key: cord-0781462-17u9hzq3
authors: Groß, Rüdiger; Conzelmann, Carina; Müller, Janis A; Stenger, Steffen; Steinhart, Karin; Kirchhoff, Frank; Münch, Jan
title: Detection of SARS-CoV-2 in human breastmilk
date: 2020-05-21
journal: Lancet
DOI: 10.1016/s0140-6736(20)31181-8
sha: 80a2835be2f887704dff161425d39bffc0d2b7e2
doc_id: 781462
cord_uid: 17u9hzq3

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Virus strain and virus propagation. Viral isolate BetaCoV/France/IDF0372/2020 was obtained through the European Virus Archive global. Virus was propagated by inoculation of 70% confluent Vero E6 (Cercopithecus aethiops derived epithelial kidney) cells in 75 cm² cell culture flasks with 100 µl SARS-CoV-2 isolate (MOI 0.1) in 3.5 ml serum-free Dulbecco's modified Eagle's medium (DMEM) containing 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, non-essential amino acids (Sigma #M7145), and 1 µg/ml trypsin. Cells were incubated for 2 h at 37°C, before adding 20 ml DMEM additionally supplemented with 2.5% heat inactivated fetal calf serum and 15 mM HEPES. Cells were incubated at 37°C in a 5% CO 2 humidified incubator and supernatant harvested at day 3 post inoculation when a strong CPE was visible. Supernatants were centrifuged for 5 min at 1,000 × g to remove cellular debris, and then aliquoted and stored at -80°C as virus stocks. Infectious virus titer was determined as plaque forming units (PFU) or tissue culture infectious dose 50 (TCID 50 ) and RNA copies were determined by RT-qPCR for CoV-N.

Collection and handling of milk samples. Breast milk was collected via pumps into sterile containers 5-10 min after feeding the newborn and after disinfecting nipples with Octenisept® wound spray. Samples of M1 were initially stored at 4°C and then frozen at day 14 at -20°C. The first two samples of M2 (day 10) were stored for 2 or 3 days at 4°C and then at -20°C. The day 12 and 13 samples were directly frozen at -20°C. Samples of day 14 (M1/M2) as well the day 25 sample were analyzed the same day they were taken without previous storage at -20°C. The remaining milk was frozen in aliquots. All samples were defrosted simultaneously, blinded, and analyzed as described below in two independent experiments (separate preparation of samples, separate RNA isolation). To avoid cross-contamination, only one tube was opened at a time. Skim milk was prepared by centrifugation for 10 min at 1,000 × g and the lower aqueous phase was used for further experiments and analysis.

RNA isolation. RNA was isolated from whole milk, skim milk and PBS using the Qiagen Viral RNA Mini Kit (Qiagen, #52906) according to the manufacturer's instructions with slight modifications from the general protocol detailed in the following. Briefly, 280 μl sample were mixed with 1120 µl lysis buffer (AVL) and incubated 20 min at room temperature to ensure lysis and inactivation of virions. Samples were then frozen at -20°C to allow processing at a later time point. Prior to RT-qPCR, samples were thawed and 5.6 μg carrier RNA added to each sample, followed by vortexing and an additional 10 min incubation at room temperature. 1120 μl ethanol was then added, samples vortexed and briefly centrifuged to remove droplets from the lid and the entire volume then stepwise loaded onto columns. All subsequent steps were performed as instructed by the manufacturer. Viral RNA was eluted in 60 μl AVE buffer, corresponding to 4.67-fold concentration from the 280 μl source fluid used for isolation. RNA was stored at -80°C.

RT-qPCR. RT-qPCR was performed using primer sets targeting N (nucleoprotein) and ORF1b (ORF1b-nsp14) as described in (1) and outlined below. Primers were purchased from Biomers.net (Ulm, Germany) and dissolved in RNAse free water (Carl Roth, #T143.3). Synthetic SARS-CoV-2-RNA (Twist Bioscience, #102024) was used as a quantitative standard to obtain viral copy numbers. To avoid contamination of samples, RT-qPCR reaction mix was first added to all wells followed by RNA samples and these wells were then covered with sealing foil. RNA standard was titrated in a separate laminar flow cabinet and added to wells last. RT-qPCR reactions were performed using TaqMan® Fast Virus 1-Step Master Mix (Thermo Fisher, #4444436) and a OneStepPlus Real-Time PCR System (96-well format, fast mode). All reactions were run in duplicates.

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Protocol adapted from "Detection of 2019 novel coronavirus (2019-nCoV) in suspected human cases by RT-PCR