Isikhokelo Sesayensi Yasehlotyeni: Xa Igagasi Lobushushu Elingama-40°C Lidibana Novavanyo Lweemolekyuli

Amaqondo obushushu aphezulu aqhubekile kulo lonke elaseTshayina kutshanje. Ngomhla wama-24 kweyeKhala, iShandong Provincial Meteorological Observatory ikhuphe isilumkiso sobushushu obuphezulu obutyheli, ixela ukuba amaqondo obushushu “afana nesauna” angama-35-37°C (111-133°F) kunye nomswakama ongama-80% kwiintsuku ezine ezizayo kwiindawo ezingaphakathi. Amaqondo obushushu kwiindawo ezifana neTurpan, eXinjiang, asondela kuma-48°C (111-133°F). IWuhan kunye neXiaogan, eHubei, ziphantsi kwesilumkiso esiorenji, apho amaqondo obushushu adlula ama-37°C kwezinye iindawo. Kobu bushushu butshisayo, ihlabathi elincinci elingaphantsi komphezulu weepipettes lifumana ukuphazamiseka okungaqhelekanga—ukuzinza kwee-nucleic acids, umsebenzi wee-enzymes, kunye nemeko yomzimba yee-reagents zonke ziphazamiseka kancinci bubushushu.

Ukukhupha i-Nucleic acid kuye kwaba yinto enzima kakhulu. Xa ubushushu bangaphandle budlula i-40°C, nokuba i-air conditioner ivuliwe, ubushushu betafile yokusebenza buhlala bungaphezulu kwe-28°C. Ngeli xesha, iisampuli ze-RNA ezishiywe kwindawo evulekileyo ziwohloka ngokuphindwe kabini kunasentwasahlobo nasekwindla. Kwi-magnetic bead extraction, isisombululo se-buffer sigcwele ngaphakathi ngenxa yokuguquguquka ngokukhawuleza kwe-solvent, kwaye iikristale zikhawuleza ukugaleleka. Ezi kristale ziya kubangela ukuguquguquka okukhulu ekusebenzeni kwe-nucleic acid capture. Ukuguquguquka kwe-organic solvents kuyanda ngaxeshanye. Kwi-30°C, ubungakanani be-chloroform volatilization buyanda nge-40% xa kuthelekiswa ne-25°C. Ngexesha lokusebenza, kuyimfuneko ukuqinisekisa ukuba isantya somoya kwi-fume hood yi-0.5m/s, kwaye usebenzise iiglavu ze-nitrile ukugcina ukusebenza kakuhle kokukhusela.

Uvavanyo lwe-PCR lujongene nokuphazamiseka kobushushu okunzima nangakumbi. Ii-reagents ezifana ne-Taq enzyme kunye ne-reverse transcriptase zinobuthathaka kakhulu kutshintsho olukhawulezileyo lobushushu. Ukuxinana kodonga lwetyhubhu emva kokususwa kwi-freezer ye--20°C kunokubangela ukulahleka komsebenzi we-enzyme engaphezulu kwe-15% ukuba kungena kwinkqubo yokusabela. Izisombululo ze-dNTP zinokubonisa ukonakala okubonakalayo emva kwemizuzu emi-5 yokuvezwa kubushushu begumbi (>30°C). Ukusebenza kwesixhobo nako kuthintelwa bubushushu obuphezulu. Xa ubushushu obuphakathi kwelebhu bungaphezulu kwe-35°C kwaye ukucocwa kobushushu kwesixhobo se-PCR kungonelanga (<50 cm ukusuka eludongeni), umahluko wobushushu bangaphakathi unokufikelela kwi-0.8°C. Oku kuphambuka kunokubangela ukusebenza kakuhle kokukhulisa kumphetho wepleyiti ye-96-well ukuba kwehle ngaphezulu kwe-40%. Iifilitha zothuli kufuneka zicocwe rhoqo (ukuqokelelwa kothuli kunciphisa ukusebenza kakuhle kokusasazwa kobushushu nge-50%), kwaye kufuneka kuthintelwe umoya opholileyo ngqo. Ngaphezu koko, xa usenza uvavanyo lwe-PCR ubusuku bonke, kuphephe ukusebenzisa isixhobo se-PCR "njengefriji yokwexeshana" yokugcina iisampulu. Ukugcinwa kwi-4°C ngaphezulu kweeyure ezi-2 kunokubangela ukuba kubekho ukufuma emva kokuba isiciko esishushu sivaliwe, nto leyo enciphisa inkqubo yokusabela kwaye inokubangela ukuba iimodyuli zesinyithi zesixhobo zibole.

Njengoko zijongene nezilumkiso eziqhubekayo zobushushu obuphezulu, iilabhoratri zemolekyuli nazo kufuneka zikhalise i-alamu. Iisampuli ze-RNA ezixabisekileyo kufuneka zigcinwe ngasemva kwefriji ye--80°C, kwaye ukufikelela kunqunyelwe kumaxesha obushushu obuphezulu. Ukuvula ucango lwefriji ye--20°C ngaphezu kwezihlandlo ezihlanu ngemini kuya kwenza kube nzima ukuguquguquka kobushushu. Izixhobo ezivelisa ubushushu obuphezulu zifuna ubuncinane i-50 cm yendawo yokusasaza ubushushu kumacala omabini nasemacaleni angasemva. Ngaphezu koko, kuyacetyiswa ukuba kuhlengahlengiswe ixesha lovavanyo: 7:00-10:00 AM kwimisebenzi enobuzwe obuphezulu njengokukhupha i-RNA kunye nokulayisha i-qPCR; 1:00-4:00 PM kumsebenzi ongengowovavanyo njengohlalutyo lwedatha. Eli cebo linokuthintela ngempumelelo iincopho zobushushu obuphezulu ukuba zingaphazamisi amanyathelo abalulekileyo.

Uvavanyo lweemolekyuli ngexesha lobushushu luvavanyo lobuchule kunye nomonde. Phantsi kwelanga lasehlotyeni elingapheliyo, mhlawumbi lixesha lokubeka phantsi i-pipette yakho kwaye wongeze ibhokisi eyongezelelweyo yomkhenkce kwiisampulu zakho ukuvumela isixhobo ukuba sikhuphe ubushushu obuninzi. Olu hlonipho lokuguquguquka kobushushu lolona mgangatho uxabisekileyo webhubhoratri ngexesha leenyanga zasehlotyeni ezishushu—emva kwayo yonke loo nto, kubushushu behlobo obungama-40°C, neemolekyuli zifuna “ummandla we-polar owenziweyo” okhuselweyo ngononophelo.