Sonication bilan SARS-CoV-2 Koronavirusni inaktivatsiya qilish protokoli

Hielscher VialTweeter - bu SARS-COV-2 koronavirusini faolsizlantirish uchun ishlatiladigan noyob ultratovushli ko'p namunali tayyorlash moslamasi. VialTweeter bir vaqtning o'zida 10 tagacha namuna flakonlarini tayyorlash imkonini beradi va shu bilan namunani ommaviy qayta ishlash uchun ideal birlikdir.

SARS-CoV-2 Koronavirusini VialTweeter yordamida inaktivatsiya qilish

Fiksatorni olib tashlaganingizdan so'ng, monoqatlamlar hujayralarni 1 ml MEM/5% FBS ichiga qirqishdan oldin fosfat-buferli sho'r suv (PBS) bilan uch marta yuvildi va Hielscher yordamida sonikatsiya qilindi (3 × 10 soniya yoqilgan, 100% quvvat va amplitudada 10 soniya). ultratovushli protsessor VialTweeter bilan UP200St biriktirma. Supernatantlar 10 daqiqa davomida 3000 × g da santrifüj qilish orqali aniqlandi. Welch va boshqalar tomonidan SARS-CoV-2 koronavirus inaktivatsiyasi uchun to'liq protokolni bu erda o'qing. (2020) quyida:

Hujayralar va viruslar

Vero E6 cells (Vero C1008; ATCC CRL-1586) were cultured in modified Eagle’s minimum essential medium (MEM) supplemented with 10% (v/v) fetal calf serum (FCS). Virus used was SARS-CoV-2 strain hCOV-19/England/2/2020, isolated by PHE from the first patient cluster in the UK on 29/01/2020. This virus was obtained at passage 1 and used for inactivation studies at passage 2 or 3.
SARS-CoV-2 inaktivatsiyasi, shuningdek reagent sitotoksikligini yo'qotish uchun ishlatiladigan reagentlar va kimyoviy moddalar uchun Welch va boshqalarning ilmiy hisobotiga qarang. (2020).

Yuvish vositalari bilan virusni inaktivatsiya qilish – Welch va boshqalar tomonidan SARS-CoV-2 Koronavirusni inaktivatsiya qilish protokoli. 2020

SARS-CoV-2 inaktivatsiyasi

Savdo mahsulotlari uchun virus preparatlari (to'qima madaniyati suyuqligi, titrlari 1 × 10 gacha⁶ 1 × 10 gacha⁸ PFU/ml) were treated in triplicate with reagents at concentrations and for contact times recommended in the manufacturers’ instructions for use, where available, or for concentrations and times specifically requested by testing laboratories. Where a range of concentrations was given by the manufacturer, the lowest ratio of product to sample was tested (i.e. lowest recommended concentration of test product). Specimen transport tube reagents were tested using a ratio of one volume of tissue culture fluid to ten volumes of reagent, unless a volume ratio of sample fluid to reagent was specified by the manufacturer. Detergents, fixatives and solvents were tested at the indicated concentrations for the indicated times. All inactivation steps were performed at ambient room temperature (18 – 25°C). For testing of alternative sample types, virus was spiked into the indicated sample matrix at a ratio of 1:9, then treated with test reagents as above. All experiments included triplicate control mock-treated samples with an equivalent volume of PBS in place of test reagent. Immediately following the required contact time, 1mL of treated sample was processed using the appropriately selected filtration matrix. Reagent removal for inactivation testing was carried out in a larger spin column format using Pierce 4mL Detergent Removal Spin Columns (Thermo Fisher), or by filling empty Pierce 10mL capacity centrifuge columns (Thermo Fisher) with SM2 Bio-Beads, Sephacryl S-400HR or Sephadex LH-20 to give 4mL packed beads/resin. For purification using Amicon filters, 2 × 500μl samples were purified using two centrifugal filters by the method previously described, then pooled together. For formaldehyde and formaldehyde with glutaraldehyde removal, one filter was used with 1× 500μl sample volume, resuspended after processing in 500μl PBS, and added to 400ul MEM/5% FBS. For inactivation of infected bir qatlamli, 12,5 sm² Vero E6 xujayralari shishalari (2,5 × 10⁶ 2,5 ml MEM/5% FBS dagi hujayralar/kolba) MOI 0,001 da infektsiyalangan va 37°C/5% CO da inkubatsiya qilingan.₂ 24 soat davomida. Supernatant olib tashlandi va hujayralar xona haroratida 5 ml formaldegid yoki formaldegid va glutaraldegid yordamida 15 yoki 60 daqiqa davomida o'rnatildi. Fiksator olib tashlandi va hujayralarni 1 ml MEM/5% FBS ichiga qirqishdan oldin mono qatlamlar PBS bilan uch marta yuvildi va VialTweeter qo'shimchasi (Hielscher Ultrasound) bilan UP200St yordamida sonikatsiya qilindi (3 × 10 soniya yoqilgan, 100% quvvat va amplitudada 10 soniya o'chirilgan). Texnologiya). Supernatantlar 10 daqiqa davomida 3000 × g da santrifüj qilish orqali aniqlandi.

Welch va boshqalarning protokoliga muvofiq ultratovushli VialTweeter yordamida SARS-CoV-2 koronavirusini inaktivatsiya qilish uchun ishlatiladigan reagent tafsilotlari. 2020

Hielscher VialTweeter-dan foydalanishni o'z ichiga olgan to'liq protokolni bu erda topishingiz mumkin:
Welch, Stephen R.; Davies, Katherine A.; Buczkowski, Hubert; Hettiarachchi, Nipunadi; Green, Nicole; Arnold, Ulrike; Jones, Matthew; Hannah, Matthew J.; Evans, Reah; Burton, Christopher; Burton, Jane E.; Guiver, Malcolm; Cane, Patricia A.; Woodford, Neil; Bruce, Christine B.; Roberts, Allen D. G.; Killip, Marian J. (2020): Inactivation analysis of SARS-CoV-2 by specimen transport media, nucleic acid extraction reagents, detergents and fixatives. Journal of Clinical Microbiology. Accepted Manuscript Posted Online 24 August 2020.

Murakkab ultratovushli dismembratatorlar va hujayralarni buzuvchilar

Ko'p namunali ultrasonikator VialTweeter biologik, biokimyoviy va klinik laboratoriyalarda namuna tayyorlash uchun ko'plab ultratovushli echimlardan biridir. Hielscher Ultrasonics sizning ilovangiz uchun ideal ultratovushli dismembratorni taklif qiladi, masalan, hujayra lizisi, hujayra ekstraktsiyasi, to'qimalarni homogenlashtirish, lizat eritish, eritish, namunani gazsizlantirish va boshqalar.
Agar siz to'g'ridan-to'g'ri yoki bilvosita sonikatsiyani afzal ko'rsangiz va ultratovush namunasini davolashning maqsadi nima bo'lsa, soatiga va kuniga qancha namunani qayta ishlashingiz kerakligini bizga xabar bering. Sizga kundalik ish tartibi uchun eng mos ultratovush qurilmasini tavsiya qilamiz!
Hielscher Ultrasonics’ digital ultrasonic processors are equipped with smart software, automatic data recording, easy pre-setting options for temperature control, sonication duration, cycle/pulse mode as well as sample illumination and browser remote control. We strive to make our ultrasonic devices as smart as possible so that your research and work routine becomes as convenient and successful as possible.
VialTweeter yordamida ultratovush namunalarini tayyorlash protokollarini o'z ichiga olgan qo'shimcha ilovalarni topish uchun shu yerni bosing!