Umhlahlandlela Wesayensi Yasehlobo: Lapho Igagasi Lokushisa elingu-40°C Lihlangabezana Nokuhlolwa Kwemolekyuli

Amazinga okushisa aphezulu aqhubekile kulo lonke elaseShayina muva nje. Ngomhla zingama-24 kuJulayi, i-Shandong Provincial Meteorological Observatory yakhipha isexwayiso sokushisa okuphezulu okuphuzi, ibikezela amazinga okushisa “afana nesauna” angu-35-37°C (111-133°F) kanye nomswakama ongu-80% ezinsukwini ezine ezilandelayo ezindaweni ezimaphakathi nezwe. Amazinga okushisa ezindaweni ezifana neTurpan, Xinjiang, asondela ku-48°C (111-133°F). I-Wuhan kanye ne-Xiaogan, e-Hubei, bangaphansi kwesexwayiso esisawolintshi, amazinga okushisa angaphezu kuka-37°C kwezinye izindawo. Kulokhu kushisa okushisayo, umhlaba we-microscopic ongaphansi kobuso bama-pipette ubhekana nokuphazamiseka okungavamile—ukuzinza kwama-nucleic acid, ukusebenza kwama-enzyme, kanye nesimo somzimba sama-reagents konke kuhlanekezelwa buthule yi-heatwave.

Ukukhishwa kwe-Nucleic acid sekuphenduke umjaho wokulwa nesikhathi. Uma izinga lokushisa langaphandle lingaphezu kuka-40°C, ngisho ne-air conditioner ivuliwe, izinga lokushisa lethebula lokusebenza livame ukundiza ngaphezu kuka-28°C. Ngalesi sikhathi, amasampula e-RNA ashiywe endaweni evulekile ewonakala ngokushesha okuphindwe kabili kunasentwasahlobo nasekwindla. Ekukhishweni kobuhlalu obuzibuthe, isixazululo sebhafa sigcwele endaweni ngenxa yokushibilika ngokushesha kwesincibilikisi, futhi amakristalu awana kalula. Lawa makristalu azodala ukushintshashintsha okukhulu ekusebenzeni kahle kokubanjwa kwe-nucleic acid. I-Volanity of organic solvents iyanda kanyekanye. Ku-30°C, inani le-chloroform volatilization likhuphuka ngo-40% uma liqhathaniswa no-25°C. Ngesikhathi sokusebenza, kuyadingeka ukuqinisekisa ukuthi isivinini somoya ku-fume hood singu-0.5m/s, futhi usebenzise amagilavu ​​e-nitrile ukuze ugcine ukusebenza kahle kokuvikela.

Ukuhlolwa kwe-PCR kubhekene nokuphazamiseka kwezinga lokushisa okuyinkimbinkimbi nakakhulu. Ama-reagents afana ne-Taq enzyme kanye ne-reverse transcriptase azwela kakhulu ekuguquguqukeni okungazelelwe kwezinga lokushisa. Ukuminyana ezindongeni zeshubhu ngemva kokususwa efrijini -20°C kungabangela ukulahleka komsebenzi wama-enzyme angaphezu kuka-15% uma ingena ohlelweni lokusabela. Izixazululo ze-dNTP zingaphinda zibonise ukonakala okuqaphelekayo ngemva kwemizuzu emi-5 nje yokuchayeka kuzinga lokushisa legumbi (>30°C). Ukusebenza kwezinsimbi nakho kuphazanyiswa amazinga okushisa aphezulu. Uma izinga lokushisa elabhorethri lingaphezu kuka-35°C futhi imvume yokukhipha ukushisa kwensimbi ye-PCR inganele (<50 cm ukusuka odongeni), umehluko wangaphakathi wezinga lokushisa ungafinyelela ku-0.8°C. Lokhu kuchezuka kungabangela ukusebenza kahle kokukhulisa emaphethelweni epuleti elinemithombo engu-96 kwehle ngaphezu kuka-40%. Izihlungi zothuli kufanele zihlanzwe njalo (ukuqoqwa kothuli kunciphisa ukusebenza kahle kokukhipha ukushisa ngo-50%), futhi isimo somoya esiqondile kufanele sigwenywe. Ngaphezu kwalokho, lapho wenza izivivinyo ze-PCR ngobusuku obubodwa, gwema ukusebenzisa ithuluzi le-PCR “njengesiqandisi sokuzenzela” ukuze ugcine amasampula. Isitoreji ku-4°C ngaphezu kwamahora angu-2 singabangela ukuminyanisa ukuthi kwakheke ngemva kokuba isivalo esishisayo sivalekile, sihlambulule isistimu yokusabela futhi sikwazi ukugqwala amamojula ensimbi ensimbi.

Njengoba zibhekene nezixwayiso eziqhubekayo zezinga lokushisa eliphezulu, amalabhorethri amangqamuzana kufanele nawo akhalise i-alamu. Amasampula e-RNA ayigugu kufanele agcinwe ngemuva kwefriji -80°C, futhi ukufinyelela kukhawulelwe ezikhathini zokushisa okuphezulu. Ukuvula umnyango wesiqandisi esingu-20°C izikhathi ezingaphezu kweziyisihlanu ngosuku kuzokwengeza ukwehla kwezinga lokushisa. Imishini ekhiqiza ukushisa okuphezulu idinga okungenani isikhala esingu-50 cm sokulahla ukushisa emaceleni omabili nangemuva. Ngaphezu kwalokho, kuyanconywa ukuhlela kabusha isikhathi sokuhlola: 7:00-10:00 AM ngemisebenzi ezwela izinga lokushisa njengokukhishwa kwe-RNA kanye nokulayisha kwe-qPCR; 1:00-4:00 PM ngomsebenzi ongewona wokuhlola njengokuhlaziya idatha. Leli su lingavimbela ngempumelelo amazinga okushisa aphezulu ukuthi angaphazamisi izinyathelo ezibalulekile.

Ukuhlolwa kwamangqamuzana ngesikhathi se-heatwave kuwukuhlola kokubili ubuciko nokubekezela. Ngaphansi kwelanga lasehlobo elingapheli, mhlawumbe sekuyisikhathi sokubeka phansi i-pipette yakho bese wengeza ibhokisi leqhwa elengeziwe kumasampuli akho ukuze uvumele ithuluzi ukuthi likhiphe ukushisa okwengeziwe. Lokhu kuhlonipha ukuguquguquka kwezinga lokushisa kuyikhwalithi eyigugu kakhulu yaselabhorethri phakathi nezinyanga zasehlobo ezishisayo—phela, ekushiseni okungaba ngu-40°C ehlobo, ngisho nama-molecule adinga “indawo yokwenziwa emaphakathi nentshonalanga” eqashwe ngokucophelela.