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Muhiimadda la socodka muunadaha deegaanka ayaa si weyn loo mahadiyay tan iyo bilowgii faafitaanka COVID-19, iyo dadaallada kormeerka qaarkood ayaa lagu sameynayaa iyadoo la adeegsanayo heerka dahabka, in kasta oo farsamooyinka ku saleysan qPCR ay qaali yihiin. Shaqadan, waxaan ku muujineynaa ogaanshaha korantada kiimikada ee amplicons ee laga helay Phi6 phage oo ka go'an shaybaarada biyaha harada ee spiked (kuwa caan ah ee SARS-CoV-2), iyadoo la adeegsanayo ENIG si loo dhamaystiro korantada PCB iyada oo aan dusha laga beddelin.Jawaabta dareemayaasha korantada waxaa si fiican loogu sifeeyay laba jajab DNA oo dhererkoodu kala duwan yahay (\({117}\,\hbox {bp}\) iyo \({503}\,\hbox {bp} Saamaynta cusbada ee PCR master mixes on methylene blue (MB) -DNA interactions.Our natiijooyinka ayaa muujinaya in dhererka jajabka DNA si weyn u go'aamiyo dareenka electrochemical iyo muujin in shaqadan in awoodda lagu ogaado amplicons dheer iyada oo aan jel sifaynta alaabta PCR waa. muhiim u ah in goobta lagu cabbiro saamiga biyaha.Xalka si otomaatig ah u buuxsamay ee culeyska fayraska ayaa si fiican u bogsanaya.
Gudbinta fayraska biyaha ka dhasha waxa loo yiqiin khatar caafimaad oo dadwayne tan iyo 1940-meeyadii, iyada oo caddayntii ugu horreysay ee gudbinta biyaha ee dabaysha iyo cagaarshow E1. Hay'adda caafimaadka Adduunka (WHO) ayaa u kala saartay dhowr jeermiska fayraska biyaha ka dhasha ee dhexdhexaadka ah iyo muhiimadda caafimaadka sare. hababka ogaanshaha waxay ku tiirsan yihiin farsamooyinka ku salaysan qPCR-dahab, kuwaas oo aad xasaasi u ah oo gaar ah, laakiin u baahan shaqaale xirfad leh si ay u tijaabiyaan shaybaarka iyagoo isticmaalaya qalab qaali ah. Si kastaba ha ahaatee, wadamada dakhligoodu hooseeyo iyo dhexdhexaadka ah (LMICs) ee leh kheyraad xaddidan, aadanaha Tijaabada muunada waxay u badan tahay inay ka hormarto la socodka muunada biyaha deegaanka.Sidaa darteed, habab kale oo qiimo jaban ayaa loo baahan yahay si waara, la socodka muunada biyaha iyo biyaha wasakhda ah ee wadamada dakhligoodu hooseeyo iyo kuwa dhexdhexaadka ah sida digniinaha ugu horeeya ee cudurada soo ifbaxaya. Si looga ilaaliyo saamaynta xun ee dhaqan-dhaqaale ee ba'an ee fayraska faafa. Dusha sare iyo isku-darka marka isku xigxiga isku midka ah uu ku jiro muunada Waxaa lagu soo warramey in ay dhexda u galayso DNA-da laba-geesoodka ah (dsDNA) marka lagu daro xidhiddeeda aan khaaska ahayn ee DNA5,6 qaabeynta dareemayaasha5,6,7,8,9.In kasta oo isdhexgalka MB ee DNA uu yahay mid aan gaar ahayn, iyo gaar ahaaneed ee dareemayaasha elektiroonigga ah waxay inta badan ku xiran tahay nadiifnimada asaasiga ah ee loo isticmaalo PCR ama xoojinta isothermal, waxay si fiican ugu habboon tahay hirgelinta dhabta ah. -time electrochemical-based qPCR ama fluorescence isothermal amplification sida beddelka cabbirka fiirsashada DNA 9 cabbiraadda PCR amplicons oo leh MB iyadoo la adeegsanayo voltammetry garaaca kala duwan (DPV) 9. Xaaladaha kale, Ramirez et al.Detection of SARS-CoV-2 ee biyaha wasakhda ah ee falcelinta RT-LAMP iyadoo la adeegsanayo MB oo leh electrodes-ka daabacan. loo isticmaalo sida situ electrodes ee madal PCR microfluidic loogu talagalay in electrochemically lagu ogaado amplicons inta lagu guda jiro falcelinta 8 .Dhammaan daraasaddan waxay u baahan yihiin wax ka beddelka dusha sare ee electrodes ah, oo tilmaamaya kororka wax soo saarka iyo kharashka hawlgalka ay sabab u tahay shuruudaha kaydinta gaarka ah ee xasiloonida ee electrodes functionalized.
Nidaamka socodka shaqada ee ogaanshaha korantada kiimikaad ee amplicon-yada laga helay walxaha fayraska ah ee ku jira muunadaha biyaha harada.
Waxaan dhawaan soo bandhignay dareenka koronto-kimikada ee SARS-CoV-2 amplicon oo leh koronto daabacan oo qiimo jaban (PCB) oo ku saleysan DPV iyo voltammetry wareeg ah (CV) oo ay keentay xayeysiinta dhismayaasha MB-DNA ee dusha sare ee koronto aan la beddelin) isbeddellada ugu sarreeya hadda11.Waxaan soo sheegnay in jajabyada DNA-da ee dhaadheer (N1-N2, \ ({943}\, \ hbox) la sameeyay iyadoo la adeegsanayo CDC-ku-talisay N1 hore iyo N2 rogayaasha marka la barbar dhigo jajabyada gaagaaban {bp} \)) waxay muujiyeen toosnaan ka wanaagsan jawaabta dareenka ( N1, \ (72 \, \ hbox {bp} \)) oo la sameeyay iyadoo la adeegsanayo N1 hore iyo N1 jaangooyooyinka asaasiga ah. -2 amplicon oo ku jira shaybaarada biyaha wasakhda ah ee la isku daray (oo lagu helay isugeynta guud ee shaybaarada RNA ee leh SARS-CoV-2 RNA).Maadaama RNA ay u nugushahay xiirid inta lagu jiro go'doominta iyo habaynta biyaha hoose,12,13 way adagtahay in lagu kordhiyo jajabyo dhaadheer muunadan kala duwan. Sidaa darteed, muujinta dareenka korantada kiimikada ee SARS-CoV-2 amplicon ee biyaha wasakhda ah waxay ku xaddidan tahay gabaga gaaban \(72 \, \ hbox {bp} \) N1 jajab.
Shaqadan, waxaan ku baarnay suurtagalnimada ENIG PCB-ku-saleysan kororka kiimikaad ee phage Phi6 xoogga saaray oo ka go'doomay muunado biyaha harada (Jaantus. 1) .Phi6 phages waxay la mid yihiin cabbirka (80-100 nm) ilaa SARS-CoV-2 iyo Sidoo kale waxay leeyihiin xuubka dufanka iyo borotiinka caaga ah.Sababahaan, bacteriophage Phi6 waa beddelka caanka ah ee SARS-CoV-2 iyo fayraska kale ee pathogenic RNA14,15.RNA oo ka soocan qaybaha phage-ga ayaa loo adeegsaday qaab-dhismeedka cDNA oo ay ku xigto. PCR si loo helo laba qaybood oo DNA ah oo ah 117 iyo 503 lammaane oo dherer ah. Marka la eego caqabada ah xoojinta \ (943 \, \ hbox {bp} \) N1-N2 jajabyada shaqadeena hore, waxaan beegsaneynaa jajabyada dhererka dhexdhexaadka ah (\(117) \,\hbox {bp} \hbox {pg}/{\upmu \hbox {l}}} \) ilaa \({20}\, {\hbox {ng}/{\upmu \hbox {l}}}\)) Labada jajab ee ku jira Joogitaanka MB, saamaynta milixdu ku leedahay jawaab celinta dareemayaasha ayaa lagu sifeeyay oo lagu ansixiyay cabbirada spectrophotometric. Waxyaalaha ugu muhiimsan ee shaqadani waa sida soo socota:
Dhererka jajabka DNA-da iyo joogitaanka milixda ee muunadda ayaa si xooggan u saameeya dareenka.Natiijooyinkayagu waxay muujinayaan in dhaqdhaqaaqa korantada ay ku xiran tahay habab kala duwan oo isdhexgalka MB, DNA, iyo dareemayaasha jawaabta voltammetric, taas oo ku xidhan diiradda DNA iyo dhererka, oo leh jajabyo dheer ayaa muujinaya dareen sare, inkastoo milixdu ay saameyn xun ku leedahay isdhexgalka elektrostatic ee u dhexeeya MB iyo DNA.
Xoojinta DNA-da ayaa go'aamisa habka isdhexgalka MB-DNA ee electrodes-ka aan la bedelin Waxaan muujineynaa in hababka kala duwan ee isdhexgalka MB-DNA ay ku xiran yihiin diiradda DNA-da ee ka hooseeya qadar yar oo ah \ {l}}} \), waxaan aragnay in jawaabta hadda jirta ee korantada ay inta badan go'aamisay xayeysiinta MB-DNA ee korantada, halka heerarka hoose ee uruurinta DNA-da sare, jawaabta hadda jirta ee korantada waxaa lagu go'aamiyay xannibaadda adag ee redox dhaqdhaqaaqa ay sabab u tahay gelinta MB ee lammaanaha saldhigga DNA.
Dareenka ENIG PCB-ku-salaysan Electrochemical ee Asiidhyada Nucleic-ka ah ee Fayraska ku jira Saamaynta Biyaha Harada Aragtida waxaa lagu ansixiyay ogaanshaha korantada kiimikada ee Phi6-ku daray \(503 \, \ hbox {bp} \) jajabyada DNA ee laga helay muunadaha biyaha ee Lake Powai, IIT Mumbai Campus Faajiga natiijada.
Dhaqangelinta kharash yar iyo suurtogalnimada is dhexgalka nidaamyada si toos ah otomaatig ah ula socodka, oligonucleotides ama aptamers ee korantada oo leh nolol shelf dheer.
Phage Phi6 waa fayraska dsRNA ee baqdin leh oo ka tirsan qoyska Cytoviridae kaas oo ku dhaca syringae Pseudomonas. Genome-ka Phi6 phage wuxuu u jiraa qaab 3 jajab ah: S ((2.95 \, \ hbox {Kb}\)), M (\(4.07) \,\hbox {Kb} waxaana si fudud loogu kori karaa shaybaarka.Phage Phi6 iyo ciidankiisa Pseudomonas syringae waxaa laga soo iibsaday Xarunta Tixraaca Felix d'Herelle ee Fayraska Bakteeriyada, Jaamacadda Laval, Kanada (lambarada buug-yaraha xarunta tixraaca waa HER-102 iyo HER-1102, siday u kala horreeyaan) .Phi6 phage iyo martida loo yahay ayaa dib loo soo nooleeyay sida ay tilmaamtay xarunta tixraaca. ml}}\) (cutubyada samaynta huurada/ millilitir).RNA waxa laga go'doomiyay qaybo phage la safeeyey iyadoo la isticmaalayo GenElute™ Universal Total RNA Purification Kit (Sigma-Aldrich) sida ku cad tilmaamaha soo saaraha. 100}\,{{\upmu \hbox {l}}}\) waa la lysed oo lysate-ka ayaa lagu raray tiirka lafdhabarta si ay u ogolaato RNA inay ku xidho tiirka resin-ka 50}\,{{\upmu \hbox {l}}}\) ay bixiso qalabka ( {-80} \, {^ {\circ } \ hbox {C}} \) ilaa isticmaal dheeraad ah.\({2}\,{\upmu \hbox {g}}\) Qalabka iScript cDNA Synthesis Kit (Bio) -Rad Laboratories) waxaa loo adeegsaday qaab-dhismeedka cDNA iyadoo la raacayo tilmaamaha soo saaraha. Si kooban, falcelinta isku-darka cDNA waxay ka kooban tahay 3 tillaabo: priming at \({25}\,{^{\circ }\hbox {C}}\ )\({5}\,{\hbox {min} } \) , qoraal gadaal ah ee \({20}\,{\hbox {min}} \) at \({46}\,{^{\circ) }\hbox {C}} \), oo rogaal celisku wuxuu ku jiraa \({95}\,{^{\circ}\hbox {C}}\) ee \({1}\,{\hbox {min) }}\) .Marka lagu ordo 1% agarose jel, cDNA waxay muujisay saddex qaybood oo u dhiganta saddexda qaybood ee RNA ee la filayo (xogta aan la muujin) . iyadoo cDNA u adeegsanaysa qaab-dhismeedka PCR ee miniPCR® mini8 wareegga kulaylka:
Aasaaska \ (117 \, \ hbox {bp} \) iyo \ (503 \, \ hbox {bp} \) waxay u dhigmaan 1476-1575 nucleotides ee qaybta M iyo 458-943 nucleotides ee qaybta L, siday u kala horreeyaan aashitada Dhammaan alaabooyinka PCR ee la xoojiyay ayaa lagu shubay korantada 1% agarose gels, iyo DNA bartilmaameedka la xoojiyay ayaa lagu sifeeyay iyadoo la adeegsanayo Qalabka Soosaarka Gel-ka ee GeneJET (Thermo Fisher Scientific).
Haro ku taal xerada IIT Mumbai (Powai Lake, Powai, Mumbai) ayaa loo isticmaalay in lagu daro qaybo phage ah.Biyaha harada waxaa lagu sifeeyay xuubka \({5}\,{\upmu \hbox {m}}) Qaybaha la hakiyay, ka dibna Phi6 phage ayaa lagu daray.Ku dar \({1}\,{\hbox {ml}}\) ee \(10^{6}\,{\hbox {PFU}/\hbox {ml}} \) ilaa \( {100}\ , {\hbox {ml}} \) biyaha harada la sifeeyey, gudaha \({4}\,{^{\circle}\hbox {C}}\) ). Waxa aanu tijaabinay laba hab oo kala duwan si aanu isugu ururino qaybaha fayraska Phi6 ee la soo booday: (1) habka roobka ee aluminium hydroxide adsorption,19 kaas oo loo ansaxiyay uruurinta fayrasyada RNA dhowr baqdin leh oo laga soo qaaday muunado deegaan, iyo (2) Habka fiirsashada fayraska ku salaysan ee polyetylen (PEG) ayaa laga soo minguuriyay daadka iyo al.20. Tan iyo markii waxtarka soo kabashada ee habka PEG-ku-salaysan la ogaaday inuu ka fiican yahay habka aluminium hydroxide, habka PEG-ku salaysan ayaa loo isticmaalay in lagu ururiyo qaybaha Phi6 ee ka soo baxa biyaha harada.
Habka PEG ee la adeegsaday ayaa ahaa sida soo socota: PEG 8000 iyo \ (\hbox {NaCl} \) ayaa lagu daray shaybaarada biyaha harada ee Phi6-spiked si loo helo 8 % PEG 8000 iyo \(0.2 \, \ hbox {M} \) \( \ hbox {NaCl}\) .Samples waxa lagu darey ruxul\({4}\,{^{\circ}\hbox {C}}\)\({4}\,{\hbox {h}}\) ), ka dibna xuddun u ah \(4700 \,\hbox {g}\) waa \({45}\,{\hbox {min}}\) .Tur supernatant oo dib u celi pellet gudaha \({1}\, {\hbox {ml}} in kit-supplied elution buffer\({40}\,{\upmu \hbox {l}}\) .Maadaama fiirsashada RNA ay ku kala duwanaan doonto muunad ilaa muunad saddex geesood ah, \({2}\,{\upmu \) hbox {l}}\) ee RNA waxa loo isticmaalaa dhamaan sadexdaba iyada oo aan loo eegin fiirsigeeda cDNA synthesis of samples. waxaa loo isticmaalay qaab-dhismeedka \({20}\,{\upmu \hbox {l}}\) PCR 35 wareegood si loo kordhiyo \ (117 \, \ hbox {bp} \) iyo \ (503 \, \ hbox { bp}\) jajabyo. Shaybaaradan waxa loo metelayaa sida "1:1″, ie iyada oo aan la qasin sida qaab-dhismeedka kontoroolka togan (PC) .Tirada PCR (qPCR) waxaa lagu sameeyay qalab Stratagene Mx3000P RT-PCR iyadoo la isticmaalayo Brilliant III Ultra-Fast SYBR Cagaaran QPCR Master Mix (Agilent Technologies) . Sifeynta wareegga wareegga (Ct) ayaa loo duubay dhammaan shaybaarrada. Intaa waxaa dheer, shaybaarrada la qasi jiray waxay ahaayeen \({1}\,{\upmu \ hbox {l}} \) iyadoo la isticmaalayo cDNA lagu qasi jiray 1:100 ee biyaha harada la sifeeyay sida \({20}\,{\upmu \hbox {l}}\) PCR ee 35 wareegood. Muunadyadan waxa loo metelayaa "1:100″.
Electrodes-ka PCB-ga waxa lagu farsameeyaa iyada oo la adeegsanayo hab-raac qiimo jaban oo ganacsi ahaan laga heli karo Electroless Nickel Immersion Gold (ENIG) iyada oo aan loo baahnayn dahabi dheeraad ah Xalka piranha ama sulfuric acid cyclic voltammetry laguma talinayo sababtoo ah waxay keeni karaan diirka lakabka khafiifka ah ee dahabka ( dhumucda \ (\ ku dhawaad \) \ (100 \, \ hbox {nm } \)) oo ay soo bandhigaan lakabyada naxaasta ee hoose ee u nugul. 21, 22, 23, 24, 25. Sidaa darteed, ku nadiifi electrodes-ka maro-free-free ah oo lagu qooyay IPA. Muunada la tijaabinayo waxaa lagu daray \({50}\,{\upmu \hbox {M} } , waxaan aragnay in dareenka iyo toosnaanta dareemayaasha la hagaajiyay iyada oo la kordhinayo xoojinta MB 11. Iyada oo ku saleysan hagaajinta laga soo sheegay shaqadeena hore, waxaan isticmaalnay \ ({50}\,{\upmu \hbox {M}}\) MB Baaritaanka korantada ee DNA-da laba-geesoodka ah (ds-DNA) waxaa lagu gaari karaa iyadoo la isticmaalayo anionic ama cationic intercalators. In kasta oo intercalators anionic ay ogaadaan DNA oo leh xulasho wanaagsan, waxay u baahan yihiin isku-duubid habeen dhaxe ah, taasoo keentay waqtiyo ogaansho dheer. Dhanka kale, isugeeyayaasha cationic sida MB waxay u baahan yihiin waqtiyo ka-soo-bax ah oo gaaban, qiyaastii \({1}\,{\hbox {h}}\) ogaanshaha korantada ee ds-DNA6. Cabbir kastaa wuxuu ku lug leeyahay bixinta muunada lagu tijaabinayo electrode \({5}\,{{\upmu \hbox {l}}}\), ka dibna ku nadiifi calal IPA-qoyan, ka hor intaadan sii wadin muunad kale.Hal cabbir.Muunad kasta waxaa lagu tijaabiyay 5 electrodes oo kala duwan haddii aan si kale loo sheegin.DPV iyo cabbirada CV waxaa lagu sameeyay iyadoo la adeegsanayo PalmSens Sensit Smart potentiostat, iyo PSTrace software ayaa loo isticmaalay qaabeynta potentiostat iyo helitaanka xogta, oo ay ku jiraan xisaabinta ugu sarreysa ee hadda jira. Goobaha soo socda ayaa la isticmaalay. ee cabbiraadaha DPV iyo CV:
DPV: Waqtiga dheellitirka = \ (8 \, \ hbox {s} \), Tallaabada korantada = \ (3 \, \ hbox {mV} \), Pulse Voltage = \ (25 \, \ hbox {mV} \) , pulse duration = \ (50 \, \ hbox {ms} \), iskaanka heerka = \ ({20} \, \ hbox {mV/s} \)
CV: Waqtiga dheellitirka = \ (8 \, \ hbox {s} \), Talaabada Voltage = \ (3 \, \ hbox {mV} \), Qiimaha xaaqid = \ ({300} \, \ hbox {mV/ s) }\)
Dhaqdhaqaaqyada ugu sarreeya ee laga helay voltammograms DNA oo isku dhafan \({50}\,{\upmu \hbox {M}}\) MB: (a) \(503\,\hbox {bp}\) DPV , (b) \ (503\,\hbox {bp}
DPV iyo CV voltammograms ayaa laga helay ENIG PCB electrodes \({50}\,{\upmu \hbox {M}}\) MB oo lagu kariyey DNA (marka la isku koobo 10–\({20}\,{\ hbox) }/{\upmu \hbox {l}}} \) ie 0.13–\({0.26}\,{\upmu \hbox {M}}\) ee \(117\,\hbox {bp}\) iyo 0.03 -\({0.06}\,{\upmu \hbox {M}}\) ee \(503\,\hbox {bp}\))).Voltammograms matale waxa lagu muujiyay sawirka S1 ee macluumaadka Dheeraadka ah.Jaantuska 2 ayaa muujinaya natiijooyinka ee cabbiraadaha DPV iyo CV (ugu sarreeya) iyadoo la adeegsanayo alaabada PCR-ga-sadiif ah.Marka la barbar dhigo cabbirada CV-ga, cabbirada DPV waxay muujinayaan dareen sare (hadda oo ah shaqada diiradda DNA) sababtoo ah qulqulka awoodda asalka ah ee cabbirada CV waxay qarinayaan qulqulka Faradaic 26. sanduuq kasta oo ku jira sanduuqa sanduuqa wuxuu ka kooban yahay cabbirro laga soo bilaabo 5 electrodes. Dhammaan cabbirada waxay isticmaalaan isku mid ah qalabka elektaroonigga ah si looga fogaado khaladaadka cabbirka sababtoo ah kala duwanaanshaha electrode-to-electrode. Waxaan aragnay isbeddel sii kordhaya ee DPV iyo CV ayaa qiyaasay qulqulka ugu sarreeya ee heerarka hoose ee DNA. , dheer (\(503 \, \ hbox {bp} \)) \, \ hbox {bp} \ marka la barbar dhigo \ (117) ) jajab. Tani waxay la socotaa isbeddelka la filayo ee xayeysiinta korantada ee laga soo sheegay shaqadeena hore. adsorption of MB-DNA complex waxay fududaynaysaa wareejinta kharashka elektaroonigga ah, taas oo gacan ka geysata kororka heerka ugu sarreeya ee hadda jira. Daraasado kale ayaa muujiyay saameynta xajmiga oligonucleotide iyo isku xigxiga ee MB-DNA intercalation27,28,29,30. guanine. -cytosine (GC) waxa ku jira labada amplicon (\(117\,\hbox {bp}\) iyo \(503\,\hbox {bp}\)) waxay ahaayeen ku dhawaad 50%, taasoo muujinaysa in u fiirsashada farqigu uu sabab u yahay. Si kastaba ha ahaatee, uruurinta DNA-da sare (\(>{2}\,{\hbox {ng}/{\upmu \hbox {l}}}\), loogu talagalay \(503\,hbox {bp} \) iyo \( >{10}\,{\hbox {ng}/{\upmu \hbox {l}}}\) ee \(117\,\hbox {bp}\)), waxaanu dhawrnaa laba cod-weyneyn Dabeecadda ugu sarreysa ee subs-yada ayaa la dhimay marka la eego cabbirada DPV iyo CV-ga labadaba.Tani waa sababta oo ah MB ayaa dheregsan oo kala dhex gala lammaane DNA ah, taasoo keentay in la joojiyo dhaqdhaqaaqa dib-u-dhaqaajinta ee kooxda la dhimi karo ee MB31,32.
在存在 \(2\,\hbox {mM}\) \({\hbox {MgCl }_2}\): (a) \(503\,\hbox {bp}\) DPV, (b) \(503) \,\hbox {bp}
Cusbada ku jirta isku dhafka PCR waxay farageliyaan isdhexgalka elektrostatic ee u dhexeeya MB iyo DNA, markaa adoo ku daraya \(2 \, hbox {mM} \) \ (\hbox {MgCl }_2 \) \ ({50} \,{\) upmu \hbox {M}}\) MB gel-la sifeeyay badeecada si loo barto saamaynta milixdu ku leedahay isdhexgalka MB-DNA hbox {ng}/{\upmu \hbox {l}}}(503\,\hbox {bp}\) iyo \(>{10}\,{\hbox {ng}/{\upmu \hbox { l}}} Heerarka DNA-da hoose, ku-darka milixdu waxay si weyn u yaraynaysaa dareenka, taas oo keentay isbeddel weyn oo hadda ah ee diiradda DNA-da. Saamaynta xun ee la midka ah ee milixdu ku leedahay isdhexgalka MB-DNA iyo isdhexgalka ayaa hore u soo sheegay cilmi-baarayaal kale33,34. \ (\ hbox { Mg}^{2+}\) cations waxay ku xidhan yihiin laf-dhabarka fosfatka xun ee DNA-da, taas oo caqabad ku ah isdhexgalka elektrostatic ee u dhexeeya MB iyo DNA. Marka la eego uruurinta DNA-da sare, xakamaynta adag ee MBs-ka firfircoon waxay keentaa qulqulka hoose ee ugu sarreeya, sidaas darteed isdhexgalka korantada si weyn ha u saameynin jawaabta dareemaha Si otomaatig ah oo otomaatig ah loogu habeeyo shaybaarada biyaha deegaanka, halkaas oo nadiifinta jel ee alaabada PCR laga yaabo inaysan macquul ahayn.
Aagga ka hooseeya qalooca nuugista ee kala duwanaanshaha hirarka dhererka 600-700 \(\hbox {nm} ) \(503\,\hbox {bp}\) leh ama aan lahayn milix (\(2\,\hbox {mM}\) \hbox {bp} \) leh iyo aan lahayn milix (\(2 \, \ hbox {mM} \) \ (\hbox {MgCl}_2 \)). {\upmu \hbox {l}}}
Si loo sii xaqiijiyo natiijooyinka kor ku xusan, waxaanu samaynay cabbirada indhaha anagoo adeegsanayna UV/Vis spectrophotometer (Thermo Scientific Multiskan GO), muunadaha \({50}\,{{\upmu \hbox {l}}}) ayaa loo adeegsaday mid kasta Cabbirka nm} \) , sida ku cad sawirka 4 ( nuugista spectrum lagu muujiyay sawirka S3 ee macluumaadka Dheeraadka ah). {l}}} \), ma jirin farqi weyn oo u dhexeeya qaadashada DNA-ka-kooban iyo muunadaha MB-kaliya (loogu talagalay \(503 \, \ hbox {bp} \) iyo \ (117 \, \ hbox {bp} \) ) jajabyada dhererka), oo tilmaamaya maqnaanshaha xannibaadda steric ee redox-firfircoon ee MB. Heerarka sare ee DNA-da, waxaan aragnay hoos u dhac tartiib tartiib ah ee calaamadda nuugista waxaanan xusnay hoos u dhac yar oo nuugista joogitaanka milixda. Natiijooyinkan waxaa loo aaneynayaa molecular isdhexgalka iyo xannibaadda steric-ka leh ee salka ku haysa isku-dhafka DNA-da.Natiijooyinkayagu waxay la jaan qaadayaan warbixinnada suugaanta ee daraasadaha spectroscopic ee isdhexgalka MB-DNA kaas oo la xidhiidha hypochromaticity iyo heerarka tamarta hoos u dhaca ee \ (\ pi \) - \ (\ pi ^ * \ 36, 37, 38
Agarose gel electrophoresis of phage Phi6: PCR alaabta dhererka \(117 \, \ hbox {bp} \) iyo \ (503 \, \ hbox {bp} \) laga bilaabo muunado biyaha harada.M-DNA calamadaha;Kontoroolka NTC-no-template, furayaasha ka kooban amplicon-yada u dhigma;Kontoroolka togan ee PC;1, 2, 3-la-miray (1:1) muunadaha biyaha harada ee saddex geesoodka ah hbox {bp} \) haadka.
Waxaan qiimeynay utility dareemaha anagoo adeegsanayna muunado biyaha harada Powai oo lagu dhuftey Phi6 phage. Heerarka RNA ee ka go'doomay muunadaha biyaha phage-spiked waxay u dhexeeyaan 15.8- \ ({19.4} \, {\upmu \ hbox {g}/\hbox { ml}}\), halka kuwa ka go'doonsan ganaaxyada fagagga la safeeyey RNA waxaa lagu qiyaasay inay yihiin \({1945}\,{\upmu \hbox {g}/\hbox {ml}}\) oo leh wax ku ool soo kabasho oo qiyaastii 1 ah %RNA waxaa loo rogay cDNA waxaana loo adeegsaday qaab template PCR iyo qPCR. Cabbirka badeecada waxaa lagu xaqiijiyay agarose gel electrophoresis (Jaantuska 5) ka hor inta aan la tijaabin dareenka. Qiimaha Ct ee la duubay inta lagu guda jiro qPCR (Shaxda 1) ayaa lagu muujiyey inay la jaan qaadayaan fiirsashada RNA ee ka go'an muunadaha biyaha ee u dhigma. ka dhaafo marinka ama calaamada asalka. kontoroolka togan iyo muunada imtixaanku waxay sii tusinaysaa in muunad kastaa uu leeyahay ku dhawaad 1% template marka la barbar dhigo kantaroolka togan Si kastaba ha ahaatee, kobcinta fayraskayada iyo borotokoolka xoojinta PCR, waxaan awoodnay inaan si guul leh u weyneyno jajabka \(503 \, \ hbox {bp} \) ee dareenka korantada.
Jaantuska 6 wuxuu muujinayaa natiijooyinka dareemayaasha korantada ee \(503 \, \ hbox {bp} \) amplicon jajabka, labaduba waxay isticmaalayaan cDNA aan la qasin sida template (1: 1) iyo 100-laab cDNA la badhxay sida template (1: 100) la sameeyay PCR , marka la barbar dhigo NTC iyo PC (fiiri sawirka S4 ee macluumaadka dheeriga ah ee voltammograms wakiilka) -to-electrode variation.Marka loo eego cabbirada CV-yada, cabbirada DPV waxay muujinayaan xallin wanaagsan si loo kala saaro shaybaarada tijaabada iyo PC-ga ee NTCs sababtoo ah, sida hore loo sheegay, qulqulka Faradaic waa qarsoodi sababtoo ah qulqulka awoodda asalka ah ee dambe. xakamaynta taban (NTC) waxay keentay CV sare iyo DPV qulqulka ugu sarreeya marka loo eego kantaroolka togan, halka shaybaarada tijaabada ah ee togan iyo kuwa aan la qasin ay muujiyeen dhererka ugu sarreeya ee qulqulka ugu sarreeya DPV. Celceliska celceliska iyo qiimaha dhexdhexaadka ah ee mid kasta oo aan la qasin (1: 1) ) Tijaabada tijaabada iyo PC si cad ayaa loo xalin karaa wax soo saarka dareemayaasha ee saamiga NTC, halka xallinta 1: 100 muunad la qasi jiray ay yar tahay. (dariiqyada aan ku tusin sawirka 5), iyo DPV iyo CV-ga u dhigma ee qulqulka ugu sarreeya waxay la mid yihiin kuwa laga filayo NTC. Natiijooyinka jajabka \ (117 \, \ hbox {bp} \) ayaa lagu muujiyey Macluumaadka Dheeraadka ah. xakamaynta ayaa keentay jawaab celin elektrokimikal ah oo ka timid dareemayaasha PCB sababtoo ah xajinta MB bilaashka ah ee elektaroonigga ah iyo isdhexgalka MB ee asaasiga ah ee oligonucleotide. Sidaa darteed, mar kasta oo muunad la tijaabiyo, xakamaynta taban waa in la maamulaa oo hadda ugu sarreysa muunadda tijaabada marka la barbar dhigo hadda ugu sarreeya ee lagu helay kantaroolka taban si loo gaaro cabbir ka duwan (qaraabo)39,40 si loo kala saaro muunadda tijaabada mid togan ama taban.
(a) DPV, iyo (b) CV-ga ugu sarreeya ee ogaanshaha korantada kiimikada ee \(503 \, \ hbox {bp} \) jajabyada muunadaha biyaha harada kontaroolada togan (PC).
Natiijooyinkayagu waxay muujinayaan habab kala duwan oo saameeya waxqabadka dareemayaasha korantada ee amplicons ee dhererka kala duwan ee DNA-da kala duwan, iyada oo la isku koobay lagu xaqiijiyay cabbirada indhaha iyadoo la adeegsanayo spectrophotometer UV/Vis. Aragtidayadu waxay hoosta ka xariiqaysaa aragtida ah in DNA-da dheer ay jajabiso ilaa \ (\ qiyaastii \) \(500 \, \ hbox {bp} \) waxaa lagu ogaan karaa dareenka sare iyo in joogitaanka milixda ee muunadu aysan ahayn feejignaanta DNA-da xasaasiga ah taas oo saameynaysa dareenka sare ( dhif ah \ ({\hbox {ng}/{\upmu) \hbox {l}}}\) iyo wixii ka sareeya) .Intaa waxaa dheer, waxaan baarnay saameynta noocyada kala duwan ee muunado, oo ay ku jiraan gel-sifeed amplicons iyo aan lagu darin milix, iyo ku darida muunado biyaha harada ee cabbirada DPV iyo CV.Waxaan aragnay in DPV ay bixisay xalin ka wanaagsan, maadaama awoodda awoodda asalka ahi ay sidoo kale saameynayso cabbirka CV-ga, taasoo ka dhigaysa mid xasaasi ah.
Kordhinta jajabyada dhaadheer waxay kuxirantahay daacadnimada RNA genomic viral. Daraasado dhowr ah ayaa muujiyay in kordhinta jajabyada dhaadheer aysan had iyo jeer waxtar lahayn sababtoo ah hoos u dhaca RNA ee deegaanka iyo suurtagalnimada kala-baxa inta lagu jiro go'doominta11,41,42,43,44 Waxaan aragnay in habka fiirsashada fayraska ee PEG-ku uu aad ugu waxtar badan yahay xoojinta phage Phi-6 ee muunada biyaha harada marka loo eego habka fiirsashada fayraska ku salaysan aluminium hydroxide. si loo kordhiyo habab dhererka gaaban oo badan iyo in la yareeyo suurtagalnimada kala-soocidda.
Tijaabooyin bayooloji ah ayaa aad u yar, sidaas darteed waxaa loo baahan yahay in la naqshadeeyo biosensor u baahan muunado yar si loo tijaabiyo. Electrodes-ka ENIG PCB ee loo isticmaalo daraasaddan kaliya waxay u baahan yihiin \({5}\,{{\upmu \hbox {l}}}\ shaybaarada tijaabada ah si loo daboolo aagga waxtarka leh ee electrodes-ka. Intaa waxaa dheer, electrode isku mid ah ayaa dib loo isticmaali karaa ka dib marka la nadiifiyo ka hor inta aan la bixin muunadda soo socota. iyo kiimikooyinka inta badan la isticmaalo. Maadaama elektrode kasta uu ku kacayo $0.55 (ama INR 40) si loo soo saaro, biosensor-kan wuxuu noqon karaa beddelka kharash-ool ah ee teknoolojiyadda ogaanshaha jira. Shaxda 2 waxay muujineysaa isbarbardhigga shaqadan iyo dareemayaasha kale ee lagu soo warramey suugaanta muddo dheer. Qaybaha DNA-da ee muunado kala duwan.
Marka la eego in nidaamyada ogaanshaha korantada ee MB-ku-salaysan ay ku tiirsan yihiin gaar ahaan PCR, xaddidaadda ugu weyn ee habkan ayaa ah suurtagalnimada kordhinta aan gaarka ahayn ee shaybaarrada kala duwan sida biyaha wasakhda ah iyo biyaha harada ama iyadoo la adeegsanayo asal-sadiif hooseeya. Hababka ogaanshaha korantada ee ogaanshaha DNA ee alaabada PCR ee aan la nadiifin iyadoo la adeegsanayo korantada ENIG PCB aan la beddelin, waxaa lagama maarmaan ah in si wanaagsan loo fahmo khaladaadka ay keeneen dNTP-yada iyo alwaaxiyeyaasha aan la isticmaalin, iyo in la wanaajiyo xaaladaha falcelinta iyo borotokoolka baaritaanka. Baahida ogsijiinta (BOD) ee muunadda biyaha ayaa sidoo kale loo baahan karaa in la cabbiro si loo hagaajiyo saxnaanta cabbirka.
Gebogebadii, waxaan soo jeedinaynaa qiime jaban koronto kiimikaad ENIG PCB shidma loogu talagalay ogaanshaha fayraska ee deegaanka (biyaha harada) muunado.Si ka duwan oligonucleotide electrodes ama substrates caadadii ee dareenka DNA oo u baahan kaydinta cryogenic si loo ilaaliyo dareenka,53,54 farsamadayadu waxay isticmaashaa PCB aan la beddelin Electrodes oo leh nolol shelf dheer oo aan lahayn shuruudo kayd ah oo gaar ah oo sidaas darteed ku habboon horumarinta xalalka cabbiraadda oo leh farsamaynta muunad otomaatig ah oo la geeyey LMICs. biosensor-ku wuxuu isticmaalaa midabyo isku-dhafan DNA-ga oo aan qaali ahayn (MB) si loo ogaado degdegga ah ee amplicons. The nonspecific amplification shaybaarada caadiga ah ee deegaanku waxay yareeyaan qaas u ah habkan dareenka sababtoo ah isku-xidhka aan khaaska ahayn ee MBs ee oligonucleotides hal-iyo laba-jibbaaran. Sidaa darteed, qeexida tijaabadani waxay ku xiran tahay hagaajinta asaasiga ah iyo xaaladaha falcelinta PCR. Intaa waxaa dheer, CV-ga iyo qulqulka ugu sarreeya ee DPV ee laga helay shaybaarrada la tijaabiyay waa in loo tarjumaa marka loo eego jawaabaha laga helay xakamaynta xun (NTC) ee imtixaan kasta. Naqshadaynta dareemayaasha korantada iyo hababka lagu soo bandhigay shaqadan waxaa lagu dhex dari karaa autosamplers si ay u horumariyaan si buuxda otomaatig ah oo hooseeya. Xalka qiimaha kaas oo ururin kara oo falanqayn kara muunadyada oo si wireless ah ugu gudbin kara natiijooyinka shaybaadhka .
Cashdollar, J. & Wymer, L. Hababka fiirsashada hore ee fayrasyada laga soo qaaday muunadaha biyaha: dib u eegis iyo falanqaynta meta ee daraasadihii dhawaa.J.Codsiga.microorganism.115, bogga 1-11 (2013).
Gall, AM, Mariñas, BJ, Lu, Y. & Shisler, JL Fayrasyada Waterborne: Caqabadaha ku wajahan biyaha nadiifka ah ee la cabbo.
Shrestha, S. et al.Wastewater epidemiology ee ilaalinta baaxadda weyn ee kharash-ool ah ee COVID-19 ee wadamada dakhligoodu hooseeyo iyo kuwa dhexe: caqabadaha iyo fursadaha.Biyaha 13, 2897 (2021).
Palecek, E. & Bartosik, M. Nucleic acid electrochemistry.Chemical.Rev.112, 3427-3481 (2012).
Tani, A., Thomson, AJ & Butt, JN Methylene buluug oo ah midabtakoor kiimiko ah oo ah oligonucleotides hal-iyo laba-jibbaaran oo aan dhaqdhaqaaq lahayn oo ku yaal substrate-ka dahabka. Falanqeeyaha 126, 1756-1759 (2001).
Wong, EL, Erohkin, P. & Gooding, JJ Isbarbardhigga Cation iyo Anion Intercalators for Electrochemical Transduction of DNA Hybridization by Long-Range Electron Transfer.Electrochemistry.comminicate.6, 648-654 (2004).
Wong, EL & Gooding, JJ Kharashka wareejinta iyada oo loo marayo DNA: biosensor kiimikaad DNA ah oo xulashada ah.
Fang, TH et al. Qalabka microfluidic PCR-waqtiga-dhabta ah oo leh ogaanshaha korantada kiimikaad ee isku-dhafka ah.
Win, BY et al. Daraasada habka calaamadaynta iyo xaqiijinta waxqabadka ee nidaamka PCR ee wakhtiga-dhabta ah ee elektiroonigga ah ee ku salaysan isdhexgalka methylene blue ee DNA. Falanqeeyaha 136, 1573-1579 (2011).
Ramirez-Chavarria, RG iyo al. Loop-dhexdhexaadin ah oo-weyneyn-ku-saleysan koronto-kimikaad-ku-saleysan koronto-kimiko si loo ogaado sars-cov-2 ee muunadaha biyaha wasakhda ah.J.Deegaanka.Chemical.Britain.10, 107488 (2022).
Kumar, M. et al. Dareenka korantada ee SARS-CoV-2 amplicons oo leh korantada PCB. Dareemaha waa la hawlgeliyay.B Chemistry.343, 130169 (2021).
Kitamura, K., Sadamasu, K., Muramatsu, M. & Yoshida, H. Ogaanshaha hufan ee SARS-CoV-2 RNA ee jajabka adag ee biyaha wasakhda ah.sayniska. guud ahaan deegaanka.763, 144587 (2021).
Alygizakis, N. et al. Hababka Analytical for SARS-CoV-2 Ogaanshaha Biyaha Waste: Hab-maamuuska iyo Aragtida Mustaqbalka.TraC trending anal.Chemical.134, 116125 (2020).
Fedorenko, A., Grinberg, M., Orevi, T. & Kashtan, N. Badbaadinta bakteeriyada baqshadda leh ee Phi6 (ku-beddelka SARS-CoV-2) ee dhibco candhuuf ah oo uumi baxay oo lagu shubay dusha muraayadaha.science.Rep.10, 1–10 (2020).
Dey, R., Dlusskaya, E. & Ashbolt, NJ Ku adkaysiga deegaanka ee SARS-CoV-2 beddelka (Phi6) ee nolol xor ah amoeba.J.Caafimaadka Biyaha 20, 83 (2021).
Mindich.63, 149-160 (1999).
Pirttimaa, MJ & Bamford, Qaab dhismeedka sare ee DH RNA phage\(\varphi\)
Bonilla, N. et al.Phages on the Tap - Nidaam degdeg ah oo hufan oo loogu talagalay diyaarinta kaydka shaybaarka ee bakteeriyada.PeerJ 4, e2261 (2016).
Waqtiga boostada: Meey-27-2022