Teaching during COVID-19 pandemic in practical laboratory classes of applied biochemistry and pharmacology: A validated fast and simple protocol for detection of SARS-CoV-2 Spike sequences

The pandemic caused by the SARS-CoV-2 virus (COVID-19) is still a major health issue. The COVID-19 pandemic has forced the university teaching to consider in high priority the switch from in-presence teaching to remote teaching, including laboratory teaching. While excellent virtual-laboratory teaching has been proposed and turned out to be very useful, the need of a real-laboratory in-presence teaching is still a major need. This study was aimed at presenting a laboratory exercise focusing (a) on a very challenging therapeutic strategy, i.e. SARS-CoV-2 diagnostics, and (b) on technologies that are playing a central role in applied biochemistry and molecular biology, i.e. PCR and RT-PCR. The aims of the practical laboratory were to determine: (a) the possibility to identify SARS-CoV-2 sequences starting from a recombinant plasmid and (b) the possibility to discriminate cells with respect to the expression of SARS-CoV-2 Spike protein. This activity is simple (cell culture, RNA extraction, RT-qPCR are all well-established technologies), fast (starting from isolated and characterized RNA, few hours are just necessary), highly reproducible (therefore easily employed by even untrained students). We suggest that this laboratory practical exercises should be considered for face-to-face teaching especially if the emergency related to the COVID-19 pandemic is maintained. The teaching protocol here described might be considered in order to perform fast but meaningful in-presence teaching, making feasible the division of crowded classes in low-number cohorts of students, allowing the maintenance of the required social distance.


SARS-CoV-2 Spike
Kan (R) The pCMV-C-GFPSpark vector has been designed for high-level stable and transient expression in most mammalian cells.
The pCMV-C-GFPSpark vector contains the following key elements: (a) Human enhanced cytomegalovirus immediate-early (CMV) promoter for highlevel expression in a wide range of mammalian cell lines.
(b) Hygromycin resitance gene for selection of transfected mammalian cells.
(c) A Kozak consensus sequence to enhance mammalian expression.
-Thaw bacteria (E.coli JM109 strain, made competent by chemical treatment) on ice for about ten minutes (use about 100 µl aliquots).
-Mix bacteria by finger vortexing.
-Add 10 ng of pCMV-C-GFPSpark by slowly pipetting (no more than a couple of times) (A).
-Put on ice for 2 minutes.
-Transfer, with a pipette, the contents of the Eppendorf in a plate (F).
-Distribute the bacteria on the plate using a loop, continue until the plate is completely dry (G).
-Then, leave the plate open under the hood for 5 minutes (H).

Transformation of chemically-competent bacterial cells with pCMV-C-GFPSpark plasmid: a short description
-Grow the bacteria carrying the pCMV-GFP plasmid in liquid LB-medium (Luria Bertani medium) at 37°C under shaking until the desired density is reached. Use a single colony as starting material. Comments.
(1) The bacterial cells divide approximately every 20 min, until they reach a plateau phase in which their concentration is about 10 9 cells/ml. (2) It is possible to monitor growth through the evaluation of the culture medium measuring OD (optical density) at 600 nm using a spectrophotometer.
(3) The goal is to create a medium with a high number of bacterial cells that contain the plasmid inside them. These cells will be lysed to expose the plasmid which will be extracted

Amplification of bacterial cells carrying the pCMV-C-GFPSpark plasmid
-Take about 50 ml of culture medium containing bacteria grown up to the plateau (turbid medium); centrifuge bacteria at 3000 rpm for 30 minutes at 4°C; remove the supernatant.
-Centrifuge for 30 minutes at 4°C, 3000 rpm; take the supernatant which contais the plasmid DNA.

Isolation/extraction of the pCMV-C-GFPSpark plasmid: a short description (I)
-Purify plasmid DNA through column purification.
-Elute the plasmid (comment: diluted in a large volume).
-Add 3.5 ml of isopropanol to precipitate the plasmid DNA (comment: isopropanol removes H 2 O molecules from DNA precipitating DNA).
-Discard the supernatant by inverting the tube and dry the tube using a lab paper.
-Add 400 µl of 75% EtOH to each eppendorf [comment: a white 'feather' (precipitated plasmid DNA) is formed (EtOH solution is used to eliminate salts from DNA)].
-Remove the ethanol with a syringe.
-Allow the pellet to dry in the air, under the wood; resuspend plasmid DNA pellet in TE (Tris EDTA pH = 8) (ready for the laboratory classes).
Isolation/extraction of the pCMV-C-GFPSpark plasmid: a short description (II)