Sep 04, 2020

Public workspaceIntegrated Virus Detection System - sample collection - prep - processing V.2

DOI
dx.doi.org/10.17504/protocols.io.bktkkwkw
Integrated Virus Detection System - sample collection - prep - processing
  • 1BVS, Inc
mrwick
Open access
DOI: dx.doi.org/10.17504/protocols.io.bktkkwkw
Protocol Citation: mrwick 2020. Integrated Virus Detection System - sample collection - prep - processing. protocols.io https://dx.doi.org/10.17504/protocols.io.bktkkwkwVersion created by mrwick
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 03, 2020
Last Modified: September 04, 2020
Protocol Integer ID: 41548
Keywords: IVDS, Integrated Virus Detection System, No Reagents, Real Time Virus Detection, Detection of unknown viruses,
Abstract
IVDS is uniquely suited to large volume sampling.Collection from saliva or sputum are straightforward – collection in disposable pipette and transfer to a small vial, dilute as needed, filter, and test, all at under 5 minutes including results.Sample collection, storage and processing are straightforward, not requiring sensitive or unstable solutions.
Guidelines
none
Materials
MATERIALS
ReagentAmmonium AcetateSigma AldrichCatalog #A1542-500G
ReagentButanolFischer ScientificCatalog #71-36-3

Safety warnings
Attention
none
Collection
Collection
Sample collection:
•Collect saliva in a disposable pipet by haveing the subject placing the squeezed pipet into subject's mouth and releasing pressure on the bulb, with patient directing the pipet to the pooled saliva in the mouth.This will collect Amount1 mL to Amount2.5 µL of sample.

Sample processing:
•~Amount1.5 µL of this sample is moved from the pipet into a 1.5 ml centrifuge tube.

•Amount200 µL of this sample is moved to a clean and empty 1.5ml centrifuge tube and mixed with Amount800 µL of RO water.
Expected result
greatly reduced viscosity, the amount of sample may be reduced if the saliva is highly viscous.


•Sample is then agitated for Duration00:00:05 to Duration00:00:15 to mix.
Expected result
even mix,


•Sample is then placed into a 3 ml disposable syringe with a o.45um MCE filter attached and the sample is then filtered into a new 1.5 centrifuge tube.
Expected result
This removes the possible mouth contaminates that can potentially clog the instrument capillary.

•This filtered sample is then placed into the IVDS sample holder.
•Instrument saturates for Duration00:00:30 to Duration00:01:00 and then initiated for instrument scan (testing).Test is 1 scan of 1.5 minutes and sample is processed at 40nl per minute. Test results are a real time read out from IVDS.Data is stored automatically.Sample is assigned a number for processing, this thesampleidwhichwillmatchsourceid.

If the sample does not have enough ionization salt naturally from the saliva, then adding ammonium acetate (Amount50 µL @ Concentration20 millimolar (mM) ) is placed in the sample and vortexed for Duration00:00:05 toDuration00:00:15 and rerun. (Determined by the voltage and Taylor cone shape.)

Instrument setup:
•Air is set to 4psi
•CO2and Air flow are set to 2.5 and 1.5 lpm respectively.
•nA is typically set to -300nA and the kV is typically set to 2.05 . These are set to balance the Taylor cone on the ESI.
•Scan is set to Duration00:01:30 and set for 1 to 2 scans as needed. (View Results)
•Other instrument settings are per standard setting in the operators guide.
•These settings do not change for the days run, regardless of the number of samples run.
After each sample processing:
•After a positive test a Amount1.5 µL of blank buffer is placed into the instrument tube holder and is run as a blank test with the same settings as a sample.This will flush the instrument and the readout will have a zero reading.If not then repeat.
•A sample that has a negative result, proceed to the next sample, a flush is not necessary.
At the end of the day run:
Clean the instrument with Potassium Hydroxide Concentration20 millimolar (mM) solution in a 1.5 ml centrifuge tube flowing into the instrument for Duration00:02:00 .
Remove clean vial and Reverse air flow for Duration00:02:00 and then return to normal.
Run a buffer sample for Duration00:01:00 to verify instrument has no counts or is clean.
Remove buffer and reverse air flow for Duration00:02:00 , return to normal air flow for Duration00:10:00 .Shut off instrument, air supply, CO2, and electrical.





Equipment
Integrated Virus Detection System
NAME
Non Specific Virus Detector
TYPE
TSI, Inc
BRAND
3081, 3480, 3080, 3772, 3032
SKU
components; 3081 - DMA, 3480 - Electrospray Generator, 3080 - Electrostatic Classifier, 3772 - Condensation Particle Counter, 3032 - vacuum pump.
SPECIFICATIONS

Expected result
a negative result will display as no counts by the cpc for the determined "size" or m/z ratio. A positive result is a cpc count at the determined "size" or m/z ratio. It is unknown to determine if there is an active infection since it is unknown what the minimum number of virions are needed to start an infection, so we simply detect them and count the virus particles and not the copies of gentic material that are a secondary detection if at all.