Monkeypox Virus Real Time PCR Kit

Kufotokozera Kwachidule:

Mphaka.Ayi	B001P-01	Kukula	48 Mayeso / Kit
Chitsanzo	Serum / Lesion Exudate	Trans.ndi Sto.Temp.	-25 ~ 15 ℃
Mtundu	Premix anali lyophilized ndipo aliquoted mu 8 strip PCR machubu
Chida Chogwiritsidwa Ntchito	Zida zenizeni za PCR monga ABI7500, Bio-Rad CFX96, SLAN-96S, QuantStudio ndi BTK-8

Titumizireni imelo

Tsatanetsatane wa Zamalonda

Zolemba Zamalonda

Zambiri zamalonda

Cholinga Chogwiritsidwa Ntchito

Amagwiritsidwa ntchito pozindikira kachilombo ka Monkeypox mu seramu ya anthu kapena zitsanzo za exudate pogwiritsa ntchito makina a PCR nthawi yeniyeni.

Mfundo Yoyesera

Chogulitsachi ndi pulogalamu yoyeserera ya PCR yochokera ku fulorosenti yochokera ku Taqman® real-time PCR.Zoyambira ndi ma probe apadera adapangidwa kuti azindikire mtundu wa F3L wa Monkeypox Virus.Ulamuliro wamkati wolunjika ku jini yosungidwa yamunthu imayang'anira zosonkhanitsira zitsanzo, kasamalidwe ka zitsanzo ndi njira yeniyeni ya PCR kuti mupewe zotsatira zabodza.Chidachi ndi premix lyophilized system, yomwe imaphatikizapo zinthu zofunika kuti azindikire kachilombo ka Monkeypox: enzyme ya nucleic acid amplification, UDG enzyme, reaction buffer, primer yeniyeni ndi probe.

Zamkatimu Zazikulu

Zigawo zoperekedwa zandandalikidwa patebulo.

Zigawo	Phukusi	Zosakaniza
Monkeypox VirusLyophilized Premix	8 machubu a PCR× 6 zikwama	Zoyamba, zofufuza, dNTP/dUTP Mix, Mg2+, Taq DNA polymerase, UDG Enzyme
MPV Positive Control	400 μL × 1 chubu	Ma DNA omwe ali ndi Target gene
MPV Negative Control	400 μL × 1 chubu	Ma DNA omwe ali ndi gawo la majini aumunthu
Dissolving Solution	1 mL × 1 chubu	Stabilizer
Satifiketi Yogwirizana	1 chidutswa	/

Operation Flow

1. ChitsanzoZosonkhanitsidwa:Zitsanzo ziyenera kusonkhanitsidwa m'machubu osabala molingana

ndi standard specifications.

2. Kukonzekera kwa Reagent (Reagent Preparation Area)

Chotsani zigawo za zida, kuzilinganiza kutentha kwa chipinda kuti mugwiritse ntchito.

3. Kukonza Zitsanzo (M'dera Lopangira Zitsanzo)

3.1 Kutulutsa kwa nucleic acid

Ndi bwino kutenga zitsanzo zamadzimadzi 200μL, Positive Control ndi Negative Control kwa nucleic acid m'zigawo, malinga ndi zofunikira ndi ndondomeko za ma virus ochotsa DNA.

3.2 Lyophilized ufa kusungunuka ndi kuwonjezera template

Konzani Monkeypox Virus Lyophilized premix malinga ndi kuchuluka kwa zitsanzo.Chitsanzo chimodzi chimafuna chubu chimodzi cha PCR chokhala ndi Lyophilized premix powder.Kuwongolera koyipa & kuwongolera kwabwino kuyenera kutengedwa ngati zitsanzo ziwiri.

(1) Onjezani 15μL Dissolving Solution mu chubu chilichonse cha PCR chokhala ndi Lyophilized premix, kenaka yikani zitsanzo zotengedwa 5μL/Negative Control/Positive Control muchubu chilichonse cha PCR motsatana.

(2) Phimbani machubu a PCR mwamphamvu, gwedezani machubu a PCR ndi dzanja mpaka ufa wa lyophilized utasungunuka kwathunthu ndi kusakanikirana, sonkhanitsani madziwo pansi pa machubu a PCR ndi centrifugation yomweyo yotsika kwambiri.

(3) Ngati ntchito wamba Real-nthawi PCR chida kudziwika, ndiye mwachindunji kusamutsa machubu PCR kudera matalikidwe;Ngati mugwiritsa ntchito BTK-8 kuti muzindikire, ndiye kuti muyenera kuchita izi: kusamutsa madzi 10 μL kuchokera ku PCR chubu kupita ku chitsime cha BTK-8.Chubu chimodzi cha PCR chimafanana ndi chitsime chimodzi pa chip.Pa ntchito ya pipetting, onetsetsani kuti pipette ndi 90 madigiri ofukula.Nsonga za aerosol chotchinga pipette ziyenera kuyikidwa pakati pa chitsime ndi mphamvu zochepa ndikusiya kukankhira pipette ikafika pa gear yoyamba (kupewa thovu).Zitsime zikadzazidwa, chotsani kachipangizo kakang'ono kuti mutseke zitsime zonse ndipo chipcho chimasamutsidwa kumalo ozindikira matalikidwe.

4. Kukulitsa kwa PCR (Detection Area)

4.1 Ikani machubu a PCR/rection chip mu thanki yochitira ndikuyika mayina azomwe zimachitika bwino molingana.

4.2 Zikhazikiko za kuzindikira fluorescence: (1) Monkeypox virus (FAM);(2) Kulamulira Kwamkati (CY5).

4.3 Thamangani ndondomeko yoyendetsa njinga yotsatirayi

Protocol ya ABI7500, Bio-Rad CFX96, SLAN-96S, QuantStudio:

Masitepe		Kutentha	Nthawi	Zozungulira
1	Pre-denaturation	95 ℃	2 mins	1
2	Denaturation	95 ℃	10 s	45
2	Kuphatikiza, kukulitsa, kupeza fulorosenti	60 ℃	30 s	45

Ndondomeko ya BTK-8:

Masitepe		Kutentha	Nthawi	Zozungulira
1	Pre-denaturation	95 ℃	2 mins	1
2	Denaturation	95 ℃	5 s	45
2	Kuphatikiza, kukulitsa, kupeza fulorosenti	60 ℃	14 s	45

5. Kusanthula zotsatira (chonde onani Instrument User Manual)

Pambuyo anachita, zotsatira adzapulumutsidwa basi.Dinani "Unikani" kuti muwunike, ndipo chidacho chidzatanthauzira zokha Ct zachitsanzo chilichonse pazotsatira.Zotsatira zoyipa ndi zabwino zowongolera zidzagwirizana ndi izi "6. Kuwongolera Ubwino".

6. Kuwongolera Kwabwino

6.1 Kuwongolera Koyipa: Palibe Ct kapena Ct> 40 mu njira ya FAM, Ct≤40 mu CY5 njira yokhala ndi curve yokhazikika.

6.2 Ulamuliro Wabwino: Ct≤35 mu njira ya FAM yokhala ndi mapindikidwe okulirapo, Ct≤40 mu CY5 njira yokhala ndi ma curve wamba.

6.3 Zotsatira zake ndizovomerezeka ngati zonse zomwe zili pamwambazi zakwaniritsidwa.Apo ayi, zotsatira zake ndizolakwika.

Kutanthauzira zotsatira

Zotsatira zotsatirazi ndizotheka:

	Mtengo wapatali wa magawo FAM	Mtengo wapatali wa magawo CY5	Kutanthauzira
1#	Palibe Ct kapena Ct> 40	≤40	Monkeypox virus negative
2#	≤40	Zotsatira zilizonse	Monkeypox HIV
3#	40; 45	≤40	Kuyesanso;ngati ikadali 40 ~ 45, nenani ngati 1 #
4#	Palibe Ct kapena Ct> 40	Palibe Ct kapena Ct> 40	Zosalondola

ZINDIKIRANI: Ngati zotsatira zolakwika zichitika, chitsanzocho chiyenera kusonkhanitsidwa ndikuyesedwanso.

Kuitanitsa Zambiri

Dzina lazogulitsa	Mphaka.Ayi	Kukula	Chitsanzo	Shelf Life	Trans.ndi Sto.Temp.
Monkeypox Virus Real Time PCR Kit	B001P-01	48 mayeso / zida	Serum / Lesion Exudate	Miyezi 12	-25 ~ 15 ℃