Hababka ogaanshaha molecular waxay awood u leeyihiin inay soo saaraan mug weyn oo aashitada nucleic ah iyada oo la kordhinayo tirada raadadka laga helo muunado.In kasta oo ay tani faa'iido u leedahay awood u yeelashada ogaanshaha xasaasiga ah, waxay sidoo kale soo bandhigaysaa suurtogalnimada wasakheynta iyada oo loo marayo faafinta hawada-weynida ee deegaanka shaybaarka.Marka la samaynayo tijaabooyin, tillaabooyin ayaa la qaadi karaa si looga fogaado wasakheynta reagents, qalabka shaybaadhka iyo booska kursiga, sababtoo ah wasakheyntan waxay dhalin kartaa natiijooyin been abuur ah (ama been abuur ah).

Si loo yareeyo suurtagalnimada wasakheynta, Dhaqanka Shaybaarka Wanaagsan waa in la sameeyaa mar kasta.Gaar ahaan, waa in laga digtoonaado arrimaha soo socda:

1. Xakamaynta reajentiyeyaasha
2. Habaynta goobta shaqada iyo qalabka
3. Isticmaalka iyo nadiifinta talada meesha molecular ee loo qoondeeyay
4. Talada guud ee bayoolajiga molecular
5. Koontaroolada gudaha
6. Buugaag

1. Xakamaynta reajentiyeyaasha

Si kooban u xidh tuubooyinka reagent ka hor inta aanad furin si aad uga fogaato abuuritaanka hawada hawada.Aliquot reagents si ay uga fogaato qaboojinta dhalaalidda iyo faddaraynta kaydka sayidku.Si cad u calaamadee oo taariikhda ku dheji dhammaan tuubooyinka reagent iyo falcelinta oo ilaali diiwaannada reagent badan iyo nambarada dufcada loo isticmaalo dhammaan tijaabooyinka.Pipette dhammaan reagen-yaasha iyo muunadaha iyadoo la isticmaalayo talooyinka shaandhada.Kahor iibsashada, waxaa lagu talinayaa in la xaqiijiyo soo saaraha in talooyinka shaandhada ay ku habboon yihiin nooca pipette ee loo isticmaalo.

2. Habaynta goobta shaqada iyo qalabka

Goobta shaqada waa in la habeeyaa si loo hubiyo in socodka shaqadu u dhaco hal jiho, laga bilaabo meelaha nadiifka ah (PCR-ka-hor) ilaa meelaha wasakhaysan (PCR-ka dambe)Taxadirka guud ee soo socda ayaa kaa caawin doona yaraynta fursada faddaraynta.Hayso qolal gaar ah, ama ugu yaraan meelo kala duwan oo jir ahaan, loogu talagalay: diyaarinta mastermix, soo saarista aashitada nucleic-ka iyo ku darista template DNA, kordhinta iyo maaraynta alaabta la xoojiyay, iyo falanqaynta alaabta, tusaale ahaan jel electrophoresis.

Goobaha qaarkood, haysashada 4 qol oo gooni ah way adag tahay.Ikhtiyaarka suurtagalka ah laakiin aan la jeclayn ayaa ah in la sameeyo diyaarinta isku-dhafka ah ee aagga xakamaynta, tusaale ahaan golaha qulqulka laminarka.Marka la eego kor u qaadista PCR ee buul leh, diyaarinta mastermix ee falcelinta wareegga labaad waa in lagu diyaariyaa aagga 'nadiif' ee diyaarinta mastermix, laakiin tallaalka badeecada PCR-ga aasaasiga ah waa in lagu sameeyaa qolka weyneynta, iyo haddii ay suurtagal tahay. meel gaar ah oo haynta (tusaale golaha socodka laminar).

Qol/goob kastaa wuxuu u baahan yahay qayb gaar ah oo si cad u calaamadsan pipettes, talooyinka shaandhaynta, mashiinnada tuubada, vortexes, centrifuges (haddii ay khusayso), qalimaan, reagen shaybaadhka guud, jaakadaha shaybaadhka iyo sanduuqyada galoofyada kuwaas oo ku sii jiri doona goobahooda shaqo.Gacmaha waa in la dhaqaa oo gacan-gashiyada iyo jaakadaha shaybaadhka la beddelaa marka loo kala guurayo meelaha loo cayimay.Reagents iyo qalabka waa in aan laga rarin meel wasakh ah loona guurin meel nadiif ah.Haddii ay dhacdo xaalad aad u daran oo loo baahan yahay in dib loo raro reagent ama qalab, marka hore waa in lagu sumeeyaa 10% sodium hypochlorite, ka dibna lagu tirtiro biyo nadiif ah.

Ogow

Xalka 10% sodium hypochlorite waa in la sameeyaa maalin kasta cusub.Marka loo isticmaalo sun-saarid, ugu yaraan wakhtiga xidhiidhka oo ah 10 daqiiqo waa in lagu dhaqmaa.
Beddelkeeda, alaabada ganacsi ahaan loo heli karo ee loo ansixiyo sida DNA-da baabi'inta wasakhowga dusha sare waxaa loo isticmaali karaa haddii talooyinka badbaadada maxalliga ah aysan oggolaan isticmaalka sodium hypochlorite ama haddii sodium hypochlorite aysan ku habboonayn wasakhaynta qaybaha macdan ee qalabka.

Sida habboon, shaqaaluhu waa inay u hoggaansamaan hab-socodka shaqo ee aan jiho lahayn oo aanay ka bixin meelaha wasakhaysan (PCR-ka dambe) oo dib ugu noqon aagagga nadiifka ah (PCR-ka-hor) isla maalintaas.Si kastaba ha ahaatee, waxaa jiri kara waqtiyo ay tani tahay mid aan laga fursan karin.Marka dhacdadan oo kale timaado, shaqaaluhu waa inay taxaddaraan inay si fiican u dhaqdaan gacmaha, beddelaan galoofyada, isticmaalaan jaakada shaybaadhka ee loo qoondeeyay oo aanay soo bandhigin wax qalab ah oo ay rabaan inay qolka dib ugala baxaan, sida buugaagta shaybaadhka.Tallaabooyinka xakamaynta noocan oo kale ah waa in lagu xoojiyaa tababarka shaqaalaha ee hababka molecular.

Isticmaalka ka dib, meelaha kursiga waa in lagu nadiifiyaa 10% sodium hypochlorite (oo ay raacaan biyo nadiif ah si ay meesha uga saaraan warankiilo haraaga ah), 70% ethanol, ama sun-saarid DNA-da oo ganacsi ahaan la heli karo.Fikrad ahaan, nalalka ultraviolet (UV) waa in lagu rakibaa si ay awood ugu yeelaan sun-saarid shucaac.Si kastaba ha ahaatee, isticmaalka nalalka UV waa in lagu xaddidaa meelaha shaqada ee xiran, tusaale ahaan armaajooyinka badbaadada, si loo xaddido soo-gaadhista UV ee shaqaalaha shaybaadhka.Fadlan u hoggaansan tilmaamaha soo-saaraha ee daryeelka laambada UV, hawo-siin iyo nadiifinta si loo hubiyo in nalalka ay waxtar leeyihiin.

Haddii la isticmaalo 70% ethanol halkii laga isticmaali lahaa sodium hypochlorite, shucaaca leh iftiinka UV ayaa loo baahan doonaa si loo dhamaystiro sun-saaritaanka.
Ha ku nadiifin vortex-ka iyo centrifuge sodium hypochlorite;beddelkeeda, ku tirtir 70% ethanol oo u muuji iftiinka UV, ama isticmaal sun-saarid baabi'inta DNA-da ganacsi.Wixii daadanaya, ka hubi warshadeeyaha wixii talo nadiifin ah oo dheeraad ah.Haddii tilmaamaha soo-saareyaashu oggolaadaan, pipette-yada waa in si joogto ah loogu jeermiyo autoclave.Haddii pipette-yada aan la is-qarxin karin, waa inay ku filnaataa in lagu nadiifiyo 10% sodium hypochlorite (oo ay ku xigto si fiican oo biyo nadiif ah loogu tirtiro) ama sun-saarid burburinta DNA-da oo ay ku xigto soo-gaadhista UV.

Nadiifinta boqolkiiba sare ee sodium hypochlorite waxay ugu dambeyntii dhaawici kartaa balaastikada pipette iyo biraha haddii si joogto ah loo sameeyo;hubi talooyinka soo saaraha marka hore.Dhammaan qalabka waxay u baahan yihiin in si joogto ah loo habeeyo iyadoo la raacayo jadwalka soo-saareyaashu ku taliyeen.Qofka la magacaabay waa inuu mas'uul ka noqdaa hubinta in jadwalka isku-habaynta la raacayo, la hayo qoraallo tafatiran, iyo sumadaha adeegga si cad loogu dhejiyay qalabka.

3. Isticmaalka iyo nadiifinta talada meesha molecular ee loo qoondeeyay

Pre-PCR: Reagent aliquoting / mastermix diyaarinta: Tani waa inay ahaataa kuwa ugu nadiifsan dhammaan meelaha loo isticmaalo diyaarinta tijaabooyinka kelli waana inay noqotaa mid loo qoondeeyay qulqulka laminarka oo ku qalabaysan iftiinka UV.Muunado, aashitada nucleic-ka ah ee la soo saaray iyo alaabada PCR ee la xoojiyay waa in aan meeshan lagu qaban.Dib-u-qaadista dib-u-qaadista waa in lagu hayaa qaboojiyaha (ama qaboojiyaha, sida ku cad talooyinka soo-saareyaasha) isla meel la qoondeeyay, oo ku habboon meel u dhow golaha socodka laminarka ama aagga hore ee PCR.Gacan-gashiyada waa in la beddelaa mar kasta marka la soo galo aagga hore ee PCR ama golaha socodka laminarka.

Meesha hore ee PCR ama kabadhka qulqulka laminarka waa in la nadiifiyaa ka hor iyo ka dib isticmaalka sida soo socota: tirtir dhammaan alaabta ku jirta golaha, tusaale ahaan pipettes, sanduuqyada caarada, vortex, centrifuge, racks tube, qalin, iwm. leh 70% ethanol ama a sun-saare DNA baabi'isa ganacsi, una ogolow in uu qalalo.Xaaladda goob shaqo oo xiran, tusaale ahaan kabadhada qulqulka laminarka, u soo bandhig daboolka iftiinka UV 30 daqiiqo.

Ogow

Ha u soo bandhigin reageniyeyaasha iftiinka UV;Kaliya u soo geli golaha wasiirada marka ay nadiif noqoto.Haddii la sameeyo PCR-ga beddelka qoraalka, waxa kale oo ay ku caawin kartaa in lagu tirtiro sagxadaha iyo qalabka xal jebiya RNases marka la taabto.Tani waxay kaa caawin kartaa inaad ka fogaato natiijooyinka beenta ah ee ka yimaada hoos u dhigista enzyme ee RNA.Ka dib markii la nadiifiyo iyo ka hor inta aan la diyaarin mastermix-ga, galoofyada waa in la beddelaa mar kale, ka dibna golaha wasiirada ayaa diyaar u ah in la isticmaalo.

Pre-PCR: Soo saarista aashitada nucleic-ka ah ee ku darista:

Aashitada nukliyeerka waa in la soosaaraa oo lagu qabtaa meel labaad oo loo qoondeeyay, iyadoo la adeegsanayo qayb gaar ah oo pipettes ah, talooyinka shaandhada, xirmooyinka tuubada, galoofyada cusub, jaakadaha shaybaadhka iyo qalabyada kale. tuubooyinka mastermix ama taarikada.Si looga fogaado wasakheynta shaybaarada aashitada nucleic-ka ah ee la baaray, waxaa lagu talinayaa in la beddelo galoofyada ka hor inta aan la maarayn kontaroolada wanaagsan iyo in la isticmaalo qayb gaar ah oo pipettes ah.Dib-u-hagaajinta PCR iyo alaabada la xoojiyey waa inaan lagu shubin meeshan.Tijaabooyinku waa in lagu kaydiyaa talaajado ama qaboojiyaal la cayimay oo isku meel ah.Goobta shaqada ee muunada waa in loo nadiifiyaa si la mid ah meesha mastermix-ka.

Kadib PCR: Kordhinta iyo maaraynta badeecada la xoojiyay

Meeshaan loo qoondeeyay waxaa loogu talagalay hababka kor-u-qaadista ka dib waana inay jir ahaan ka soocna aaggii hore ee PCR.Waxay badanaa ka kooban tahay heerkulbeeyeyaal iyo goobo-waqtiga-dhabta ah, iyo sida ugu habboon waa inay lahaataa golaha socodka qulqulka laminarka si loogu daro wareegga 1 ee badeecada PCR ee wareegga 2, haddii PCR-ga buulkiisa la samaynayo.Dib-u-hagaajinta PCR iyo aashitada nucleic-ka ah ee la soo saaray waa inaan meeshan lagu maamulin mar haddii khatarta wasakhdu ay sarreyso.Meeshani waa inay lahaataa gacmo-gashiyo gaar ah, jaakadaha shaybaadhka, saxannada iyo rafaadka tuubada, tuubooyinka, talooyinka shaandhada, weelasha iyo qalab kale.Tuubooyinka waa in ay noqdaan kuwo dhexe ka hor inta aan la furin.Goobta shaqada ee muunada waa in loo nadiifiyaa si la mid ah meesha mastermix-ka.

Kadib PCR: Falanqaynta alaabta

Qolkan waxaa loogu talagalay qalabka lagu ogaado alaabta, tusaale ahaan taangiyada electrophoresis jel, baakadaha korontada, transilluminator UV iyo nidaamka dukumentiga jel.Meeshani waa inay lahaataa gacmo-gashiyo gaar ah, jaakadaha shaybaadhka, saxanadda iyo rafaadka tuubada, tuubooyinka, talooyinka shaandhada, weelasha iyo qalab kale.Ma jiro reagen kale oo meeshan la keeni karo, marka laga reebo dheega rarida, calamadaha molecular iyo jel agarose, iyo qaybaha kaydinta.Goobta shaqada ee muunada waa in loo nadiifiyaa si la mid ah meesha mastermix-ka.

Qoraal muhiim ah

Sida habboon, qolalka PCR-ka ka horreeya waa in aan la gelin isla maalintaas haddii shaqada hore loogu qabtay qolalka PCR-ka dambe.Haddii tani ay tahay mid gebi ahaanba aan laga fursan karin, hubi in gacmaha marka hore si fiican loo dhaqo iyo in jaakado shaybaar gaar ah lagu xidho qolalka.Buugaagta shaybaadhka iyo warqadaha waa in aan la gelin qolalka PCR ka hor haddii lagu isticmaalay qolalka PCR ka dambeeya;haddii loo baahdo, qaado daabacaadda nuqul ka mid ah borotokoolka/muunadaha aqoonsiga, iwm.

4. Talada guud ee bayoolajiga molecular

Isticmaal galoofyada aan budada lahayn si aad uga fogaato xakamaynta baaritaanka.Farsamada tuubooyinka saxda ah ayaa muhiim u ah yaraynta wasakheynta.Dhuumaha aan saxda ahayn waxay keeni kartaa in ay daadiyaan marka dareeraha la daadinayo iyo abuurista hawada hawada.Dhaqanka wanaagsan ee tuubooyinka saxda ah waxaa laga heli karaa xiriirinta soo socota: Tusaha Gilson ee tuubooyinka, Fiidiyowyada farsamada pipetting-ka Anachem, Tuubooyinka Centrifuge ka hor inta aan la furin, oo si taxadar leh u fur si aad uga fogaato qulqulka.Xir tuubooyinka isla marka la isticmaalo ka dib si aad uga fogaato soo bandhigida wasakhowga.

Markaad samaynayso falcelino badan, diyaari hal mastermix oo ay ku jiraan reagents caadi ah (tusaale biyo, dNTPs, buffer, primers iyo enzyme) si loo yareeyo tirada wareejinta reagent loona yareeyo khatarta faddaraynta.Waxaa lagu talinayaa in la sameeyo mastermix barafka ama block qabow.Isticmaalka Enzyme Start Hot wuxuu kaa caawin karaa inuu yareeyo wax soo saarka alaabada aan gaarka ahayn.Ka ilaali dib-u-hawlgalayaasha ay ku jiraan baarayaasha fluorescent iftiinka si aad uga fogaato xaalufka.

5. Koontaroolada gudaha

Ku dar si wanaagsan loo sifeeyay, kontaroolada togan iyo kuwa taban ee la xaqiijiyay, oo ay weheliso kontorool aan qaabayn ku jirin dhammaan fal-celinnada, iyo jaangooyooyin dhowr-dhibcood leh oo falcelinno tiro leh.Xakamaynta togan waa in aanay noqon mid aad u xoogan oo ay keenayso khatar faddarayn.Ku dar kontaroolada soo saarista togan iyo kuwa taban marka la samaynayo soo saarista nucleic acid.

Waxaa lagu talinayaa in tilmaamo cad lagu dhejiyo mid kasta oo ka mid ah aagagga si ay isticmaalayaashu uga warqabaan xeerarka anshaxa.Shaybaarada ogaanshaha ee lagu ogaanayo heerarka aadka u hooseeya ee DNA ama RNA ee shaybaarada kiliinikada ayaa laga yaabaa inay rabaan inay qaataan cabbirka amniga dheeriga ah ee lahaanshaha habab hawo kala duwan oo leh cadaadis hawo oo xoogaa togan qolalka PCR-ka-hor iyo cadaadiska hawo yar oo xun ee qolalka PCR-ka dambe.

Ugu dambeyntii, sameynta qorshaha hubinta tayada (QA) waa mid waxtar leh.Qorshaha noocan oo kale ah waa inuu ku jiraa liisaska kaydka sayidkiisa reagent iyo kaydka shaqada, xeerarka kaydinta agabka iyo reagents-ka, warbixinta natiijooyinka xakamaynta, barnaamijyada tababarka shaqaalaha, algorithms-ka cilad-baadhista, iyo talaabooyinka hagaajinta marka loo baahdo.

6. Buugaag

Aslan A, Kinzelman J, Dreelin E, Anan'eva T, Lavander J. Cutubka 3: Dejinta shaybaadhka qPCR.Dukumeenti hagitaan ah oo loogu talagalay tijaabinta biyaha madadaalada iyadoo la adeegsanayo USEPA qPCR habka 1611. Lansing- Michigan State University.

Caafimaadka Dadweynaha England, NHS.Heerarka UK ee baarista microbiology: Dhaqanka Shaybaadhka Wanaagsan marka la samaynayo qiimaynta kor u qaadida molecular).Hagida Tayada.2013;4(4):1–15.

Miffin T. Dejinta shaybaadhka PCR.Qabow Spring Harb Protoc.2007;7.

Schroeder S 2013. Dayactirka joogtada ah ee centrifuges: nadiifinta, dayactirka iyo jeermiska ka-hortagga centrifuges, rotors iyo adapters (warqad cad No. 14).Hamburg: Eppendorf;2013.

Viana RV, Wallis CL.Dhaqanka Shaybaadhka Wanaagsan ee Clinical Practice (GCLP) ee tijaabooyinka ku salaysan molecular ee loo isticmaalo shaybaarada ogaanshaha, gudaha: Akyar I, tifaftiraha.Xakamaynta tayada ballaaran.Rijeka, Croatia: Intech;2011: 29-52.