku-boxplot liqukethe izilinganiso ezivela kuma-electrode angu-5. Zonke izilinganiso zisebenzisa isethi efanayo yama-electrode ukugwema amaphutha okulinganisa ngenxa yokuhluka kwe-electrode-to-electrode.Sibone ukuthambekela okukhulayo ku-DPV kanye ne-CV elinganisa imisinga ephezulu yokugxila okuphansi kwe-DNA , yinde (\(503\,\hbox {bp}\)) \,\hbox {bp}\ uma kuqhathaniswa \(117) ) incezu.Lokhu kuhambisana nokuthambekela okulindelekile kokufakwa kwe-electrode okubikwe emsebenzini wethu wangaphambili. i-adsorption ye-MB-DNA complex kusiza ukudluliswa kwezindleko ku-electrode, okufaka isandla ekwandeni kwenani eliphakeme lamanje.Olunye ucwaningo luye lwabonisa umphumela wesayizi we-oligonucleotide nokulandelana kwe-MB-DNA intercalation27,28,29,30.I-guanine Okuqukethwe kwe-cytosine (GC) kwama-amplicon amabili (\(117\,\hbox {bp}\) kanye \(503\,\hbox {bp}\)) kwakucishe kube ngu-50%, okubonisa ukuthi ukubhekwa Umahluko ufanelekile kubude be-amplicon.Nokho, ngokugxila okuphezulu kwe-DNA (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), kokuthi \(503\,\hbox {bp} \) kanye \( >{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) ye-\(117\,\hbox {bp}\)), sibona ama-amplification amabili ukuphakama okuphezulu kwama-subs kuncishisiwe kuzo zombili izilinganiso ze-DPV ne-CV.Lokhu kungenxa yokuthi i-MB igcwele futhi ihlangana phakathi kwamapheya esisekelo e-DNA, okuholela ekuvinjweni kwe-steric komsebenzi we-redox weqembu elincishiswayo ku-MB31,32.
在存在 \(2\,\hbox {mM}\) \({\hbox {MgCl }_2}\): (a) \(503\,\hbox {bp}\) DPV, (b) \(503 \,\hbox {bp}\) CV, (c) \(117\,\hbox {bp}\) DPV,(d) \(117\,\hbox {bp}\) CV.
Usawoti okhona kumamiksi ayinhloko we-PCR uphazamisa ukusebenzisana kwe-electrostatic phakathi kwe-MB ne-DNA, ngakho-ke ngokwengeza \(2\,\hbox {mM}\) \(\hbox {MgCl }_2\) ngokuthi \({50} \,{\ upmu \hbox {M}}\) MB umkhiqizo ohlanzwe ijeli ukuze kufundwe umthelela kasawoti ekusebenzisaneni kwe-MB-DNA. Njengoba kubonisiwe kuMfanekiso 3, siqaphele ukuthi ekugxilweni okuphezulu kwe-DNA (\(>{2}\,{\ hbox {ng}/{\upmu \hbox {l}}}\) (503\,\hbox {bp }\) kanye \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}}\) kokuthi \(117\,\hbox {bp} \)), ku-DPV naku-CV Ukwengezwa kukasawoti akuzange kube nomthelela omkhulu ezilinganisweni (bheka Umfanekiso S2 kokuthi Ulwazi Olwengeziwe ukuze uthole ama-voltammogram amele). Nokho, at ukugxila kwe-DNA ephansi, ukufakwa kukasawoti kunciphisa kakhulu ukuzwela, okubangela kungabikho ushintsho olubalulekile okwamanje ngokugxila kwe-DNA.Imiphumela emibi efanayo kasawoti ekusebenzisaneni kwe-MB-DNA kanye nokuhlangana kuye kwabikwa ngaphambili ngabanye abacwaningi33,34.\(\hbox { Mg}^{2+}\) ama-cations abophezela kumgogodla we-phosphate ongemuhle we-DNA, ngaleyo ndlela avimbe ukusebenzisana kwe-electrostatic phakathi kwe-MB ne-DNA. Emazingeni aphezulu e-DNA, ukuvinjelwa okuqinile kwama-MB asebenzayo e-redox kuholela ekuphakameni okuphansi, ngakho ukusebenzisana kwe-electrostatic ayithinti kakhulu impendulo yenzwa.Iphuzu elibalulekile ukuthi le-biosensor ifaneleka kangcono ukuthola ukugxila kwe-DNA ephakeme (akuvamile \({\hbox {ng}/{\upmu \hbox {l}}}\) noma ngaphezulu), ukucubungula okuzenzakalelayo kwamasampula amanzi emvelo, lapho ukuhlanzwa kwejeli kwemikhiqizo ye-PCR kungase kungenzeki.
Indawo engaphansi kwejika lokumuncwa lobubanzi be-wavelength 600–700 \(\hbox {nm}\) yokugxilisa okuhlukahlukene kwe-DNA ehlanganiswe ne \({50}\,{\upmu \hbox {M}}\) MB: ( a ) \(503\,\hbox {bp}\) enosawoti nangaphandle kwayo (\(2\,\hbox {m}\) \(\hbox {MgCl}_2\)), (b) \( 117\, \hbox {bp}\) enosawoti nangaphandle kwayo (\(2\,\hbox {mM}\) \(\hbox {MgCl}_2\)).\({0}\,{\hbox {pg}/) {\upmu \hbox {l}}}\) Ukugxiliswa kwe-DNA okuhambisana \({50}\,{\upmu \hbox {M}}\) MB amasampuli Awekho i-DNA.
Ukuze siqhubeke siqinisekisa imiphumela engenhla, senze izilinganiso zamehlo sisebenzisa i-UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), amasampula \({50}\,{{\upmu \hbox {l}}}}\) asetshenziswa endaweni ngayinye. Isilinganiso.Isiginesha yokumuncwa iyancipha ngokugxila okukhulayo kwe-DNA, njengoba kungabonakala kuthrendi yendawo engaphansi kwejika lokumunya lobubanzi be-wavelength \(600\,\hbox {nm}\) ukuya \(700\,\hbox { nm}\) , njengoba kuboniswe ku-Fig. 4 (i-spectrum yokumuncwa eboniswe ku-Fig. S3 ku-Supplementary Information).Kumasampuli anomumo we-DNA ngaphansi kuka-\({1}\,{\hbox {ng}/{\upmu \hbox {l}}}\), awubanga khona umehluko omkhulu ekuthathweni phakathi kwamasampuli aqukethe i-DNA kanye ne-MB kuphela (ye-\(503\,\hbox {bp}\) kanye \(117\,\hbox {bp}\) ) izingcezu zobude), okubonisa ukungabikho kokuvinjelwa kwe-steric kwe-redox-active MB.Ekugxilweni okuphezulu kwe-DNA, sabona ukwehla kancane kancane kwesignali yokumunca futhi saphawula ukwehla okuncane kokumunca lapho kukhona usawoti.Le miphumela kuthiwa ibangelwa amangqamuzana. ukusebenzisana kanye nokuvimbela i-steric ne-base stacking kuma-DNA hybrids.Imiphumela yethu iyahambisana nemibiko esezincwadini zezifundo ze-spectroscopic ze-MB-DNA intercalation ezihlobanisa i-hypochromaticity namaleveli wamandla ancishisiwe kokuthi \(\pi\)–\(\pi ^*\ ) izinguquko ze-elekthronikhi ngenxa yokuxhumanisa Izendlalelo 36, 37, 38.
I-Agarose gel electrophoresis of phage Phi6: Imikhiqizo ye-PCR yobude \(117\,\hbox {bp}\) kanye \(503\,\hbox {bp}\) evela kumasampula amanzi echibi.M-DNA marker;I-NTC-no-template control, ama-primer aqukethe ama-amplicon ahambisanayo;Ukulawula okuhle kwe-PC;1, 2, 3-angaxutshiwe (1:1) amasampula amanzi echibi eline-spiked nge-triplicate.Ibhendi ibonakala kokuthi \(\cishe 50\,\hbox {bp}\) ngenxa ye-oligonucleotides engasetshenziswanga ku-\(503\,\ hbox {bp}\) umzila.
Sihlole ukusetshenziswa kwenzwa sisebenzisa amasampuli wamanzi e-Powai Lake afakwe nge-Phi6 phage. Ukugxiliswa kwe-RNA okuhlukanisiwe kumasampula amanzi ane-phage-spiked kusuka ku-15.8–\({19.4}\,{\upmu \hbox {g}/\hbox { ml}}\), kuyilapho lezo ezihlukanisiwe ekumisweni kwephage okuhlanjululwe I-RNA yayilinganiselwa ukuthi \({1945}\,{\upmu \hbox {g}/\hbox {ml}}\) ngokusebenza kahle kokuthola kabusha okungaba ngu-1 I-%.RNA yahlehliswa yabhalwa ku-cDNA futhi yasetshenziswa njengesifanekiso se-PCR kanye ne-qPCR.Usayizi womkhiqizo waqinisekiswa yi-agarose gel electrophoresis (Umfanekiso 5) ngaphambi kokuhlolwa ngenzwa.Lawa masampuli awahlanzwanga ijeli ngakho aqukethe zonke izingxenye ze-PCR njenge kanye nama-amplicons of interest.Amanani e-Ct arekhodwe ngesikhathi se-qPCR (Ithebula 1) aboniswe ukuze ahlobane nokugxilwa kwe-RNA ehlukanisiwe kumasampula amanzi anezipikili ahambisanayo. Inani le-Ct lembula inani lemijikelezo edingekayo ukuze isignali ye-fluorescent yeqa umkhawulo noma isignali yangemuva. Amanani aphezulu e-Ct abonisa ukugxiliswa kwesifanekiso esiphansi futhi ngokuphambene nalokho. Amanani e-Ct amasampula e-NTC ayephezulu ngendlela elindelekile. Umehluko kumanani \(\ cishe 3\) Ct phakathi ukulawula okuhle kanye nesampula yokuhlola kubonisa ukuthi isampula ngayinye yokuhlola inethempulethi ecishe ibe ngu-1% uma kuqhathaniswa nokulawula okuhle.Sike saxoxa ngaphambili ngokuthi ama-amplicon amade aholela ekuzweleni okungcono. yokuhlushwa kwegciwane eliphansi kanye nokuwohloka kwe-RNA.Nokho, ngephrothokholi yethu yokunothisa igciwane kanye nephrothokholi yokukhulisa i-PCR, sikwazile ukukhulisa ngempumelelo \(503\,\hbox {bp}\) isiqeshana senzwa ye-electrochemical.
Umfanekiso 6 ubonisa imiphumela yenzwa ye-electrochemical ye-amplicon yesiqephu \(503\,\hbox {bp}\), kokubili kusetshenziswa i-cDNA engaxutshiwe njengesifanekiso (1:1) kanye ne-cDNA ehlanjululwe izikhathi eziyi-100 njengesifanekiso (1:100 ) eyenziwe nge-PCR. , uma kuqhathaniswa ne-NTC ne-PC (bheka Umfanekiso we-S4 kuLwazi Olwengeziwe lwe-voltammograms emele). Ibhokisi ngalinye ebhokisini lebhokisi elisemfanekisweni wesi-6 liqukethe izilinganiso ezivela kumasampula amathathu kuma-electrode angu-5. Ama-electrode afanayo asetshenziselwa ukukala wonke amasampula ukuze kugwenywe amaphutha ngenxa ye-electrode -ukuhluka kwe-electrode.Uma kuqhathaniswa nezilinganiso ze-CV, izilinganiso ze-DPV zibonisa ukulungiswa okungcono ukuhlukanisa amasampula okuhlola kanye ne-PC ku-NTCs ngoba, njengoba kushiwo ngaphambili, imisinga ye-Faradaic ifihliwe ngenxa yama-capacitive currents angemuva ekugcineni. ukulawula okunegethivu (NTC) kuphumele ekuphakameni okuphezulu kwe-CV ne-DPV okuhlobene nokulawula okuhle, kuyilapho amasampula okuhlola amahle nangahluziwe abonisa ukuphakama okufanayo okuphezulu kwamagagasi aphakeme we-DPV. ) isampula yokuhlola kanye ne-PC ingaxazululwa ngokucacile kusukela ekuphumeni kwenzwa yesampula ye-NTC, kuyilapho ukulungiswa kwesampula ehlanjululwe engu-1:100 kuchazwa kancane.Ngokwehlanjululwa kwe-cDNA okuphindwe kayi-100, asizange sigcine noma yimaphi amabhande ngesikhathi se-gel electrophoresis (imizila engabonisiwe kuMdwebo 5), kanye ne-DPV ehambisanayo kanye nama-CV asezingeni eliphezulu ayefana nalawo alindelwe ku-NTC. Imiphumela yesiqephu \(117\,\hbox {bp}\) ikhonjiswe kokuthi Ulwazi Olwengeziwe.Okunegethivu ukulawula kubangele impendulo ye-electrochemical evela kunzwa ye-PCB ngenxa yokukhangiswa kwe-MB yamahhala ku-electrode kanye nokusebenzisana kwe-MB ne-primer-stranded oligonucleotide yokuqala. inani eliphakeme lamanje lesampula yokuhlola uma liqhathaniswa nenani eliphakeme lamanje elitholwe ukulawula okungalungile ukuze kuzuzwe isilinganiso esihlukile (esihlobene)39,40 ukuze kuhlukaniswe isampula yokuhlola njengephozithivu noma inegethivu.
(a) I-DPV, kanye (b) nenani eliphakeme le-CV yokutholwa kwe-electrochemical \(503\,\hbox {bp}\) izingcezu kumasampula amanzi echibi. Amasampula okuhlola akalwa ngokuphindwe kathathu futhi aqhathaniswa nezilawuli ezingenazo izifanekiso (NTC) kanye izilawuli ezinhle (PC).
Esikutholile kukhombisa izindlela ezihlukene ezithinta ukusebenza kwezinzwa ze-electrochemical zama-amplicon anobude obuhlukene kuma-DNA ahlukene, nokugxila okuqinisekiswe izilinganiso zokubona kusetshenziswa i-UV/Vis spectrophotometer.Okusikuqapheleyo kugcizelela ukuqonda ukuthi izingcezu ze-DNA ende ezifika \(\approx\) \(500\,\hbox {bp}\) ingatholwa ngokuzwela okuphezulu nokuthi ubukhona bukasawoti kusampula abuthinti ukuzwela kwe-DNA okuthinta ukuzwela okuphakeme (akuvamile \({\hbox {ng}/{\upmu) \hbox {l}}}\) nangaphezulu).Ngaphezu kwalokho, siphenye umthelela wezinhlobo ezahlukene zamasampuli, okuhlanganisa ama-amplicon ahlanzwe ijeli anosawoti owengeziwe nangaphandle kwawo, kanye nokwengezwa kwamasampula amanzi echibi ezilinganisweni ze-DPV ne-CV.Siqaphele ukuthi i-DPV ihlinzeke ngokulungiswa okungcono, njengoba i-capacitive current yangemuva iphinde ithinte isikali se-CV, iyenze ingazweli kangako.
Ukukhulisa izingcezu ezinde kuncike ekuthembekeni kwe-RNA yegciwane lesandulela ngculaza.Izifundo ezimbalwa zibonise ukuthi ukukhuliswa kwezingcezu ezinde akusebenzi kahle ngaso sonke isikhathi ngenxa yokuwohloka kwe-RNA emvelweni kanye namandla okuhlukanisa phakathi kokuhlukaniswa11,41,42,43,44 .Siqaphele ukuthi indlela yokugxilisa igciwane esekelwe ku-PEG yayisebenza kangcono ekugxiliseni i-phage Phi-6 spiked kumasampula amanzi echibi kune-aluminium hydroxide-based based concentration method.Ikhono lokubona izingcezu ze-DNA ezinde libonise ukunqoba imfuneko ye-multiplex PCR. ukukhulisa izifanekiso eziningi zobude obufushane futhi unciphise amathuba okucaciswa okuphambene.
Amasampula ezinto eziphilayo ayivelakancane, ngakho kunesidingo sokuklama i-biosensor edinga amasampula amancane ukuze ahlolwe.Ama-electrode e-ENIG PCB asetshenziswe kulolu cwaningo adinga kuphela \({5}\,{{\upmu \hbox {l}}}\\" ) amasampula okuhlolwa ukuze ahlanganise indawo esebenzayo yama-electrode.Ukwengeza, i-electrode efanayo ingasetshenziswa kabusha ngemva kokuhlanza ngaphambi kokukhipha isampula elilandelayo.Amasampula akhulisiwe awadingi ukungezwa kwanoma yimaphi amakhemikhali ngaphandle kwe-methylene blue, engabizi. kanye namakhemikhali asetshenziswa kakhulu.Njengoba i-electrode ngayinye ibiza cishe u-$0.55 (noma INR 40) ukuze yenziwe, le biosensor ingaba enye indlela engabizi kakhulu kubuchwepheshe obukhona bokubona.Ithebula lesi-2 libonisa ukuqhathaniswa kwalo msebenzi nezinye izinzwa ezibikwe ezincwadini isikhathi eside. Izingcezu ze-DNA kumasampuli ahlukahlukene.
Njengoba kunikezwe ukuthi amaphrothokholi okuthola i-electrochemical asuselwa ku-MB ancike ekucacisweni kwe-PCR, umkhawulo omkhulu wale ndlela amandla okukhulisa okungaqondile kumasampula ahlukahlukene njengamanzi angcolile namanzi echibi noma ukusebenzisa iziqalo zokuhlanzeka okuncane. Ukuze kuthuthukiswe ukuzwela Izindlela zokuthola i-electrochemical ukuthola i-DNA yemikhiqizo ye-PCR engahlanzekile kusetshenziswa ama-electrode e-ENIG PCB angashintshiwe, kuyadingeka ukuqonda kangcono amaphutha enziwe ama-dNTP angasetshenzisiwe nama-primers, nokuthuthukisa izimo zokusabela kanye nezinqubo zokuhlola.Imingcele eyengeziwe ye-physicochemical efana ne-pH, izinga lokushisa, kanye nebhayoloji. isidingo se-oxygen (BOD) sesampula samanzi singase sidinge ukulinganiswa ukuze kuthuthukiswe ukunemba kwesilinganiso.
Sengiphetha, siphakamisa inzwa ye-electrochemical ENIG PCB eshibhile yokutholwa kwegciwane kumasampula emvelo (amanzi echibini).Ngokungafani nama-electrode e-oligonucleotide angenakunyakaziswa noma ama-substrates angokwezifiso enzwa ye-DNA edinga isitoreji se-cryogenic ukuze silondoloze ukuzwela,53,54 inqubo yethu isebenzisa i-PCB engaguquki. ama-electrodes aneshelufu ende futhi angenazo izidingo ezithile zokulondoloza futhi afaneleka ukuthuthukiswa kwezixazululo zokulinganisa ngokucubungula isampula okuzenzakalelayo okufakwe kuma-LMICs.I-biosensor isebenzisa odayi be-DNA-intercalating redox (MB) abangabizi kakhulu ukuze bathole ngokushesha ama-amplicon aqondiwe.Ukukhulisa okungaqondile okuvamile kumasampula emvelo kunciphisa ukucaciswa kwale ndlela yokuzwa ngenxa yokubophezela okungaqondile kwama-MBs kuma-oligonucleotides anemicu eyodwa nephindwe kabili.Ngakho-ke, ukucaciswa kwalokhu kuhlolwa kuncike ekusetshenzisweni kahle kwezimo zokuqala nezimo ze-PCR.Ngaphezu kwalokho, i-CV kanye namagagasi aphezulu e-DPV atholwe kumasampula ahloliwe kufanele ahunyushwe ngokuhlobene nezimpendulo ezitholwe ekulawuleni okungalungile (NTC) ekuhlolweni ngakunye.Imiklamo yenzwa ye-electrochemical nezindlela ezethulwe kulo msebenzi zingahlanganiswa nama-autosamplers ukuze kuthuthukiswe okuzenzakalelayo ngokuphelele futhi okuphansi. -isixazululo sezindleko esingaqoqa futhi sihlaziye amasampula futhi sidlulisele imiphumela ngaphandle kwentambo ibuyele elabhorethri .
I-Cashdollar, J. & Wymer, L. Izindlela zokugxila kokuqala kwamagciwane avela kumasampula amanzi: ukubuyekezwa kanye nokuhlaziywa kwemeta yezifundo zakamuva.J.Application.microorganism.115, pp. 1-11 (2013).
Gall, AM, Mariñas, BJ, Lu, Y. & Shisler, JL Amagciwane atholakala emanzini: Izithiyo emanzini okuphuza aphephile.PLoS Pathogens.11, E1004867 (2015).
I-Shrestha, S. et al.I-Wastewater epidemiology yokugada kwesilinganiso esikhulu esingabizi kakhulu se-COVID-19 emazweni anemali engenayo ephansi naphakathi: izinselele namathuba.Water 13, 2897 (2021).
I-Palecek, E. & Bartosik, M. Nucleic acid electrochemistry.Chemical.Rev.112, 3427–3481 (2012).
U-Tani, A., Thomson, AJ & Butt, JN Methylene blue njenge-electrochemical discriminator ye-oligonucleotides enemicu eyodwa nephindwe kabili enganyakazi kuma-substrates egolide.Umhlaziyi 126, 1756–1759 (2001).
Wong, EL, Erohkin, P. & Gooding, JJ Ukuqhathaniswa kweCation kanye ne-Anion Intercalators for Electrochemical Transduction of DNA Hybridization by Long-Range Electron Transfer.Electrochemistry.comminicate.6, 648-654 (2004).
I-Wong, EL & Gooding, JJ Ukudluliswa kwenkokhelo nge-DNA: i-electrochemical DNA biosensor.anus.Chemical.78, 2138–2144 (2006).
U-Fang, TH et al.Idivayisi ye-PCR yesikhathi sangempela ye-microfluidic enokutholwa kwe-electrochemical ngesikhathi esisodwa.inzwa ye-biological.Bioelectronics.24, 2131–2136 (2009).
Win, BY et al.Signing mechanism study kanye nokuqinisekiswa kokusebenza kwesistimu ye-PCR yesikhathi sangempela ye-electrochemical esekelwe ekusebenzisaneni kwe-methylene blue ne-DNA.Umhlaziyi 136, 1573–1579 (2011).
Ramirez-Chavarria, RG et al.Loop-mediated isothermal-based electrochemical sensor yenzwa yokutholwa kwe-sars-cov-2 kumasampula amanzi angcolile.J.Imvelo.Chemical.Britain.10, 107488 (2022).
I-Kumar, M. et al.Inzwa ye-Electrochemical ye-SARS-CoV-2 amplicon ene-PCB electrode.Inzwa iyasebenza.B Chemistry.343, 130169 (2021).
Kitamura, K., Sadamasu, K., Muramatsu, M. & Yoshida, H. Ukutholwa ngempumelelo kwe-SARS-CoV-2 RNA engxenyeni eqinile yamanzi angcolile.science.general environment.763, 144587 (2021).
I-Alygizakis, N. et al.Izindlela Zokuhlaziya zokutholwa kwe-SARS-CoV-2 Emanzini Angcolile: Iphrothokholi Nemibono Yesikhathi esizayo.I-TraC ehamba phambili ku-anal.Chemical.134, 116125 (2020).
Fedorenko, A., Grinberg, M., Orevi, T. & Kashtan, N. Ukusinda kwe-bacteriophage Phi6 emboziwe (i-surrogate ye-SARS-CoV-2) kumaconsi amathe ahwamukile afakwe endaweni yengilazi.science.Rep.10, 1–10 (2020).
U-Dey, R., Dlusskaya, E. & Ashbolt, NJ Ukuphikelela Kwezemvelo kwe-SARS-CoV-2 Surrogate (Phi6) ku-amoeba ephilayo mahhala.Impilo Yamanzi 20, 83 (2021).
Mindich, L. Ukupakishwa okunembile kwezingcezu ezintathu ze-genomic ze-RNA bacteriophage enemicu ekabili\(\varphi\)6.microorganism.Moore.biology.Rev.63, 149–160 (1999).
I-Pirttimaa, MJ & Bamford, Isakhiwo sesibili se-DH RNA phage\(\varphi\)6 indawo yokupakisha.RNA 6, 880–889 (2000).
Bonilla, N. et al.Phages on the Tap – Iphrothokholi esheshayo nesebenzayo yokulungisa izitoko zaselabhorethri ze-bacteriophages.PeerJ 4, e2261 (2016).
Isikhathi sokuthumela: May-27-2022