Detection of different variants of SARS-CoV-2 RNA (genome) on inanimate surfaces in high-touch public environmental surfaces

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease started in late 2019 and still continues as a global pandemic, spreading among people around the world. There is limited knowledge about the role of contaminated environmental surfaces, especially high-touch public surfaces, in the transmission of the disease. The objective of the present investigation was detection of different variants (Delta, UK, and Omicron) of SARS-CoV-2 RNA (genome) on inanimate surfaces in high-touch public environmental surfaces in different seasons. Automated teller machines of banks (ATM), point-of-sale (POS) machine, gas station pump nozzles, and escalator handrails of malls were selected as high-touch environmental surfaces in public places. Overall, 75 samples were collected from these places and examined for the presence of SARS-CoV-2 RNA (genome), and 21 samples (28%) were positive. Although the role of fomite transmission of COVID-19 is understood, more studies should be conducted to determine the virus survival rate as well as the required efforts to prevent the spread of SARS-CoV-2 such as frequent cleaning and the use of efficient disinfectants on environmental surfaces, especially high-touch public places. In conclusion, the results address the importance of touching contaminated inanimate objects as well as transmission through environmental surfaces, and they could be used to establish an effective protocol to prevent indirect environmental transmission of SARS-CoV-2, slow down the spread of the virus, and reduce the risk of infection.


Sample collection.
The sampling locations included two large shopping centers, 10 automated teller machines (ATM) of banks, four gas station pump nozzles, and five mobile point-of-sale (POS) machines.A total of 75 samples were taken in three seasons: spring, summer and autumn, which coincided with the arrival of the above-mentioned variants.Sampling was done at three points in time (simultaneously with confirmation of diagnosis of the new variant in the region by sending random samples to the National Reference Laboratory).Due to the small number of large shopping centers and fuel stations in the city, sampling was done from all these centers (census), but in the case of banks and mobile POS machines, sampling was done randomly (simple random sampling) from all parts of the city with more emphasis on crowded places with higher density.Swab sampling was done in the same way as in the study of Faezeh et al. 1 .Briefly, swab was moistened with Viral Transport Medium (VTM).Using a separate sterile collection swab and VTM for each surface, the swab was applied to an area measuring 10 × 10 cm, then the swab was returned to the VTM.After sample collection, they were transported to the reference laboratory in cool boxes (ensure the respect of the cold chain using the thermometer) while employing standard precautions and proper cold chain.In order to control the bias, all the samples were obtained at a certain time of the day (5-7 PM).The staff carrying out the sampling were fully clothed in gloves and face shields.In order to check the relationship between the level of contamination and climatic variables, temperature (°C) and relative humidity (%) were also monitored and recorded during environmental surface sampling using the portable weather station (Kimo).

Viral genome extraction.
The viral RNA was extracted using a Roje Technologies kit (Pishgam, Iran) and was stored in a deep freezer (− 70 °C).Subsequently, a real-time (RT)-PCR test was used to determine the presence of SARS-CoV-2 RNA in the collected environmental surface samples.

Detection of virus infections by RT-PCR. According to the manufacturer's instructions (Pishtaz Teb
Zaman, Iran), 5 μL extracted RNA was added to 15 μL of One-Step qRT-PCR master mix supplemented with a modified RdRp and N gene primer/probe sets.All real-time RT-PCR experiments were carried out by the Applied Biosystems Step One plus RT-PCR System.In the kit that was used, complementary DNA synthesis and PCR were performed through a continuous thermal-cycling program.Thermal-cycling program consists of four steps: Step one: one cycle of reverse transcription at 50 °C for 20 min; step two: one cycle of cDNA initial denaturation at 95 °C for 3 min; step three: 45 cycles consisting of two stages, a denaturation stage at 95 °C for 10 s and annealing, extension, and fluorescence measurement stage at 55 °C for 40 s; step four: one cycle of cooling at 25 °C for 10 s.According to the kit's instructions, a positive reaction is detected by cycle threshold (Cq) values < 40 cycles, and PCR was repeated when the Cq values were between 40 and 45 cycles.In order to ensure that valid results were reported, negative and positive controls were run during each experiment.Moreover, the limit Identification of SARS-CoV-2 variants.Due to the long-term impact of COVID-19 and emergence of more aggressive new variants, the progressive diagnosis of these variants can be used in diagnosis, infection prevention, and control plans.Qualitative TaqMan real-time PCR assay was used to determine the dominant variant of SARS-CoV-2.Geneva kit with a triple target gene was used to confirm Omicron and Delta variants.GA SARS-CoV-2 one-step RT-PCR testing kit covers three target genes specific to SARS-COV-2, N, S, ORF1a genes, and human RNase P gene.The sensitivity and specificity of the detection and differentiation of the two deletions in the S and ORF1a regions of the target kit have been reported as 100%.The limit of detection (LOD) for N gene was 100 copies/mL, it was 50 copies/mL for S gene, and 200 copies/mL of sample for ORF1a gene.Moreover, IPI SARS-CoV2 UK variant screening kit was used for detecting the UK variant by multiple spike protein mutations (deletion 69-70, deletion 144).The diagnostic sensitivity and specificity of this kit are 100%.The experiments performed in the laboratory for detecting SARS-CoV-2 RNA genome in the samples from public environmental surfaces are illustrated in Fig. 1.

Ethical Approval.
The authors would like to thanks Health and Environment research center, Ilam University of Medical Sciences, Ilam.

Results and discussion
A total of 75 (21 positive environmental surface samples) contaminated environmental surfaces in different hightouch public places were examined, and the obtained findings as well as the detected positive/negative cases and sampling sites are presented in Tables 1, 2 and/or on different types of environmental surfaces.In addition, during the site sampling, the meteorological parameters including temperature (°C) and RH (%) were monitored, and the related results are demonstrated in Tables 1, 2, 3 and 4. The results of previous works on contaminated environmental/inanimate surfaces during the COVID-19 pandemic were compared with the results obtained in this work, and the findings are shown in Table 5.
In recent studies, it has been reported that using different molecular procedures, the SARS-CoV-2 RNA can be found in different contaminated environmental surfaces and facilities including hospital and non-hospital settings 1,9,14,27,28 .Although the evidence demonstrated that the highest detection rates of SARS-CoV-2 could be found in healthcare facilities and hospital settings with COVID-19 patients, other high-touch inanimate surfaces in public places can be important sources and transmission routes for COVID-19.This is due to the fact that most infected people are not aware of their disease, especially in the initial stages of the disease, and therefore spread the SARS-CoV-2 in public places 1,9,14,27,28 .Therefore, inanimate environmental surfaces can considered as one of the most important routes for SARS-CoV-2 transmission 17,18,18,19,29 .
Keeping this in mind, a total of 75 inanimate environmental surface samples were collected and examined for detection of the virus genome in three seasons (winters of 2021 and 2022, and summer of 2021), which are the same seasons that officials declared that different variants of SARS-CoV-2 were prevalent.This was carried out in order to survey the contamination of frequently-touched inanimate surfaces in public places.It is declared that in winter of 2021, the UK variant of concern (VOC) 202,012/01 (lineage B.1.1.7),in summer of 2021, the Delta (B.1.617.2) variant, and in winter of 2022, the Omicron variant were the predominant variants of SARS-CoV-2.The above-mentioned samples from inanimate surfaces in public places were taken from different locations including Bank ATMs, POS machines, gas station pump nozzles, and escalator handrails in malls.The laboratory evidence confirmed that viable SARS-CoV-2 RNA could be detected on frequently-touched inanimate environmental surfaces, from 3 h in bioaerosols to 8-72 h on dry surfaces depending on the type of material of inanimate surfaces 8,18,30,31 .However, other studies reported that SARS-CoV-2 can survive up to 28 days on environmental and inanimate surfaces contingent upon characteristics of the environment and surface material.Therefore, this evidence confirmed that the viability of SARS-CoV-2 is higher and longer than other genera of coronaviruses including SARS-CoV-1 and MERS-CoV 6,8,18,32 .
The data related to detection of different variants of SARS-CoV-2 on frequently-touched environmental surfaces of different ATMs in various public places and seasons is demonstrated in Table 1.As it can be seen in Table 1, 10 out of 30 samples were positive and SARS-CoV-2 RNA was detected on different ATMs.The matter of survival of SARS-CoV-2 on different surfaces has been controversial in various studies (from hours to a few days).Riddell et al. 33 showed that SARS-CoV-2 can be potentially infectious for longer than believed 33 .Van Doremalen et al. reported that SARS-CoV-2 stayed infectious in aerosols for hours, and up to days on different surfaces 31 .According to the studies, it has been determined that survival of SARS-CoV-2 in aerosols is about 3 h, and depending on the type of surface material, it can survive from hours to days.Moreover, apart from surface material, conditions such as temperature and humidity are effective on survival and infectiousness 7 .It is worth noting that since the surface material of ATMs is comprised of glass and plastic, it can be concluded that SARS-CoV-2 may survive longer on these types of environmental surfaces.On the other hand, most of the positive samples are related to the winter and summer of 2021, when the UK and Delta variants were dominant.Therefore, it can be concluded that these variants are probably more persistent on glass and plastic and are more resistant against temperature and RH.These results in accordance to Noorimotlagh et al. 18 .
The data related to detection of different variants of SARS-CoV-2 RNA on high-touch environmental surfaces of different POS machines in public places and seasons is demonstrated in Table 2.As it can be seen in Table 2, only one out of 15 environmental surface samples was positive and RNA of SARS-CoV-2 was detected.The one detected positive sample belonged to the winter of 2021 when the UK variant was predominant.It can be assumed that the contamination on this POS machine was low because only operators used it and it was not touched by other people.
Table 3 provides the data related to detection of different variants of SARS-CoV-2 RNA on frequently-touched environmental surfaces of different gas station pump nozzles in various public places and seasons.As it can be seen in Table 3, seven out of 24 surface samples were positive and SARS-CoV-2 RNA was detected in those locations.The positive samples were related to the winter and summer of 2021 when the UK and the Delta variants were predominant.Therefore, it can be concluded that these variants could persist in different meteorological conditions.The Delta variant persists in high temperatures (up to 44 °C).In a similar study, Karami et al. 34 reported that five out of 36 inanimate surface samples (pump nozzle) were positive regarding the presence of SARS-CoV-2.However, they investigated the nozzle surfaces before and after the corona restrictions and after the quarantine, and all the samples were negative 34 .It is worth noting that the current study was performed after the quarantine conditions when different variants of COVID-19 were prevailing.Therefore, gas station pump nozzles could be a potential source of transmission of COVID-19.The results obtained herein are in agreement with other studies 8,10,14,35 .
Escalator handrails in malls are an important environmental surface for transmission of SARS-CoV-2.Therefore, the presence of SARS-CoV-2 on them was investigated as an essential and frequently-touched environmental surface in different public places and seasons, and the results are tabulated in Table 4.As it can be seen in Table 4, three environmental surface samples were positive in terms of the presence of SARS-CoV-2 RNA.The positive samples are associated with the winter of 2021 (when the UK variant was dominant) and the summer of 2021 (when the Delta variant was dominant).Therefore, it can be concluded that these variants might survive in different meteorological conditions (various temperatures and RH).
The comparison of some similar investigations on detection of SARS-CoV-2 RNA on high-touch environmental surfaces of public places is demonstrated in Table 5.As illustrated in Table 5, most of the studies were carried out in healthcare settings and there are limited studies focusing on high-touch environmental surfaces in public places.The present study is one of the few studies focused on the spread of SARS-CoV-2 RNA on inanimate environmental surfaces, which have a great impact on the spread of COVID-19 in the world.As it can be seen in Tables 1, 2, 3 and 4

RT-Polymerase Reaction (PCR)
Omicron O O r ro on n Delta D D public places including bank ATMs, POS machines, gas station pump nozzles, and escalator handrails in malls, in various seasons with different meteorological conditions.The schematic presentation of the environmental surface transmission chain of COVID-19 via public environmental surfaces is presented in Fig. 2. Therefore, the results of this study suggested that preventive measures such as using suitable disinfectants in the desired duration are of utmost importance to prevent and limit the spread of COVID-19.

Conclusion
The present work focused on detection of SARS-CoV-2 RNA (genome) on high-touch public surfaces in different seasons, and was concerned with three variants (Delta, UK, and Omicron).A comprehensive sampling was performed and 75 samples were collected from different high-touch environmental surfaces in public places including bank ATMs, POS machines, gas station pump nozzles, and escalator handrails in malls and they were examined for the presence of SARS-CoV-2 RNA (genome), from which 21 (28%) samples were positive.Although the role of fomite transmission of COVID-19 is understood, more studies are required for determining the survival rate of the virus, and there should be continuous attempts to prevent the spread of SARS-CoV-2 including frequent cleaning and using efficient disinfectants on environmental surfaces, especially high-touch public places.In conclusion, the results of this study address the importance of the possibility of transmission by touching contaminated inanimate and environmental surfaces and they could be used to establish an effective protocol to prevent the indirect environmental transmission of SARS-CoV-2, interrupt the virus spread, and reduce the risk of infection.

Figure 1 .
Figure 1.The illustration of experiments to detect of SARS-CoV-2 RNA genome in public environmental surfaces samples in the laboratory.
, SARS-CoV-2 RNA was detected on different high-touch environmental surfaces of

Figure 2 .
Figure 2. Schematic presentation of the environmental surfaces transmission chain of COVID-19 disease via public environmental surfaces.

Table 2 .
Detection of different variants of SARS-CoV-2 on frequently-touched environmental surfaces of different POS Machine in various public places and seasons.*P1, P2, P3, P4 and P5 are POS Machine 1, 2, 3, 4 and 5, respectively in the different public places.Significance values are [bold].

Table 3 .
Detection of different variants of SARS-CoV-2 on frequently-touched environmental surfaces of different Gas station pump nozzles in various public places and seasons.

Table 4 .
Detection of different variants of SARS-CoV-2 on frequently-touched environmental surfaces of different Escalator handrails of malls in various public places and seasons.* M1 and M2 are Mahestam Mall and Chahardah masoum Mall respectively in the different public places.Significance values are [bold, italics].