Sau: Wang Xiaoyan, Zhao Eryu

Chav tsev: Jiaozhou Tsev Kho Mob, Tsev Kho Mob Dongfang koom nrog Tongji University

Tam sim no, hom qauv tseem ceeb rau kev kuaj pom tus kab mob ua pa nucleic acid yog caj pas swab.Muaj ntau hom kev siv cov qauv kev khaws cia, txhua yam yuav tsum tau muab tso rau hauv tub yees lossis khov rau kev thauj mus los thiab khaws cia;Nws yog ib qho nyuaj rau kev tswj tag nrho cov txheej txheem ntawm qhov kub thiab txias thaum lub sij hawm sau thiab kev thauj mus los, thiab nws yog ib qho nyuaj los xyuas kom meej cov kev tswj kom zoo ua ntej kuaj cov qauv.^[1-2].

RNase (RNase) yog ib qho endonuclease uas hydrolyzes RNA, feem ntau severing phosphodiester bonds ntawm nucleotides.Lub RNase molecule yog ruaj khov heev, muaj disulfide daim ntawv cog lus nyob rau hauv cov qauv, thiab nws cov kev ua si tsis xav kom muaj divalent cations, yog li RNase tsis yooj yim denatured, thiab nws yog ib qho yooj yim rau renature txawm tias tom qab kub los yog siv cov denaturants.RNases muab faib ua endogenous thiab exogenous sawv daws yuav.Cov RNases endogenous tuaj yeem tso tawm tib lub sijhawm thaum cov hlwb tawg.Yog li ntawd, tshem tawm lub luag haujlwm ntawm endogenous RNases yog ib kauj ruam tseem ceeb hauv RNA extraction txheej txheem.Exogenous RNases tau nthuav dav.RNases muaj nyob rau hauv huab cua, tib neeg daim tawv nqaij, plaub hau thiab qaub ncaug, uas yog ib qho tseem ceeb vim li cas rau qhov yooj yim degradation ntawm RNA.^[3].

Cov ntaub ntawv nug

CANS-CL02-A009 "Daim Ntawv Thov Kev Kho Mob Lub Chaw Kho Mob Zoo thiab Cov Lus Qhia Txog Kev Pom Zoo hauv Kev Tshawb Fawb Txog Molecular" nyob rau hauv cov kev xav tau qhia tias cov qauv dej tsim nyog yuav tsum tau tsim raws li daim ntawv thov;Disposable airtight ntim:

RNase/Classification

(1) RNase A

Ribonuclease A (RNase A), muab los ntawm bovine pancreas, yog ib qho endoribonuclease uas tuaj yeem tawm tsam 3′ kawg ntawm pyrimidine residues ntawm RNA, txiav cytosine lossis uracil tsim los ntawm nucleotides uas nyob ib sab.Phosphodiester daim ntawv cog lus, cov khoom kawg ntawm cov tshuaj tiv thaiv yog 3′ pyrimidine nucleotide thiab ib qho oligonucleotide nrog 3′ pyrimidine nucleotide tom kawg.

(2) RNase T1

Ribonuclease T1 (RNase T1) yog muab los ntawm Aspergillus orjzae, tshwj xeeb tshaj yog ua rau ntawm 3′-terminal phosphate ntawm guanine, thiab qhov chaw cleavage yog nruab nrab ntawm 3′ phosphate ntawm guanine thiab 5′ hydroxyl ntawm nucleotides uas nyob ib sab.Cov khoom kawg ntawm cov tshuaj tiv thaiv yog 3′guanylic acid thiab oligonucleotide fragments nrog 3′guanylic acid thaum kawg.

(3) RNase H

Ribonuclease H (RNase H) yog thawj zaug pom los ntawm calf thymus cov ntaub so ntswg, thiab nws encoding noob tau cloned rau hauv Escherichia coli.Nws tuaj yeem degrade DNA tshwj xeeb: RNA strands hauv RNA hybrid duplexes, ua rau oligonucleotides thiab mononucleotides nrog 3′-OH thiab 5′-monophosphate kawg, nws tsis tuaj yeem degrade ib leeg- lossis ob-kab DNA lossis RNA.

RNase

Muaj nuj nqi thiab siv

Ribonuclease tuaj yeem ua rau lub zog degradation ntawm ribonucleic acid (RNA) thiab tuaj yeem ua kom muaj zog.Tshuaj pleev ib ce yog siv topically kho mob thiab mob pob qij txha.Raws li cov lus ceeb toom, ribonuclease tuaj yeem hloov pauv cov cell metabolism, inhibit tus kab mob synthesis, inhibit qhov kev loj hlob ntawm tus kab mob khaub thuas hauv vitro, thiab inhibit tsim cov tshuaj tiv thaiv thiab kab mob herpes hauv qaib embryos.Kev kho mob siv ribonuclease txhua hnub intramuscular txhaj ntawm 180 mg, muaj txiaj ntsig zoo rau kev kho mob ntawm tus kab mob encephalitis, ribonuclease tuaj yeem ua rau lub degradation ntawm ribonucleic acid (RNA), thiab tam sim no tuaj yeem ua cov khoom sib txuas.Tshuaj pleev ib ce yog siv topically kho mob thiab mob pob qij txha.Raws li cov lus ceeb toom, ribonuclease tuaj yeem hloov pauv cov cell metabolism, inhibit tus kab mob synthesis, inhibit qhov kev loj hlob ntawm tus kab mob khaub thuas hauv vitro, thiab inhibit tsim cov tshuaj tiv thaiv thiab kab mob herpes hauv qaib embryos.Kev kho mob siv ribonuclease txhua hnub intramuscular txhaj ntawm 180 mg yog qhov zoo rau kev kho mob ntawm tus kab mob encephalitis.

RNase

Inhibitor txhais

Lub ntsiab lus ntawm RNase inhibitor unit: Tus nqi ntawm cov enzyme yuav tsum tau inhibit 50% ntawm cov haujlwm ntawm 5ng RNase A yog ib chav.

Yuav ua li cas RNase Inhibitors ua haujlwm

Guanidine isothiocyanate:

Guanidine isothiocyanate yog ib qho organic compound nrog cov mis mos molecular C2H6N4S.Feem ntau yog siv rau hauv cov tshuaj lom neeg, tshuaj reagents, thiab lwm yam. Guanidine isothiocyanate yog cov protein lysing tus neeg sawv cev, thiab nws feem ntau yog siv los ua cov ntsiab lus tseem ceeb ntawm cov tshuaj lysis hauv molecular diagnostic reagents.Nws muaj peev xwm lyse cov ntaub so ntswg, rhuav tshem cell qauv thiab dissociate nucleic acid los ntawm nucleoprotein, thiab muaj zog denaturation rau RNase.Nws yog qhov zoo tshaj plaws RNase inhibitor tam sim no.

TRIzol yog ib qho tshiab tag nrho RNA extraction reagent uas tuaj yeem ncaj qha rho tag nrho RNA los ntawm cov hlwb lossis cov ntaub so ntswg.Nws muaj cov tshuaj xws li phenol thiab guanidine isothiocyanate, uas tuaj yeem cuam tshuam cov hlwb sai thiab inhibit nucleases tawm los ntawm cov hlwb.

(Txawm li cas los xij, guanidine isothiocyanate yog qhov txaus ntshai rau cov kws tshawb nrhiav kev noj qab haus huv.)

RNasin:

Acid glycoprotein muab rho tawm los ntawm nas lub siab lossis tib neeg blastoderm.Rnasin yog ib qho tsis sib tw inhibitor ntawm RNase, uas tuaj yeem khi rau ntau yam RNases kom inactivate lawv.

(Ntau cov ntsiab lus: https://www.foreivd.com/foreasy-rnase-inhibitor-product/)

Hkub kub:

Kub kub kuj yog ib qho kev txhais tau tias ntawm protein denaturation.

Diethylpyrocarbonate (DEPC):

DEPC yog ib qho muaj zog tab sis tsis tiav RNase inhibitor, uas tuaj yeem cuam tshuam RNase kev ua haujlwm los ntawm kev sib txuas nrog cov amino acid imidazole nplhaib ntawm RNase active pab pawg rau denature proteins.

Vanadyl ribonucleoside complex:

Lub complex tsim los ntawm vanadium oxide ions thiab nucleosides, uas khi rau RNase nyob rau hauv daim ntawv ntawm kev hloov tshuaj, uas yuav luag tag nrho inhibit qhov kev ua ntawm RNase.

lwm yam:

SDS, urea, diatomaceous ntiaj teb, thiab lwm yam kuj muaj qee yam cuam tshuam rau RNase.

Kws tshuaj xyuas

Li Yujie Chief Technician

Tus thawj coj ntawm Laboratory Department, Jiaozhou Tsev Kho Mob, Dongfang Tsev Kho Mob Affiliated rau Tongji University

Txhawm rau tiv thaiv biodegradation ntawm exogenous RNase, lub qhov ncauj qhov ntswg, hnab looj tes thiab kaus mom yuav tsum tau hnav thiab hloov ntau zaus thaum lub sij hawm RNA extraction txheej txheem.Tag nrho cov khob iav yuav tsum tau ci hauv qhov cub kom qhuav ntawm 200 ° C rau ntau tshaj 2 teev.Cov khoom siv rau ci, xws li yas, yuav tsum tau kho nrog dej DEPC, thiab tom qab ntawd ntxuav nrog dej distilled.Cov reagents lossis cov cuab yeej siv rau kev rho tawm, khaws cia thiab kev txheeb xyuas ntawm RNA yuav tsum tau mob siab rau RNA, thiab thaj chaw ua haujlwm RNA ywj pheej yuav tsum tau teeb tsa.

cov ntaub ntawv:

[1] Smith-Vaughan HC, Binks MJ, Beissbarth J, et al.Cov kab mob thiab cov kab mob hauv nasopharynx tam sim ntawd ua ntej pib muaj kab mob ua pa qis hauv cov me nyuam hauv paus txawm Australian [J].Eur J Clin Microbiol Infect Dis, 2018,37 (9): 1785-1794.

[2] Tsev Kho Mob Tiv Thaiv Kab Mob ntawm Tuam Txhab Tiv Thaiv Tshuaj Tiv Thaiv.Cov lus qhia rau kev sau thiab tshuaj ntsuam xyuas cov kab mob microbial [J].Suav Journal of Hospital Infection, 2018(20): 3192-3200.

[3] "Tsev Kho Mob Kaum txhiab Vim Li Cas Molecular Biology Test Volume"