Xirmada PCR ee Fayraska Busbuska
Faahfaahinta alaabta
Isticmaalka la damacsan yahay
Waxaa loo isticmaalaa in lagu ogaado Virus-ka Monkeybux ee serum bini'aadamka ama muunadaha dheecaanka nabarrada iyadoo la isticmaalayo hababka PCR ee waqtiga dhabta ah.
Mabaadi'da Imtixaanka
Alaabtani waa nidaamka qiimaynta PCR ee Taqman® ee ku salaysan fluorescent.Aasaasayaal gaar ah iyo baaritaanno ayaa loogu talagalay in lagu ogaado hidda-wadaha F3L ee Virus-ka Monkeybux.Xakamaynta gudaha ee lagu beegsanayo hiddo-wadaha dhawrsoon ee bini'aadamka ayaa kormeeraya ururinta muunada, muunad maaraynta muunada iyo habka PCR ee wakhtiga dhabta ah si looga fogaado natiijooyinka beenta ah.Qalabkani waa nidaam lyophilized premix ah oo si buuxda u diyaarsan, kaas oo ay ku jiraan agabka loo baahan yahay ogaanshaha Virus-ka Monkeybux: nucleic acid amplification enzyme, UDG enzyme, falcelinta falcelinta, curiye gaar ah iyo baadhitaan.
Tusmada ugu muhiimsan
Qaybaha la bixiyay waxay ku taxan yihiin shaxda.
| Qaybaha | Xidhmada | Walaxda |
| Fayraska BusbuskaLyophilized Premix | 8 xariijimo PCR tubooyinka× 6 boorso | Qalabkii hore, baarayaasha, dNTP/dUTP Mix, Mg2+, Taq DNA polymerase, UDG Enzyme |
| MPV Control Togan | 400 μL × 1 tube | Taxane DNA oo ka kooban hidde-sidaha Target |
| MPV Xakamaynta Xun | 400 μL × 1 tube | Tixanaha DNA-da oo ka kooban qaybta hiddo-wadaha aadanaha |
| Kala dirida Xalka | 1 ml × 1 tube | Xasilinta |
| Shahaadada waafaqsanaanta | 1 gabal | / |
Socodka Hawlgalka
1. TusaaleUrurinta:Muunada waa in lagu ururiyaa tuubooyinka nadiifka ah si waafaqsan
oo leh tilmaamo farsamo oo caadi ah.
2. Diyaarinta Reagent (Aagga Diyaarinta Reagent)
Ka soo saar qaybaha qalabka, ku dheelitir heerkulka qolka si loogu isticmaalo heegan.
3. Habaynta Muunada (Aagga Habaynta Muunada)
3.1 Soo saarista aashitada nukliyeerka
Waxaa lagu talinayaa in la qaado muunado dareere ah 200μL, Kontoroolka Wanaagsan iyo Xakamaynta Xun ee soo saarista aashitada nucleic, iyadoo loo eegayo shuruudaha iyo hababka u dhigma ee xirmooyinka soo saarista DNA-ga.
3.2 Kala diri budada Lyophilized iyo ku darida qaab-dhismeedka
U diyaari Fayraska Busbuska ee loo yaqaan 'Lyophilized premix' iyadoo loo eegayo tirada shaybaarada.Hal muunad ayaa u baahan hal tuubo PCR oo ay ku jiraan budada premix Lyophilized.Xakamaynta xun iyo xakamaynta togan waa in loola dhaqmaa sidii laba muunado.
(1) Ku dar 15μL Kala dirida Tubo kasta oo PCR ah oo ay ku jiraan Lyophilized premix, ka dibna ku dar 5μL muunado la soo saaray/ Xakamaynta Xun/Kontoroolka Wanaagsan ee tuubada PCR kasta siday u kala horreeyaan.
(2) Dabool tuubooyinka PCR si adag, ku jiidhi tuubooyinka PCR gacanta ilaa budada lyophilized ay si buuxda u milmayso oo la qaso, ku soo ururi dareeraha xagga hoose ee tuubooyinka PCR adigoo isticmaalaya xarun dhexe oo degdeg ah.
(3) Haddii loo isticmaalo qalabka PCR-ga ee caadiga ah si loo ogaado, ka dib si toos ah ugu wareeji tuubooyinka PCR aagga cod-weyneysiinta;Haddii loo isticmaalo BTK-8 si loo ogaado, ka dibna u baahan tahay in la sameeyo hawlaha soo socda: ku wareejin 10 μL dareere ah tuubada PCR si ay chip falcelinta ceelka BTK-8.Hal tubbo oo PCR ah waxay u dhigantaa hal ceel oo chip-ka ku yaal.Inta lagu jiro hawlgalka tuubooyinka, hubi in pipette-ku yahay 90 darajo toosan.Talooyinka pipette aerosol barrier pipette waa in lagu dhejiyaa bartamaha ceelka xoog dhexdhexaad ah oo jooji riixida pipette marka ay gaarto qalabka ugu horreeya (si looga fogaado xumbo).Ka dib marka ceelasha la buuxiyo, soo saar xuubka xuubka falcelinta si uu u daboolo dhammaan ceelasha ka dibna jajabka ayaa loo wareejiyaa meesha lagu ogaanayo.
4. Kordhinta PCR (Aagga ogaanshaha)
4.1 Geli tuubooyinka PCR/chip falcelinta gudaha haanta falcelinta oo si fiican u deji magacyada falcelin kasta sida ay u kala horreeyaan.
4.2 Goobaha ogaanshaha fluorescence: (1) Fayraska busbuska (FAM);(2) Xakamaynta Gudaha (CY5).
4.3 Samee borotokoolka baaskiilka ee soo socda
Borotokoolka ABI7500, Bio-Rad CFX96, SLAN-96S, QuantStudio:
| Talaabooyinka | Heerkulka | Waqtiga | Wareegtada | |
| 1 | Denaturation ka hor | 95℃ | 2 daqiiqo | 1 |
| 2 | Denaturation | 95℃ | 10 s | 45 |
| Annealing, fidinta, helitaanka fluorescence | 60℃ | 30 s | ||
Hab-maamuuska BTK-8:
| Talaabooyinka | Heerkulka | Waqtiga | Wareegtada | |
| 1 | Denaturation ka hor | 95℃ | 2 daqiiqo | 1 |
| 2 | Denaturation | 95℃ | 5 s | 45 |
| Annealing, fidinta, helitaanka fluorescence | 60℃ | 14 s | ||
5. Falanqaynta natiijooyinka (fadlan tixraac Buugga Isticmaalaha Qalabka)
Falcelinta ka dib, natiijooyinka si toos ah ayaa loo keydin doonaa.Guji "Falanqeynta" si aad u falanqeyso, qalabkuna wuxuu si toos ah u tarjumi doonaa qiyamka Ct ee muunad kasta ee tiirka natiijada.Natiijooyinka xakamaynta taban iyo kuwa togan waa inay u hoggaansamaan kuwan soo socda "6. Xakamaynta tayada ".
6. Xakamaynta tayada
6.1 Xakamaynta taban: Ma jiro Ct ama Ct>40 ee kanaalka FAM, Ct≤40 ee kanaalka CY5 oo leh qalooca dhawaaqa caadiga ah.
6.2 Xakamaynta Wanaagsan: Ct≤35 ee kanaalka FAM oo leh qalooca cod-weyneysiinta caadiga ah, Ct≤40 ee kanaalka CY5 oo leh qalooca cod-weyneysiinta caadiga ah.
6.3 Natiijadu waa ansax haddii dhammaan shuruudaha kor ku xusan la buuxiyo.Haddii kale, natiijadu waa been.
Fasiraadda Natiijada
Natiijooyinka soo socda ayaa suurtagal ah:
| Qiimaha Ct ee kanaalka FAM | Ct qiimaha kanaalka CY5 | Tafsiir | |
| 1# | Ma jiro Ct ama Ct>40 | ≤40 | Fayraska busbuska oo xun |
| 2# | ≤40 | Natiijo kasta | Fayraska Busbuska ayaa togan |
| 3# | 40-45 | ≤40 | Dib u tijaabi;Haddii ay weli tahay 40 ~ 45, u soo sheeg sida 1 # |
| 4# | Ma jiro Ct ama Ct>40 | Ma jiro Ct ama Ct>40 | Aan sax ahayn |
FIIRO GAAR AH: Haddii ay dhacdo natiijo aan sax ahayn, muunadda ayaa loo baahan yahay in la qaado oo mar kale la tijaabiyo.
Macluumaadka Dalabka
| Magaca Alaabta | BisadMaya | Cabbirka | Tijaabada | Nolosha shelf | Trans& Sto.Heerkulka |
| Xirmada PCR ee Fayraska Busbuska | B001P-01 | 48 tijaabo/xirmo | Serum/ Lesion Exudate | 12 bilood | -25～-15℃ |
