Anion Exchange Resin–Based Detection of Viruses from Bioaerosols

Resumo

Source: Schaeffer, J. W., Chandler, et al. Detection of Viruses from Bioaerosols Using Anion Exchange Resin. J. Vis. Exp. (2018).

This video demonstrates the detection of viruses from bioaerosols using an anion exchange resin. A liquid impinger containing the resin is used to capture viruses from the air. The resin is collected and treated with a virus lysis buffer containing carrier RNA. The lysate is transferred to a microcentrifuge tube for viral RNA isolation, followed by qRT-PCR to amplify and confirm the presence of the virus.

Protocolo

1. Nucleic Acid Extraction and quantitative reverse transcription polymerase chain reaction or qRT-PCR Detection

1. Isolate nucleic acids directly from anion exchange resin and, for comparative purposes, from the liquid used within the liquid impingers. In this example, an RNA isolation procedure is described, but a similar protocol could be used for DNA viruses.
   1. Nucleic acid isolation from the anion exchange resin.
      1. Transfer all of the anion exchange resin-containing liquid of the liquid impinger to a sterile 50 mL conical tube.
      2. Allow the resin to settle, and slowly decant the liquid sample from the anion exchange resin. Use a 1 mL pipette tip to remove all of the remaining liquid from the anion exchange resin.
      3. From a viral RNA isolation kit, add 560 µL of viral lysis buffer containing carrier RNA directly to the anion exchange resin and incubate for 10 min at room temperature with periodic mixing.
      4. Using a 1 mL pipette tip, transfer the viral lysate (the viral lysis buffer) to a sterile 1.5 mL conical tube and proceed with RNA isolation using a viral RNA isolation kit (see Table of Materials) in accordance with the manufacturer's instructions, except that RNA is eluted in a total volume of 60 µL of elution buffer.
   2. Nucleic acid isolation from liquid impingers.
      1. Pipette 140 µL of the liquid sample into a sterile 1.5 mL conical tube and perform RNA isolation using the viral RNA isolation kit in accordance with the manufacturer's instructions, with the exception of eluting RNA in a total volume of 60 µL of elution buffer.
         NOTE: 140 µL is the maximum sample volume that can be processed with the specific viral RNA isolation kit employed here in a single-step preparation.
2. Perform qRT-PCR (quantitative Reverse Transcription Polymerase Chain Reaction) for the detection of Male-specific 2 (MS2) fRNA coliphage and influenza A and B viruses. In this demonstration, 5 µL of nucleic acid eluates are used for qRT-PCR.

Materiais

Lista de materiais utilizados neste artigo

Nome	Empresa	Número de catálogo	Comentários
Escherichia coli bacteriophage MS2 (ATCC 15597-B1)	American Type Culture Collection	ATCC 15597-B1
FluMist Quadrivalent	AstraZeneca	Contact manufacturer	Viral constitutents of this vaccine are subject to change on an annual basis
CFX96 Touch Real-Time PCR Detection System	Bio-Rad	1855195
Primers and probes	Integrated DNA Technologies	NA
0.2 µM sterile filter	NA	NA
1 L Pyrex bottles or equivalent	NA	NA
1 mL Pipett tips	NA	NA
1 mL Pipettor	NA	NA
50 mL serological pipet	NA	NA
PCR tubes	NA	NA
Pipet-aid or equivalent	NA	NA
QIAamp Viral RNA Mini Kit	Qiagen	52904
QuantiTect Probe RT-PCR Kit	Qiagen	204443
Amberlite IRA-900 chloride form	Sigma-Aldrich	216585-500G
Phosphate buffered saline	Sigma-Aldrich	P5368-10PAK
Water (molecular biology grade)	Sigma-Aldrich	W4502-1L
Eppendorf DNA LoBind Microcentrifuge Tubes	ThermoFisher	13-698-791
Falcon 50 mL Conical Centrifuge Tubes	ThermoFisher	14-432-22
Falcon Polypropylene Centrifuge Tubes	ThermoFisher	05-538-62
SuperScript III Platinum One-Step qRT-PCR Kit w/ROX	ThermoFisher	11745100
SKC Biosampler 20 mL, 3-piece glass set	SKC Inc.	225-9593
Vac-u-Go sample pumps	SKC Inc.	228-9695
Collison nebulizer (6-jet)	BGI Inc.	NA
HEPA capsule	PALL	12144
Q-TRAK indoor air quality monitor 8554	TSI Inc.	NA
Alnor velometer thermal anemometer AVM440-A	TSI Inc.	NA
SidePak AM510 personal aerosol monitor	TSI Inc.	NA
Bioaerosol chamber	NA	NA