VIRAL RNA EXTRACTION AND AMPLIFICATION

Name: LUKE JOVANNI B. TAOC Rating:

Laboratory Section: MCB104 – A7B12 Date: DECEMBER 1 , 2023

General Instructions:
For the viral RNA procedure/protocol, visit this link: Viral RNA Extraction for testing COVID-19
(using MagListo™) - YouTube
For the RT-qPCR procedure, refer to this video (start at 4:32): RT-qPCR for diagnosing COVID-
19 (former 2019-nCoV) - YouTube

1. What are the general steps in the isolation of viral RNA?

According from the information I obtained from watching the Youtube Video, Viral RNA
Extraction for testing COVID-19 (using MagListo™) - YouTube , This is the generalized step-by-
step process for isolating the viral RNA using the MagListo™ Viral DNA/RNA Extraction Kit:

 Preparation:
o Dissolve Proteinase K in nuclease-free water.
o Mix Poly(A) with ER Buffer and combine with VB Buffer.
o Prepare absolute ethanol for specific buffers.
o Pre-heat ER Buffer to 60°C.
 Viral RNA Isolation:
o Combine Proteinase K solution with the sample in a tube.
o Add VB Buffer, mix thoroughly, and incubate at 56-60°C for 10 minutes.
o Introduce absolute ethanol, Magnetic NanoBeads Solution, and vortex mix well.
o Employ a Magnetic Separation Rack to separate beads from the supernatant.
o Discard supernatant and wash beads using VWM1 and RWB2 Buffers via
magnetic separation.
o Remove residual ethanol:
 a. (Washing Bead) Wash with WE Buffer and remove supernatant.
 b. (Drying Bead) Wash with 80% ethanol, dry beads at 60°C, and remove
supernatant.
o Add ER Buffer, mix, and incubate at 56-60°C.
o Separate beads using a Magnetic Separation Rack and transfer the supernatant
containing RNA to a new tube.
These steps provide a broad overview of the process, including mixing, incubation, magnetic
separation, and careful handling to obtain purified viral RNA. The specifics may vary slightly
depending on the kit and the samples being processed.
 Figure 1. A schematic diagram on the isolation of the viral RNA using the MagListo™ Viral DNA/RNA Extraction Kit
(Source: https://eng.bioneer.com/literatures/manual/MagListo/EN_QM_MagListo_5M_Viral_DNA_RNA_Extraction_Kit.pdf)

2. In the PCR-based testing method, what genes were targeted to detect SARS-CoV-2?
Refer to the link/video.

The RNA specific to B-βCOV targets the E gene using the FAM Reporter dye/channel. For
SARS-COV-2 RNA, it targets the S gene utilizing the Cy5/618-660 Reporter dye/channel. The
Internal control targets IC genes employing the HEX/465-533 Reporter dye/channel.
(Source:https://media.tghn.org/medialibrary/2020/10/SOP011b-ITM_CTP_Realtime_PCR.docx)

3. What possible circumstances can lead to a false-positive and a false-negative result?

These are the possible circumstances that can lead to a false-positive and false-negative result
False Positive Results:

o Technical Problems: These include contamination during sampling (e.g., a swab

touching a contaminated surface), contamination by PCR amplicons, contamination of
reagents, sample cross-contamination, and cross-reactions with other viruses or genetic
material.
(Source: https://www.thelancet.com/journals/lanres/article/PIIS2213-2600%2820%2930453-7/fulltext#:~:text=Technical%20problems%20including
%20contamination%20during,positive%20results)

o Incorrect Test Execution: Improper handling of the test kit or failing to follow
instructions carefully, especially in the case of rapid antigen tests, can lead to false
positives.
(Source: https://www.mayoclinic.org/tests-procedures/covid-19-diagnostic-test/about/pac-20488900)

False Negative Results:

o Incorrect Test Administration: A common reason for false negatives is the incorrect
administration of the test. This includes inadequate swabbing of the nose or throat, not
swabbing for long enough, or swabbing too timidly. Tests performed by healthcare
personnel are more likely to detect the virus compared to self-administered tests.
(Source: https://covidblog.oregon.gov/the-science-behind-false-negative-covid-19-tests/)

o Viral Particle Shedding Variability: Individuals might not be shedding virus particles in
their nose at the time of the test. The virus can infect different parts of the body, meaning
someone could be shedding more virus in their throat or gut than in their nose, which
would result in a negative test if only a nose swab is taken.
(Source: https://covidblog.oregon.gov/the-science-behind-false-negative-covid-19-tests/)

o Type and Sensitivity of the Test: The risk of false negatives depends on the type and
sensitivity of the COVID-19 diagnostic test. PCR tests are generally more sensitive and
accurate compared to rapid antigen tests.
(Source: https://www.mayoclinic.org/tests-procedures/covid-19-diagnostic-test/about/pac-20488900)

o Thoroughness of Sample Collection: How thoroughly the sample is collected can

significantly impact the test result. An inadequate sample collection can lead to a false
negative.
(Source: https://www.mayoclinic.org/tests-procedures/covid-19-diagnostic-test/about/pac-20488900)

o Accuracy of Lab Analysis: The precision with which the lab analyzes the sample also
plays a critical role in determining the accuracy of the test result.
(Source: https://www.mayoclinic.org/tests-procedures/covid-19-diagnostic-test/about/pac-20488900)

4. You are tasked to prepare the following components for a one-step RT-qPCR
procedure. Calculate the volume of the components (a-f) in a 20 uL total reaction volume
for 1 RNA sample (1X).
Components Initial/Stock Final Volume
Concentration Concentration (μL)
1X
1 Extracted RNA 5 uL

2 Combined 6.7 uM 500 nM 1.5 uL

Primer/Probe Mix primers/1.7 uM (forward and
probe reverse
primers)/125
 nM probe
3 dNTPs 10 mM 400 nM a

4 Buffer 5X 1X b

5 DTT 100 mM 10 mM c

6 Taq DNA polymerase 0.4 mg/mL 10 ng/uL d

7 Reverse transcriptase 0.02 mg/mL 1 ng/uL e

Nuclease-free water* ------ ------ f

*Adjust volume to 20 uL by adding nuclease-free water

SOLUTIONS AND ANSWERS: