alaabta

1. 1.Asalkii hore

Nitrofurans waa antibiyootig ballaadhan oo synthetic ah, kuwaas oo inta badan lagu isticmaalo wax soo saarka xayawaanka si ay u helaan bakteeriyada ugu fiican iyo daawooyinka farmashiyaha.Waxa kale oo loo isticmaali jiray kobcinta korriinka doofaarka, digaaga iyo wax soo saarka biyaha.Daraasado muddo dheer lagu sameeyay xayawaanka shaybaadhka ayaa muujiyay in dawooyinka waalidka iyo dheef-shiid kiimikaadkoodu ay muujiyeen astaamaha kansarka iyo mutagenic.Tani waxay keentay mamnuucida nitrofuran ee daaweynta xoolaha loo isticmaalo wax soo saarka cuntada.Daawooyinka nitrofuran furaltadone, nitrofurantoin iyo nitrofurazone ayaa laga mamnuucay isticmaalka wax soo saarka xoolaha ee EU 1993, isticmaalka furazolidone waa la mamnuucay 1995.

Falanqaynta haraaga dawooyinka nitrofuran waxay u baahan tahay in lagu saleeyo ogaanshaha dheef-shiid kiimikaadka ku xidhan nudaha ee dawooyinka waalidka nitrofuran.Maadaama daawooyinka waalidku ay si degdeg ah u dheefshiidaan, iyo unugyada nitrofuran metabolites ee ku xiran waxay sii hayn doonaan waqti dheer, dheef-shiid kiimikaadkan waxaa loo isticmaalaa bartilmaameedka ogaanshaha xadgudubka nitrofuran, oo ay ku jiraan Furazolidone metabolite (AOZ), Furaltadone metabolite (AMOZ). ), Nitrofurantoin metabolite (AHD) iyo Nitrofurazone metabolite (SEM).

AOZ-hadhaaga waxaa inta badan go'aamiya LC-MS ama LC-MS/MS.Enzyme immunoassays, marka la barbar dhigo hababka chromatographic, waxay muujinayaan faa'iidooyin la taaban karo oo ku saabsan dareenka, xadka ogaanshaha, qalabka farsamada iyo wakhtiga loo baahan yahay.(Qiimaha wakhtiga: 45min)

1. 2.Mabaadi'da Imtixaanka

Xirmadan ELISA waxaa loogu talagalay in lagu ogaado AOZ oo ku salaysan mabda'a immunoassay Enzyme aan tartan toos ahayn.Ceelasha microtiter-ka waxaa lagu dahaadhay antigen-ku-xidhan ee BSA-ga.AOZ muunada waxay la tartameysaa antigen-ka lagu dahaadhay saxanka microtiter ee difaaca jirka lagu daray.Ka dib marka lagu daro konjugate enzyme, substrate chromogenic ayaa la isticmaalaa waxaana calaamada lagu cabbiraa spectrophotometer.Nuugista ayaa si ka duwan u dhiganta fiirsashada AOZ ee muunadda.

1. 3.Codsiyada

Qalabkan waxa loo isticmaali karaa qiyaas ahaan iyo falanqaynta tayada ee hadhaaga AOZunugyo anima(muruqa, beerka iwm), malab.

1. 4.Falcelin-celin

Furazolidone metabolite (AOZ) ………………………………….. 100%

Dheef-shiid kiimikaadka Furaltadone (AMOZ) …………………………………………………

Nitrofurantoin metabolite (AHD)……………………………… <0.1%

Nitrofurazone metabolite (SEM) …………………………………………………….

Furazolidone …………………………………………………………………………………………………

Furatada

Nitrofurantoin………………………………………………………………………. <1%

Nitrofurazone………………………………………………………………………………………………………………………………………………………………………………………….

1. 5.Qalabka Loo Baahan Yahay

5.1Qalabka

----Mikrotiter saxan spectrophotometer (450nm/630nm)

---- Qalabka uumi-baxa rotary-ga ama nitrogen-ka lagu qalajiyo

----Homogenizer /caloosha

----Shaker

---- Isku darka Vortex

----Centrifuge

---- dheelitirnaanta falanqaynta (inductance: 0.01g)

---- pipette qalin jabiyay: 10ml

----guluubka tuubada caagga ah

----Dharka mugga leh: 100ml

---- Dhalada: 10ml

----Polystyrene centrifuge tube: 2ml, 50ml

----Mikropipettes: 20ul-200ul, 100ul-1000ul,

250ul-multipipette

5.2Reagents

----Ethyl acetate (AR)

----n-hexane (ama n-heptane) (AR)

----Dipotassium hydrogen phosphate trihydrate

(K₂HPO₄.3H₂O) (AR)

----Hydrochloric acid (HCl, AR)

---Methanol

----Sodium hydroxide (NaOH, AR)

----Biyo dheecan

1. 6.Qaybaha Kit

l saxan Microtiter dahaarka leh antigen, 96 ceel

l xalalka caadiga ah (6 dhalo, 1ml/dhalo)

0ppb,0.025ppb,0.075ppb,0.225ppb,0.675ppb,2.025ppb

l Xakamaynta heerka caadiga ah: (1ml/ dhalo).......100ppb

l Enzyme isku dhafan 1ml ...... dabool cas

l Isku-darka Enzyme 10ml …………………

l Substrate A 7ml………………………………………………………….. dabool cad

l Substrate B 7ml…………………………………………………………

l Jooji xal 7ml………………………………………………

l 20 × xal dhaqid oo xoog leh 40ml

…………………………………………………………

l 2× Xalka soo saarista xooga leh 60ml….koofi buluug ah

l 2-Nitrobenzaldehyde 15.1mg………………………

1. 7.Diyaarinta Reagents

Xalka 1: reagent derivative:

Ku dar 10ml oo methanol ah dhalada oo leh 2-Nitrobenzaldehyde oo milan.(marka la eego qiyaasta 10mM).

Xalka 2: 0.1MK₂HPO₄xal:

Miisaanka 22.8g K₂HPO₄.3H₂O ilaa 1L oo biyo miirmay si ay u milmaan.

Xalka 3: 1M HCl xal

Milaan 8.3ml hydrochloric acid oo leh biyo deionized ilaa 100ml.

Xalka 4:1M NaOH xal

Ku mil 4g NaOH oo leh 100ml oo biyo ah.

Xalka 5Xalka soo saarista:

Ku qas 2 × xal ka-saarid xoog leh oo leh biyo deionized ee saamiga mugga 1: 1.Xalkan waxa lagu kaydin karaa 1bilood at 4℃, kaas oo loo isticmaali doono in lagu soo saaro muunado.

Xalka 6xal dhaqid:

Ku walaaq 20x xal dhaqid oo xoog leh oo leh biyo deionized ee saamiga mugga 1:19, kaas oo loo isticmaali doono in lagu dhaqo taarikada.Xalkan la qasi karo waxaa lagu kaydin karaa 1 bil at 4 ℃.

1. 8.Tusaalaha Diyaarinta

8.1Ogeysiis iyo taxaddar ka hor inta aan la hawlgelin:

a) Fadlan isticmaal talooyinka hal-mar ah tijaabada, oo beddel talooyinka marka aad nuugayso reagen kala duwan.

b) Hubi in dhammaan qalabka tijaabada ahi ay nadiif yihiin.

c) reagent derivative waxaa lagu kaydin karaa at 2-8 ℃ muddo saddex bilood ah;

d) Xalka HCl waxa lagu kaydin karaa heerkulka qolka 3 bilood;

e) Xalka NaOH waxa lagu kaydin karaa 3 bilood heerkulka qolka;

f) Tijaabooyin aan la daweyn ku hay meel baraf ah (-20 ℃);

g) Shaybaarada la daweeyay waxa lagu kaydin karaa 24saac 2-8℃ gudcurka.

8.2 Nudaha xoolaha iyo shaybaarka beerka:

----Isku ekaynta shaybaarada leh homogenizer;

---- Miisaanka 1.0 ± 0.05g ee muunada nudaha isku midka ah ee la isku daray 50ml tuubo senterfuge polystyrene.Ku dar 4ml oo biyo ah, 0.5ml 1M HCl xal iyo 100ul derivative reagent (eeg xalka 1).Rux ilaa 2 daqiiqo

---- Incubated at 37 ℃ habeenkii (qiyaastii 16h);

---- Ku dar 5ml 0.1MK₂HPO₄ (xal2), 0.4ml 1M NaOH (xal4) iyo 5ml ethyl acetate.Si kulul u rux ilaa 30s;

---- Centrifuge heerkulka qolka (20-25 ℃) 10min, ugu yaraan 3000g;

---- Qaado 2.5ml ee wajiga dabiiciga ah ee ka sarreeya 10ml tuubo dhalo nadiif ah, ku qallaji 50-60℃ gaaska nitrogen ama uumiga wareega;

---- Ku milaan hadhaaga engegan 1ml n-hexane (ama n-heptane), vortex ilaa 30s, ku dar 1ml soo saarista (xal5), vortex 1min, si buuxda u qas.

---- Centrifuge heerkulka qolka (20-25^oC) 5min, ugu yaraan 3000g;

---- Ka saar wejiga organic ka sarreeya;Qaado 50μl ee wajiga biyaha substrate si aad u tijaabiso.

8.4 Malabka

---- miisaamay 1.0 ± 0.05g ee muunada malabka ee isku midka ah una gal 50ml tuubo centrifuge polystyrene;

----Ku dar 4ml oo biyo ah, 0.5ml 1M HCl (xal3) iyo 100 μl reagent derivative (xal1);si buuxda u rux ilaa 2min;

----Incubate at 37℃ habeenkii (qiyaastii 16h);

----Ku dar 5ml 0.1MK₂HPO₄ (xal2), 0.4ml 1M NaOH (xal4) iyo 5ml ethyl acetate, si aad ah u rux ilaa 30s;

----Centrifuge heerkulka qolka (20-25 ℃) 10min, ugu yaraan 3000g;

---- Qaado 2.5ml ee wajiga dabiiciga ah ee ugu sarreeya 10ml tuubo dhalada nadiif ah, ku qallaji 50-60℃ gaaska nitrogen ama uumiga wareega;

----Ku milmaan hadhaaga engegan 1ml n-hexane (ama n-heptane), vortex ilaa 30s, ku dar 1ml soo saarista (xal5), vortex 1min, si buuxda u qas.

----Centrifuge heerkulka qolka (20-25^oC) 10min, ugu yaraan 3000g;

---- Ka saar wejiga dabiiciga ah ee sarreeya;Qaado 50μl ee wajiga biyaha substrate si aad u tijaabiso.

1. 9.Habka qiimaynta

9.1Ogaysii kahor qiimaynta

9.1.1 Hubi in dhammaan reagen-yada iyo microwells ay dhamaantood ku jiraan heerkulka qolka (20-25 ℃).

9.1.2 Ku soo celi dhammaan reagen-yaasha intiisa kale 2-8 ℃ isla markiiba ka dib isticmaalka.

9.1.3 Si sax ah u dhaqida ceelasha yaryar waa tallaabo muhim ah oo geedi socodka qiimaynta ah;waa qodobka muhiimka u ah soo saarista falanqaynta ELISA.

9.1.4 Ka fogow iftiinka oo dabool microwells inta lagu jiro soo-baxa.

9.2 Tallaabooyinka Qiimaynta

9.2.1 Ka saar dhammaan reagennada heerkulka qolka (20-25 ℃) in ka badan 30min, isku mid ka hor inta aanad isticmaalin.

9.2.2 Soo saar microwells-ka loo baahan yahay oo ku soo celi inta soo hadhay bacda qufulka 2-8℃ isla markiiba.

9.2.3 Xalka dhaqidda xoogga leh iyo xalka soo saarista xoogga leh waa in lagu diiriyaa si ay ugu jiraan heerkulka qolka kahor isticmaalka.

9.2.4Tirada:La tiriyey boos kasta oo microwell ah iyo dhammaan heerarka iyo muunadaha waa in lagu socodsiiyaa si nuqul ah.Diiwaangeli jaangooyooyinka iyo tusaalooyinka boosaska.

9.2.5Milicsiga xalka antibody-ga oo xoogaysan: ku milo xalalka enzym-ku-koobnaanta leh ee isku-dhafka enzyme-ka ee saamiga mugga ee 1:10

9.2.6Ku dar xal caadi ah / muunad iyo xalka isku xidhka enzyme: ku dar 50µl oo xal caadi ah ama muunad diyaarsan ceelasha u dhigma, ku dar 50µl enzym conjugate xal.Si tartiib ah isku qas adigoo gacanta ku ruxaya saxanka oo ku dabool 30min at 25℃ dabool.

9.2.7Dhaq: Si tartiib ah uga saar daboolka oo ku shub dareeraha ceelasha oo ku raaci microwells 250µl xal dhaqid oo la badhxay (xal6) inta u dhaxaysa 10s ilaa 4-5 jeer.Biyaha haraaga ku nuugo warqad nuugista (xumbada hawada inteeda kale waxaa lagu baabi'in karaa caarad aan la isticmaalin).

9.2.8Midabaynta: Ku dar 50µl xal substrate A iyo 50ul substrate B ee ceel kasta.Si tartiib ah ugu qas adiga oo gacanta ku ruxaya saxanka oo ku dabool 15min at 25℃ dabool (eeg 12.8).

9.2.11Cabbir: Ku dar 50µl ceel kasta ee xalka joogsiga.Si tartiib ah ugu qas adiga oo saxanka gacanta ku ruxaya oo cabbir nuugista 450nm (Waxay soo jeedisay in lagu cabbiro laba-wavelength 450/630nm. Akhri natiijada 5min gudahood ka dib marka lagu daro xalka joojinta.)

10 Natiijooyinka

10.1nuugista boqolkiiba

Qiimaha celceliska ee qiyamka nuugista ee loo helay halbeegyada iyo shaybaarrada waxaa loo qaybiyaa qiimaha nuugista ee heerka koowaad (heer eber) waxaana lagu dhuftey 100%.Heerarka eber ayaa sidaas laga dhigay 100% qiimaha nuugista waxaa lagu soo xigtay boqolkiiba.

=

B —— heerka nuugista (ama muunad)

B0 —— nuugista heerka eber

10.2Qalooca caadiga ah

---- Si loo sawiro qalooca caadiga ah: Qaado qiimaha nuugista ee jaangooyooyinka sida dhidibka y, semi logarithmic ee fiirsashada AOZ heerka caadiga ah (ppb) sida x-dhidibka.

----U-fiirsashada AOZ ee muunad kasta (ppb), kaas oo laga akhrin karo qalooca cabbirka, waxaa lagu dhuftaa qodobka u dhigma ee muunad kasta oo la raaco, waxaana la helayaa uruurinta dhabta ah ee muunadda.

Fadlan ogow:

Software gaar ah ayaa loo sameeyay dhammaan dhimista xogta, kaas oo la bixin karo haddii la codsado.

Qodobka milanka…………………………………………………………

10.Dareenka, saxnaanta iyo saxnaanta

Dareenka: 0.025ppb

Xadka ogaanshaha:

Nudaha (muruqa, beerka) ………………………………………………… 0.1ppb

Malab------------------------------------------------- -0.1ppb

Saxnimada:

Unugyada xoolaha (muruqa iyo beerka) ………………………………………… 100± 20%

Malabka………………………………………………………………………. 100± 20%

Saxnaanta:CV ee xirmada ELISA waa in ka yar 10%.

11.Ogaysiis

12.1 Celceliska qiyamka nuugista ee loo helay halbeegyada iyo shaybaarada waa la dhimi doonaa haddii reagents-yada iyo muunadaha aan lagu maamulin heerkulka qolka (20-25 ℃).

12.2 Ha u ogolaan microwells in ay qallalaan inta u dhaxaysa tillaabooyinka si aad uga fogaato dib-u-soo-noqod guul-darraysan oo ay ku dhaqaaqdo tallaabada xigta isla markiiba ka dib marka aad taabato haysashada microwells.

12.3 Si tartiib ah u rux reagenti kasta ka hor inta aanad isticmaalin.

12.4 Haraggaaga ka fogee xalka joogsiga, waayo waa 0.5MH₂SO₄xal.

12.5 Ha isticmaalin xirmooyinka waqtigoodii dhacay.Ha beddelin reajentiyeyaasha dufcadaha kala duwan, haddii kale waxay hoos u dhigi doontaa dareenka.

12.6 Xaaladda kaydinta: Ku hay xirmooyinka ELISA 2-8℃, ha barafaysan.Xir taarikada nasashada microwell, Ka fogow iftiinka qorraxda tooska ah inta lagu jiro dhammaan soo-baxyada.Daboolka taarikada microtiter ayaa lagula talinayaa.

12.7 Xalka substrate-ka waa in laga tagaa haddii uu midabyo.Reagents-yadu way xumaan karaan haddii qiimaha nuugista (450/630nm) ee heerka eber uu ka yar yahay 0.5 (A450nm <0.5).

12.8 Dareen-celinta midabaynta waxay u baahan tahay 15min ka dib marka lagu daro xalka substrate;Waxaad ku dheerayn kartaa wakhtiga soo-jiidashada ilaa 20min ama ka badan haddii midabku aad u fudud yahay in la go'aamiyo., Weligaa ha dhaafin 25min. Taas lidkeeda, u soo gaabin wakhtiga kuleejka si habboon.

12.9 Heerkulka falcelinta ugu fiican waa 25 ℃.Heerkulka sare ama ka hooseeya wuxuu u horseedi doonaa isbeddelada dareenka iyo qiyamka nuugista.

12.Xaaladda kaydinta iyo muddada kaydinta

Xaaladda kaydinta: 2-8 ℃.

Muddada kaydinta: 12 bilood.