TITLE: MAVRICS: A Robust and Safe Magnetic Nanoparticle based  RNA Extraction Method Compatible with Phenol-chloroform Inactivated Infectious Samples
AUTHORS: Mo  Li, Gerardo  Ramos-Mandujano

[DESCRIPTION]
Diagnosis and surveillance of emerging pathogens such as SARS-CoV-2 depend on nucleic acid isolation from clinical and environmental samples. Under normal circumstances, samples would be processed using commercial proprietary reagents in Biosafety 2 (BSL-2) or higher facilities. A pandemic at the scale of COVID-19 has caused a global shortage of proprietary reagents and BSL-2 laboratories to safely perform testing. Therefore, alternative solutions are urgently needed to address these challenges. We developed an open-source method called Magneticnanoparticle-Aided Viral RNA Isolation of Contagious Samples (MAVRICS) that is built upon reagents that are either readily available or can be synthesized in any molecular biology laboratory with basic equipment. Unlike conventional methods, MAVRICS works directly in samples inactivated in acid guanidinium thiocyanate-phenol-chloroform (e.g., TRIzol), thus allowing infectious samples to be handled safely without biocontainment facilities.

[STEPS]
1. Silica magnetic nanoparticles (SiMNP) synthesis.
SiMNP synthesis was done following the published protocols in BOMB.bio: BOMB magnetic core nanoparticles synthesis and BOMB coating ferrite MNPs with silica oxide.

  

2. COVID-19 patient samples.
Oropharyngeal or nasopharyngeal swabs are steeped in 1 mL acid guanidinium thiocyanate-phenol-chloroform (AGPC, e.g., TRIzol Reagent or TRI reagent).
 
1 min 

3. Making Bis-Tris Buffer 50 mL 
3.1.  Dissolve 14.33 g guanidinium hydrochloride and 104.6 mg Bis-Tris in  50 mL of 90% ethanol.10 min 
 

 


3.2. Adjust pH (<6.5) with HCl, and adjust the volume with 90% of ethanol to 50 mL.
5 min 

4. Magnetic-nanoparticle-Aided Viral RNA Isolation of Contagious Samples.

4.1.  In an Eppendorf tube add  200 µL  clinical sample, 200 µL Bis-Tris buffer, mix well by vortexing.1 min 
 

 


4.2. Add 40 µL  SiMNP, mix at 1300 rpm 5 min 
 

4.3. Spin the tube for 2-3 seconds, settle the SiMNPs on a magnetic stand and remove the supernatant.2 min 
 


4.4. Mix  200 µL  of AGPC (TRIzol or TRI reagent) and 200 µL Bis-Tris buffer, add to the SiMNPs, mix well by vortexing.1 min 
4.5. Settle the SiMNPs on a magnetic stand and remove the supernatant.2 min 
 


4.6. Add 400 µL of 90% ethanol and mix well,  spin for 2-3 seconds, settle the siMNPs on a magnetic stand and remove the supernatant.2 min 
 

4.7. Repeat Setp 4.6 three more times for a total of 4 ethanol washes6 min 

4.8. After removing the supernatant from the last ethanol wash, dry the SiMNPs on a heat block at 50°C. Keep the lid open, no shaking. Do not elute before the SiMNPs are dried.  20 min

 

4.9.  To elute the RNA, add 40 µL nuclease-free water, and mix at 1300 rpmfor 5 min at room temperature.
4.10. Settle the SiMNPs on a magnetic stand and transfer the eluted RNA to a new RNase-free tube.2 min 

 

4.11. Analyze RNA concentration and purity using a Qubit fluorometer or Nanodrop.10 min 

4.12. Store RNA at -80℃ or use immediately.
5. Reverse transcription (RT): use 4 µL of eluted RNA and follow the instructions for SuperScript™ IV Reverse Transcriptase adding the RNase H incubation step. 60 min 

 


6. Real-time PCR: For each 10 µL qPCR reaction mix  1.5 µL cDNA,  0.5 µL SARS-CoV-2 (2019-nCoV) CDC qPCR Probe Assay, 5 µL TaqMan Fast Advanced Master Mix, and 1.5 µL nuclease-free water. Run qPCR on a Biorad CFX384 Touch Real-Time PCR Detection System (or similar instrument) using the following program: 50°C for 2 min, 95°C for 2 min followed by 45 cycles of 95°C for 5 sec and 59°C for 30 sec. 80 min