BIOL 2P02

The identification of SARS-CoV-2 by the utilization of surface sampling, RNA isolation, and

reverse transcription quantitative polymerase chain reaction (RT-qPCR)

Submitted by:

Shaini Ayodya Thelasingha Mudiyanselage

Lab Partners:

Jay Parikh

Phimdao Sathienthirakul

Min Kai Yu

Lab TA: Anel Turgambayeva

LAB #6

October 11, 2023

The data was collected from the door handle to a men’s washroom in Cairns’ bulding, Brock

university. The RNA data collected from the Nanodrop states that the concentration is 2.6 ng/µl and

the ratio is 1.33. Because the ratio was not in between ~1.8 to 2.0, the quality of the RNA sample is

considered “not pure” (Dara et al., 2022).

From the PCR data collected, the cq value is at the 27th cycle (Figure 1). The exponential phase of

the RNA goes from 27Th cycle to the 31st Cycle. The Threshold line is at 230 Relative Fluorescence

Units (RFU). From all the samples, no signals were detected other than from the Positive controls of

N protein and RNaseP (RP). The N protein detected on the positive control from FAM fluorescence

is 25.64664 and the RP detected on the positive control from HEX fluorescence is 27.5968.
Discussion:

Molecular diagnostics has emerged as a leading approach in the global efforts to combat the

COVID-19 pandemic. Specifically, the utilization of reverse transcriptase-polymerase chain

reaction (RT-PCR) and its quantitative variation (qRT-PCR) has been widely regarded as the

benchmark method for diagnosing COVID-19 (Gupta et al., 2021). In the majority of persons

experiencing symptomatic COVID-19 infection, the presence of viral RNA in the nasopharyngeal

swab, as quantified by the cycle threshold (Ct), becomes detectable as early as the first day of

symptom manifestation and reaches its highest level during the initial week of symptom onset. The

Ct, or cycle threshold, refers to the number of replication cycles needed for a fluorescent signal to

be generated. A lower Ct value indicates a larger quantity of viral RNA (Nettleton, 2021). The data

collected was considered “not pure” because of the concentration being 1.33. Based on the data

provided, there was no detection of the SARS-CoV-2 in the CPR test that was done.

On the results obtained, the data do not seem odd. Just the purity of the RNA is low but

because there are not many Covid-19 cases present in Niagara region at this present time, the results

that were detected could be true. One way we could get a better RNA extract in future would be to

do the procedure a little bit faster because the master mix needs to be in the Ice bin soon.

The utilization of fluorescent reporter molecules in the real-time reverse transcription

polymerase chain reaction (RT-PCR) enables the monitoring of amplification product generation

during each cycle of the PCR reaction (Bustin et al., 2005). For the Reverse Transcriptase Real

Time Polymerase Chain Reaction (RT-qPCR), denaturation at 95°C, annealing at 60°C and

extension at 72°C in needed. It is not possible to amplify the nucleotide sequence present in mRNA

instead of genomic DNA because SARS-CoV-2 is an RNA based Virus.

In the amplification plot, there is an exponential phase and a non exponential phase. One can

determine how many cycles that is by looking at the C q value. The threshold line refers to the level
at which a reaction attains a fluorescence intensity that surpasses the background levels, hence

enabling its detection.

Sample Ct GAPDH Ct ΔCt ΔΔCt Fold

difference

Control 30.49 23.63 6.86 0 0

Drug 1 27.03 22.66 4.37 -2.49 0.1780

Drug 2 26.25 24.60 1.65 -5.24 0.02702

Drug 3 35.83 23.01 12.82 5.96 62.25

The observed fold differences in gene expression resulting from various pharmacological

treatments indicate notable increases or decreases. It is evident that the administration of medication

1 and drug 2 results in a reduction in the fold change and therefore, the expression of the gene when

compared to the control condition. Conversely, in the context of drug 3, a substantial elevation in

the fold change of gene expression is seen when compared to the control condition.
 Reference:

Bustin SA, Benes V, Nolan T, Pfaffl MW. Quantitative real-time RT-PCR--a perspective. J

Mol Endocrinol. 2005 Jun;34(3):597-601. doi: 10.1677/jme.1.01755. PMID: 15956331.

Chaimayo C, Kaewnaphan B, Tanlieng N, Athipanyasilp N, Sirijatuphat R, Chayakulkeeree

M, Angkasekwinai N, Sutthent R, Puangpunngam N, Tharmviboonsri T, Pongraweewan O,

Chuthapisith S, Sirivatanauksorn Y, Kantakamalakul W, Horthongkham N. Rapid SARS-CoV-2

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Figure 1. Real time PCR graph

Table 1. qPCR data

Starting
Quantity
Well Fluor Target Content Sample Cq (SQ)
N
B02 FAM Protein Unkn Elevator NaN NaN
N
B03 FAM Protein Unkn Stairwell Handl NaN NaN
N Women's Wash
B04 FAM Protein Unkn Ha NaN NaN
N Men's Wash
B05 FAM Protein Unkn Hand NaN NaN
N
B06 FAM Protein Unkn Railing NaN NaN
N Handwash
B07 FAM Protein Unkn Statio NaN NaN
N
B08 FAM Protein Unkn Cheek Swab NaN NaN
N
C02 FAM Protein NTC NaN NaN
 N
C03 FAM Protein Pos Ctrl 25.64664 NaN
B02 HEX RP Unkn Elevator NaN NaN
B03 HEX RP Unkn Stairwell Handl NaN NaN
Women's Wash
B04 HEX RP Unkn Ha NaN NaN
Men's Wash
B05 HEX RP Unkn Hand NaN NaN
B06 HEX RP Unkn Railing NaN NaN
Handwash
B07 HEX RP Unkn Statio NaN NaN
B08 HEX RP Unkn Cheek Swab NaN NaN
C02 HEX RP NTC NaN NaN
C03 HEX RP Pos Ctrl 27.5968 NaN