Volume 75, No.1: 2023 Siriraj Medical Journal

https://he02.tci-thaijo.org/index.php/sirirajmedj/index

Original Article

SMJ

Nattamon Niyomdecha, Ph.D., Sirinart Chomean, Ph.D., Chollanot Kaset, Ph.D.

Department of Medical Technology, Faculty of Allied Health Sciences, ammasat University, Rangsit Campus, Pathumthani, ailand.

SARS-CoV-2 Detection on Articially Contaminated

Surfaces by Rapid Antigen Test

ABSTRACT

Objective: Evaluation of an antigen-based rapid test for detection of severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2) on articially contaminated objects in comparison with a real-time reverse transcription-polymerase

chain reaction (RT-qPCR) standard method.

Materials and Methods: Articial surface contamination with inactivated SARS-CoV-2 was tested on ten dierent

objects comprising fruits and common materials. ree contamination levels with virus titers of 10

, 10

, and 10

pfu/100 µl were studied. Each object was spiked with 200 µl of virus suspension, samples were then collected by

swabbing and evaluated by rapid antigen test and RT-qPCR. Additionally, 3- and 5-day contamination with SARS-

CoV-2 at 10

pfu/100 µl was tested for some materials.

Results: e detection rate obtained by the rapid antigen test with 10

, 10

, and 10

pfu/100 µl of SARS-CoV-2 was

10%, 90%, and 90%, respectively for the tested objects. RT-qPCR showed a detection rate of 100% at all virus titers.

Furthermore, both rapid antigen test and RT-qPCR were able to detect the 3- and 5-day extended contamination

with SARS-CoV-2.

Conclusion: e collected data suggests that the evaluated rapid antigen test is suitable for detection of SARS-CoV-2

adhered to non-human samples as a screening method. is simple method can reduce costs and turnaround time

when compared to a standard molecular assay. It may be applied to enhance safety policies for COVID-19 prevention

in public health and international export-businesses.

Keywords: SARS-CoV-2; COVID-19; Rapid antigen test; RT-qPCR, screening method; surface contamination

(Siriraj Med J 2023; 75: 7-12)

Corresponding author: Nattamon Niyomdecha

E-mail: nattamon@tu.ac.th

Received 01 September 2022 Revised 21 September 2022 Accepted 23 September 2022

ORCID ID:http://orcid.org/0000-0002-5364-6716

http://dx.doi.org/10.33192/Smj.2022.2

All material is licensed under terms of

the Creative Commons Attribution 4.0

International (CC-BY-NC-ND 4.0)

license unless otherwise stated.

INTRODUCTION

Severe acute respiratory syndrome coronavirus 2

(SARS-CoV-2) causes the pandemic coronavirus disease

2019 (COVID-19). Transmission of infectious SARS-

CoV-2 to the human respiratory tract occurs through two

major pathways: by aerosols/droplets in direct person-

to-person contact and via exposure to contaminated

fomites in indirect contact. Viable SARS-CoV-2 has

been shown to survive on dierent surfaces for days or

weeks depending on temperature, relative humidity, and

light.

High safety standards are a must in the food industry,

including in food processing and distribution to maintain

consumer trust and condence in its products. However,

infected food workers, either unaware of hygiene guidelines

or not following them, might contaminate food during

processing and packaging by touching it with contaminated

hands or via infectious droplets released when talking,

coughing, or sneezing.

SARS-CoV-2 contamination

of food products and packaging materials can lead to

serious economic loss in food export businesses. For

example, China, known for its strict COVID-19 policy,

Volume 75, No.1: 2023 Siriraj Medical Journal

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temporarily banned durian from ailand due to several

positive SARS-CoV-2 detection results during random

testing.

Although the contact with SARS-CoV-2 adhered

on food, including fruits and vegetables, or food packaging

materials is highly unlikely to cause COVID-19, such

contaminations must always be tracked, particularly in

the actual context where the virus is spreading in the

countries.

Real-time reverse transcription polymerase chain

reaction (RT-qPCR) is recognized as the gold standard

method for the detection of SARS-CoV-2 in clinical

and non-clinical samples. However, it is limited by a

long turnaround time due to nucleic acid extraction

and amplication and requires trained sta, expensive

instruments, and a laboratory setting with adequate

biosafety. ese resources are not always available in all

countries and in this case a rapid antigen test might be

an alternative to RT-qPCR. While it is less sensitive, it

is faster, easier to perform, more aordable and allows

for decentralized testing at eld areas.

At the present time, data on the use of rapid antigen

tests to detect SARS-CoV-2 in food or environmental

samples are limited. us, this study aimed to evaluate the

performance of an antigen-based rapid test for detection

of SARS-CoV-2 on articially surface-contaminated

objects in comparison with a RT-qPCR standard method.

MATERIALS AND METHODS

Inactivated SARS-CoV-2 virus preparation

An inactivated clinical isolate of SARS-CoV-2/01/

human/Jan2020/Thailand was used in this study. It

represented the original Wuhan strain isolated from

a confirmed COVID-19 patient at Bamrasnaradura

Infectious Diseases Institute, Nonthaburi, ailand.

e inactivated virus was prepared by two methods,

heating and UV-C radiation. Stock SARS-CoV-2 virus

of 10

pfu/ml was divided into two sets for incubation

at 65°C, 15 min, and for exposure by UV-C for 15 min.

Subsequently, the virus was inoculated onto Vero E6

cells to conrm the complete inactivation of the virus

by absence of cytopathic eects (CPE).

All processes involving inactivated SARS-CoV-2

were performed under Enhanced BSL-2 (BSL-2+) in

accordance with the biosafety guidelines. e project

was approved by the ammasat University Institutional

Biosafety Committee (101/2564).

Articial-surface contamination and sample collection

Serial dilutions of 10

, 10

, and 10

pfu/100 µl were

prepared from the stocks of heat- and UV-C-inactivated

SARS-CoV-2. Samples of pooled inactivated virus at

each dilution were prepared by combining 100 µl each of

heat- and UV-C-inactivated SARS-CoV-2. Ten dierent

objects comprising common fruits and packaging materials

were selected for analysis. ey were durian, rambutan,

orange, apple, leather, parcel box, fruit foam net, foam

box, foil, and plastic.

Inoculation and swab processes were performed

by dierent persons. Pooled inactivated virus of each

dilution was randomly spiked, by making tiny drops

with pipette like droplets from sneezing, onto the entire

surface of each object and the objects were then completely

dried at room temperature. e objects were collected

by randomly swabbing without knowledge of previous

inoculation site at an area of 100 to 225 cm

or entire

area for smaller ones at day 0, 3 and 5. Two swabs were

used for SARS-CoV-2 detection by rapid antigen test

and RT-qPCR.

SARS-CoV-2 testing

Nucleocapsid (NP) protein antigen of SARS-CoV-2

was detected by a Rapid Surface Ag 2019-nCov Kit

(Prognosis Biotech, Larissa, Greece). Briey, the collected

swab was placed in extraction buer for 30 seconds and

was then discarded. Aerwards, a test strip was immersed

into the extraction buer for 10 min. Detection of SARS-

CoV-2 resulted in visible colored bands at both Test

(T) and Control (C) lines. As shown in the test manual,

cross-reactivity with 4 dierent human coronavirus

strains is not found, and the limit of detection (LOD) is

2.5 ng/ml of NP or 5.75 x 10

TCID

/ml of inactivated

SARS-CoV-2.

Collected swabs for RT-qPCR assay were kept in

HiViral

transport medium (HiViral

Transport Kit,

HiMedia, Mumbai, India). Swabs were vortexed and

200 µl of HiViral

transport medium was used to extract

RNA by using a PureLink viral RNA/DNA mini kit

(Cat no. 12280050, Invitrogen, USA) according to the

manufacturer’s instructions. e concentration of the

puried RNA was measured as ng/µl and the RNA was

kept at −80°C before RT-qPCR detection. Following

the manufacturer’s instructions and interpretations,

SARS-CoV-2 RNA targeting ORF1ab, N, and E genes

was detected by an ANDiS FAST SARS-CoV-2 RT-qPCR

Detection Kit (Cat no. 3103010069, 3DMed, Germany).

Positive (SARS-CoV-2) and negative (human

coronavirus strain OC43) controls were used to validate

results in all experiments.

Statistical analysis

Descriptive analysis as mean, standard deviation

(SD), detection rate (%) was performed and compared

between rapid antigen test and RT-qPCR at each viral

dilution.

Niyomdecha et al.

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Original Article

SMJ

RESULTS

Detection of SARS-CoV-2 by rapid antigen test

and RT-qPCR on artificially contaminated objects

was compared and the results obtained on the day of

inoculation and sample collection (day 0) are shown in

Table 1 and Fig 1. RT-qPCR, the gold standard method,

had a higher sensitivity than the rapid antigen test and

detected SARS-CoV-2 contamination on all objects at

all virus dilutions. e detection rate obtained with the

rapid antigen test was 10%, 90% and 90% at 10

, 10

and 10

pfu/100 µl, respectively (Fig 1). e sensitivity

of the rapid test was poor at the lowest virus titer but

was much improved at 10

and 10

pfu/100 µl. Likewise,

the intensity of the detected T-band seemed to depend

on the virus titer (Fig 2). However, we observed that

the type of material aected the detection. Detection of

SARS-CoV-2 contamination was most dicult for both

methods on the parcel box made from paper. Indeed,

even contamination with virus titer at 10

and 10

pfu/

100 µl showed negative results when detected by the

rapid test. Although it could be detected by RT-qPCR,

the Ct values of all target genes were shied over ten

cycles (Table 1). Additionally, plastic was the only object

out of the ten spiked objects that could be detected by

the rapid test at 10

pfu/100 µl SARS-CoV-2.

Next, we investigated the detection rate aer the

articially contaminated objects were le for 3 and 5

days. All objects spiked with 10

pfu/100 µl SARS-CoV-2

could be detected by rapid test and RT-qPCR aer 3 and

5 days (Table 2). e results were consistent with the

same day testing (day 0).

Further comparison of RT-qPCR and rapid antigen

test showed the latter to have a limit for detection of

SARS-CoV-2 NP when the Ct values (mean±SD) of

RT-qPCR targeting the ORF1ab, N, and E genes were

in the range of 30.77±3.74, 27.02± 3.64, 26.54±9.72,

respectively (Table 1).

DISCUSSION

is study used pooled heat and UV-C inactivated

SARS-CoV-2 to contaminate ten dierent materials. Heat-

inactivation at 65°C for 15 min will denature viral proteins

but not the genomic RNA, while UV-C-inactivation for

15 min has a deleterious eect on the RNA but not on

the viral structure.

us, the pooled inactivated SARS-

CoV-2 used in this study allowed parallel application

of the two detection methods, i.e., antigen-based rapid

test and nucleic acid-based RT-qPCR and minimized

the risk of false negative results.

e used rapid chromatographic immunoassay

intended for qualitative detection had a lower sensitivity

in SARS-CoV-2 detection in comparison to the gold

standard method RT-qPCR. Our data showed that the

limit of detection of the rapid antigen test was at 10

pfu/100 µl. At this amount of virus RT-qPCR showed

average Ct values for ORF1ab, N, and E genes, across the

analyzed samples in the range of 30.77±3.74, 27.02±3.64,

26.54±9.72, respectively.

However, the results of the rapid test showed that

detection sensitivity depended on the kind of investigated

material. SARS-CoV-2 NP could be still detected at 10

pfu/100 µl on plastic, whereas it could not be detected

at a titer as high as 10

pfu/100 µl on other materials like

parcel box. Interestingly, Ct values from SARS-CoV-2

detection by RT-qPCR showed the highest value at all

virus titers on parcel box. Previous research supports

these ndings.

8-9

Most of the enveloped viruses like

SARS-CoV-1 or inuenza virus were found to survive

and persist in stable form longer on plastic and stainless

steel (1–7 days) than on paper and tissue (3–8 h).

9-11

SARS-CoV-2 was found to be inactivated much faster

on paper than on plastic. No virus could be detected

aer 3 hours of being inoculated on paper.

8,10

Corpet

hypothesized that dryness would inactivate SARS-CoV-2

like found on water absorbent porous materials.

Since

an enveloped virus has a lipid bilayer membrane that

needs water on both sides to maintain an intact structure

dryness might lead to oxidation of lipids and Maillard

reactions of proteins.

While smooth and waterproof

materials would protect the virus by keeping the moisture

from micro-droplets of water on the surface.

is would

explain the stability of SARS-CoV-2 on non-absorbent

materials, including durian, leather, and plastic on which

it could be detected aer many days by both, rapid test

and RT-qPCR.

Taken together, our pilot study on artificially

contaminated objects suggests that the used rapid antigen

test would be a valuable method for screening of dierent

materials. In comparison to RT-qPCR it is easier to

perform, would cost less, save time, and is suitable for

a large number of samples. Its application may enhance

safety policies in public health and international export-

businesses. However, the limitations in this study were

using only articial samples under controlled conditions

and no testing with control group of inoculation with non-

infected uid on samples that might develop interpretation

bias on an antigen-based rapid test. us, these concerns

should be considered for future study. Real-world samples

should be done with and always in comparison with a

gold standard RT-qPCR assay.

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TABLE 1. Comparison of SARS-CoV-2 detection results on articially contaminated objects by rapid antigen test and

RT-qPCR on same day testing (day 0).

Cycle threshold (Ct) value (Interpret result) of RT-qPCR detection

Rapid

Samples

ORF1ab N gene E gene

Internal Conclusion antigen

control result

test result

A. Virus titer at 10

pfu/100 µl

SARS-CoV-2

11.94 (+) 12.27 (+) 9.38 (+) 34.03 (+) + +

HCOV-OC43

> 40 (-) > 40 (-) > 40 (-) > 40 (-) - -

Durian 27.1 (+) 23.47 (+) 25.83 (+) > 40 (-) + +

Rambutan 28.84 (+) 26.49 (+) 27 (+) > 40 (-) + Weak +

Orange 26.55 (+) 23.05 (+) 25.12 (+) > 40 (-) + +

Apple 24.14 (+) 21.52 (+) 22.6 (+) > 40 (-) + +

Leather 21.46 (+) 20.27 (+) 20.07 (+) > 40 (-) + +

Parcel box 37.56 (+) 34.2 (+) 35.62 (+) 39.71 (+) + -

Fruit foam net 25.44 (+) 22.8 (+) 24.33 (+) 38.89 (+) + +

Foam box 25.49 (+) 23.11 (+) 24.12 (+) 39.79 (+) + +

Foil 24.95 (+) 21.96 (+) 25.33 (+) > 40 (-) + +

Plastic 23.57 (+) 20.61 (+) 23.12 (+) > 40 (-) + +

Mean±SD (Positive-Ct) 26.51±4.37 23.75±4.07 25.31±4.10 11.84±19.06

B. Virus titer at 10

pfu/100 µl

SARS-CoV-2 16.35 (+) 16.05 (+) 15.16 (+) 37.19 (+) + +

HCOV-OC43 > 40 (-) > 40 (-) > 40 (-) > 40 (-) - -

Durian 31.57 (+) 28.57 (+) 29.93 (+) 38.15 (+) + Weak +

Rambutan 32.63 (+) 30.32 (+) 31.54 (+) > 40 (-) + Weak +

Orange 31.17 (+) 26.45 (+) 29.16 (+) 38.64 (+) + Weak +

Apple 36.06 (+) 28.69 (+) > 40 (-) > 40 (-) + Weak +

Leather 27.36 (+) 22.63 (+) 27.24 (+) 37.43 (+) + Weak +

Parcel box 36.95 (+) 34.45 (+) 35.63 (+) > 40 (-) + -

Fruit foam net 30.13 (+) 26.71 (+) 30.2 (+) 38.19 (+) + Weak +

Foam box 29.46 (+) 25.62 (+) 28.96 (+) 35.99 (+) + Weak +

Foil 26.06 (+) 23.33 (+) 26.48 (+) > 40 (-) + Weak +

Plastic 26.32 (+) 23.42 (+) 26.27 (+) > 40 (-) + +

Mean±SD (Positive-Ct) 30.77±3.74 27.02±3.64 26.54±9.72 18.84±19.87

C. Virus titer at 10

pfu/100 µl

SARS-CoV-2 19.89 (+) 19.30 (+) 18.44 (+) > 40 (-) + +

HCOV-OC43 > 40 (-) > 40 (-) > 40 (-) > 40 (-) - -

Durian 36.99 (+) 32.73 (+) 35.11 (+) 38.32 (+) + -

Rambutan 31.79 (+) 29.71 (+) 31.21 (+) > 40 (-) + -

Orange 34.39 (+) 30.10 (+) 33.49 (+) > 40 (-) + -

Apple 28.46 (+) 25.27 (+) 27.67 (+) > 40 (-) + -

Leather 32.39 (+) 28.17 (+) 32.75 (+) > 40 (-) + -

Parcel box 37.48 (+) 36.47 (+) 36.27 (+) > 40 (-) + -

Fruit foam net 35.49 (+) 29.06 (+) > 40 (-) > 40 (-) + -

Foam box 33.45 (+) 30.03 (+) 33.04 (+) 37.40 (+) + -

Foil 31.97 (+) 28.31 (+) 32.31 (+) 37.58 (+) + -

Plastic 32.01 (+) 28.97 (+) 32.11 (+) > 40 (-) + Weak +

Mean±SD (Positive-Ct) 33.44±2.73 29.88±2.98 29.40±10.58 11.33±18.24

“+” when Ct value ≤ 40, and “-” when Ct value ≥ 40.

Conclusion results were interpreted following the manufacturer’s instruction. In brief, positive when at least 2/3 of SARS-CoV-2 specic

RNA targets were detected without relying on internal control detection.

Positive control from inactivated SARS-CoV-2.

Negative control from inactivated human coronavirus strain OC43 (HCOV-OC43).

Niyomdecha et al.

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Original Article

SMJ

Fig 1. Comparison of detection rate (%) between

rapid antigen test and RT-qPCR.

e relative number of positive results per total

samples at each tested virus titer

is shown as percentage of detection.

Fig 2. Test line intensity of rapid antigen test.

From le to right, 10

, 10

, and 10

pfu/100 µl

SARS-CoV-2 virus titers were

evaluated by rapid antigen tests. e observed

test line intensity depended on the virus titer.

e results were interpreted as positive, weak

positive, and negative, respectively.

TABLE 2. Comparison of SARS-CoV-2 detection results on articially contaminated objects by rapid antigen test

and RT-qPCR aer 3 and 5 days of inoculation.

Cycle threshold (Ct) value (Interpret result) of RT-qPCR detection

Rapid

Samples ORF1ab N gene E gene Internal Conclusion antigen test

control result

result

Day 3-Virus titer at 10

pfu/100 µl

Durian 21.91 (+) 20.88 (+) 21.14 (+) > 40 (-) + +

Leather 30.08 (+) 26.27 (+) 31.09 (+) > 40 (-) + +

Plastic 24.60 (+) 21.11 (+) 25.54 (+) 33.21 (+) + +

Day 5-Virus titer at 10

pfu/100 µl

Durian 24.77 (+) 23.27 (+) 23.73 (+) > 40 (-) + +

Leather 28.97 (+) 24.96 (+) 30.08 (+) > 40 (-) + +

Plastic 23.88 (+) 20.53 (+) 24.84 (+) 39.01 (+) + +

“+” when Ct value ≤ 40, and “-” when Ct value ≥ 40.

Conclusion results were interpreted following the manufacturer’s instruction. In brief, positive when at least 2/3 of SARS-CoV-2 specic

RNA targets were detected without relying on internal control detection.

Volume 75, No.1: 2023 Siriraj Medical Journal

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CONCLUSION

is study suggests the rapid antigen test as a rst

screening assay to identify SARS-CoV-2 contamination

on various material types. It would reduce the demand

for the expensive and time-consuming RT-qPCR assay

in non-clinical samples.

ACKNOWLEDGEMENTS

e authors thank Professor Dr. Prasert Auewarakul

and Miss Chompunuch Boonarkart from the Department

of Microbiology, Faculty of Medicine Siriraj Hospital,

Mahidol University, who kindly provided the inactivated

SARS-CoV-2 virus for the experiments.

Conict of interest statement: e authors do not have

any conict of interest to declare.

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