Development of a new field-deployable RT-qPCR workflow for COVID- 19 detection

Background: Outbreaks of coronavirus disease 2019 (COVID-19) have been recorded in different countries across the globe. The virus is highly contagious, hence early detection, isolation, and quarantine of infected patients will play an important role in containing the viral spread. Diagnosis in a mobile lab can aid to find infected patients in time. Methods: Here, we develop a field-deployable diagnostic workflow that can reliably detect COVID-19. Instruments used in this workflow can easily fit in a mobile cabin hospital and also be installed in the community. Different steps from sample inactivation to detection were optimized to find the fastest steps and portable instruments in the detection of COVID-19. Each step was compared to that of the normal laboratory diagnosis setup. Results: From the results, our proposed workflow (80 min) was two times faster compared to that of the normal laboratory workflow (183 min) and a maximum of 32 samples could be detected at each run. Additionally, we showed that using 1% Rewocid WK-30 could inactivate the novel coronavirus directly without affecting the overall detection results. Comparison of our workflow using an in-house assay to that of a commercially acquired assay produced highly reliable results. From the 250 hospital samples tested, there was a high concordance 247/250 (98.8%) between the two assays. The in-house assay sensitivity and specificity were 116/116 (100%) and 131/134 (97.8%) compared to that of the commercial assay. Conclusion: Based on these results, we believe that our workflow is fast, reliable, adaptable and most importantly, field-deployable.


Background
Coronavirus disease 2019  is caused by the serve acute respiratory syndrome coronavirus 2 . In December 2019, the first cases of human infection with the COVID-19 were identified in Wuhan, China [1][2][3]. Most of the cases were linked to a local seafood market in Wuhan which is believed to be the source of the SARS-CoV-2 virus outbreak [2][3][4][5]. Since its identification, scientists have characterized the virus [3,6,7] and reported clinical symptoms associated with COVID-19 [2,3,8,9]. Controlling the virus has been a top priority in areas affected both in China and across the globe. As of April 8 th , 2020, the World Health Organization daily situation report on the novel coronavirus disease recorded a total of 1,403,622 (87,279 new) confirmed cases and 85,495 (8,902 new) total deaths resulting from COVID-19 globally [10]. Cases of the epidemic outbreak have also been reported in nearly all countries across the globe [10,11]. With the daily rise in number of deaths, suspected, and confirmed cases across the globe, methods towards rapid diagnosis and detection of the virus disease are of key importance in fighting the pandemic.
From the beginning of the pandemic, different diagnostic approaches have been used in an attempt to tame and understand the spread of infections resulting from SARS-CoV-2 [1,3,8,[12][13][14][15]. Among these approaches includes the development of specific viral nucleic acid assays that use real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to diagnose new cases of COVID-19 [1,3,[16][17][18]. RT-qPCR applications in diagnostics however are not a new concept. Since its advent, RT-qPCR has become a well-established technique for the diagnosis of various microorganisms owing to its many advantages including rapidity, sensitivity, ease of application, and scalability [19]. Many companies have come up with different qPCR instruments that are labbased and also field deployable. In the past coronavirus outbreaks [20], RT-qPCR has also been used extensively to help in the identification of different cases and help in the diagnosis pipeline of the virus outbreaks [21][22][23]. However, in these outbreaks including the ongoing COVID-19, no research has tried to describe an optimized system and workflow suitable for point of care testing, field deployment, and one that can easily fit in a mobile cabin hospital to detect and diagnose the viral disease. The development of efficient and quicker methods for the detection of viral nucleic acids is said to play an important role in fighting the ongoing coronavirus outbreak [12]. Additionally, the development of a workflow that can fit in a mobile hospital can guarantee comprehensive healthcare to anybody, anytime and anywhere [24][25][26][27].
In light of these limitations, we sought to develop a practical workflow that is fast and can be easily adapted in different facilities. The workflow also includes a field-able approach for the diagnosis in the community and mobile cabin hospitals set-up. The workflow was compared to the standard workflow using the conventional RT-qPCR diagnosis of the ongoing coronavirus outbreak. The bacterial strains were cultured in Luria broth for 24 hours before being transferred to different falcon tubes. The original colony forming units of each bacteria was then determined before adding WK-30. After colony forming units determination, each bacteria was mixed with WK-30 to final concentrations of 0.5% and 1% WK-30. After mixing each bacteria with WK-30, the tubes were left to sit for 5, 10, 20, and 30 minutes at room temperature. 10 µL was subsequently collected at each time point and plated onto solid LB media plates. All the plates were then incubated at 37 ℃ for 24 hours. Post incubation, the plates were checked to determine the presence of any colonies. RNA extraction. Four methods of RNA extraction were evaluated to get the fastest method of extraction. The goal was to find a portable extraction method that can easily be used in the field setup. The four methods included using the Qiagen Viral RNA extraction kit (spin protocol and vacuum protocol), QIAxtractror Automated extraction, and Purifier™ Modesty automated RNA extraction. Each time the extraction methods were used, the manufacturers' recommendations were followed. To determine if WK-30 had an effect in the extraction process, seven hospital samples that had previously tested positive by RT-qPCR were treated with WK-30 to a final concentration of 1% and the RNA extracted. The same samples were also diluted using RNase-free water as a control to a final concentration of 1% for testing the normal extraction procedure. The resultant cycle threshold (Ct) values of the two tests were determined using the portable MyGo Pro real-time PCR instrument (IT-IS Life Science Ltd., Mahon, Cork, Ireland).

Sample detection
Different approaches were also considered for sample detection.

RT-qPCR quantification instruments.
To find a suitable qPCR instrument that is field-deployable and suitable for POC testing, we used a portable MyGo Pro Real-time PCR instrument readily available in our laboratory to optimize our workflow. The machine detection time was compared to that of the routinely used CFX-96 Touch™ Real-time PCR (CFX-96, Bio-Rad Laboratories, Hercules, CA, USA) instrument suitable for laboratory benchtop diagnosis. RT-qPCR assay composition and reagent preparation. Two assays were used for time comparison and workflow tests. The two assays comprised of a commercially acquired kit for RT-qPCR and an in-house assay developed using locally available reagents to perform RT-qPCR. The commercially acquired kit was labelled New Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescence PCR method) produced by the Zhongshan Daan Gene Company, Guanzhou, China. The kit is a one-step RT-qPCR kit designed to target the open reading frame 1ab and nucleocapsid protein genes of the novel coronavirus sequence. The nucleocapsid protein gene was labelled with a FAM reporter dye while the open reading frame 1ab gene was labelled with a VIC reported dye. The kit also had an endogenous internal control dye labelled Cy5. The 25 µL reaction mixture consisted of 20 µL of freshly prepared mix and a 5 µL RNA template. The one-step RT-qPCR protocol was run using the Bio-Rad's CFX-96 instrument under the following conditions: 50 ℃ for 15 min, 95 ℃ for 15 min, followed by 45 cycles of 95 ℃ for 15 sec and reading at 55 ℃ for 45 sec respectively.
The in-house assay was a one-step RT-qPCR assay composed of two sets of primers and probes. A set of primers was designed to target the receptor binding domain of the novel corona virus sequence while the other set was designed to detect an endogenous internal control. The primers and probe targeting the receptor binding domain sequence include: forward primer CTCAAGTGTCTGTGGATCACG; reverse primer CCTGTGCCTGTTAAACCATTG; and probe 5'-FAM-ACAGCATCAGTAGTGTCAGCAATGTCTC-BHQ1-3'. The internal control primers and probe sequence: forward primer AGATTTGGACCTGCGAGCG; reverse primer GAGCGGCTGTCTCCACAAGT; and probe 5'-FAM-TTCTGACCTGAAGGCTCTGCGCG-BHQ1-3'. Before addition, all the primer and probe concentrations were adjusted to 10 µM. The composition of the inhouse assay included 0.8 µL of each primer, 1 µL of each probe, 2 µL 5× PrimeScript RT Master, 1.6 µL dNTP, 0.5 µL Taq polymerase, 3.7 µL RNase free water, and 5 µL RNA template to a final volume of 20 µL. All the in-house one-step RT-qPCR reaction mixtures were then amplified using the MyGo Pro Real-time PCR instrument under the following conditions: 50 ℃ for 10 min, 95 ℃ for 10 sec, followed by 40 cycles of 95 ℃ for 5 sec and reading at 60 ℃ for 30 sec respectively.

Assay specificity and performance
For specificity testing of the in-house assay, an in-silico test using the basic local alignment search tool available online from National Center for Biotechnology Information was used. To test whether there will be a remarkable difference between the in-house assay and the commercial kit, 250 hospital samples were extracted and used for detection. The commercially available kit was used as a reference kit for validating the workflow of the in-house assay. All the commercial kit tests were done using the Bio-Rad's CFX-96 real-time PCR instrument while all the in-house assay tests were done using the portable MyGo Pro real-time PCR instrument.

Data analysis
All RT-qPCR Ct values were generated using the specific PCR instruments and read on the accompanying software. Analytical comparisons for clinical samples were performed online using the MedCalc statistical software (https://www.medcalc.org/calc/diagnostic_test.php). Bacterial colony forming units was determined by counting visible colonies on the respective culture plates.

Sample inactivation
Two approaches were used to determine the fastest sample inactivation procedure that may be suitable for field applications and POC testing. The normal routine sample inactivation procedure which involved heating the swab samples suspended in VTM tubes, took up to 40 minutes to inactivate the sample. The inactivation process also needed a heating source and cooling source for inactivation. Hence, this was not readily suitable for field deployment. Therefore, a direct approach using the biocidal surfactant WK-30 was explored. The surfactants' ability to kill different bacteria and SARS-CoV-2 was established.
The virucidal effect of 1% WK-30 against SARS-CoV-2 was tested in vitro using Vero E6 cells as shown in Figure 1. From the results, it was clear that 1% WK-30 had no effect on Vero E6 cells even after 48 hours of incubation as shown in Figure 1A. However, incubating the cells with 1.6 × 10 6 PFU/mL SARS-CoV-2 proved to be lethal. All the cells were dead after 48 hours incubation with clear cytopathic effects as shown in Figure 1B. Most of the cells were alive with no clear cytopathic effects after 48 hours incubation with both 1% WK-30 and 1.6 × 10 6 PFU/ml SARS-CoV-2 as shown in Figure 1C. Additionally, the Ct values were quantified at time 0 hours and 48 hours of incubation. The Ct greatly dropped from Ct 19.51 at time 0 hours to Ct 34.46 at time 48 hours. From these results, 1% WK-30 showed some level of protection to the cells. Also, the interaction of 1% WK-30 with SARS-CoV-2 for 1 min at room temperature can kill all of the SARS-CoV-2.
For the bactericidal effects, five bacteria were used. From the results (Table 1), all the colonies were reduced with increased exposure time to WK-30. E. coli, E. faecalis, and A. bauminii were completely inhibited by 0.5% WK-30 at all-time points. However, S. aureus and P. aeruginosa showed some level of resistance to 0.5% WK-30. S. aureus was the most resistant bacteria because even after 30 min of exposure, some colonies still grew post incubation. Of note, 1% WK-30 however was able to kill all the bacteria as no colonies were observed post incubation. 1% WK-30 had a maximum bactericidal effect to all the bacterial strains. From these results, 1% WK-30 was both bactericidal and virucidal hence suitable for use in the field as it requires no extra sample treatments and reduces the time needed for inactivation and sample transportation.

RNA extraction
Different methods were compared to find the fastest, portable and easy-to-use RNA extraction mechanism in a field setup. Factors including time, the number of sample handling steps, and any additional instrument needed were also determined as shown in Table 2. The comparison revealed that using the Purifier™ Modesty instrument was faster and needed fewer sample handling steps compared to other methods. The instrument was easily portable and fitted with an ultraviolet lamp that can help in decontamination and ensuring a clean environment. Due to these advantages, the instrument was found suitable for field deployment. Since 1% WK-30 was found to be virucidal, we tested if the solution would have an effect in the extraction process when using the Purifier™ Modesty instrument. The result of addition of 1% WK-30 to the virus was tested in comparison of using 1% RNase free water in the same sample to replace WK-30. The results as shown in Table 3 and Figure 2 proved that 1% WK-30 had no remarkable effect in the extraction process and the results were highly comparable.

Detection time
To find the shortest time that could detect samples faster and reliably, we modified our protocol to run for a total time of 47 min 17 sec using the MyGo Pro instrument. This time was 64 min 2 sec shorter compared to that of the commercially acquired New Coronavirus 2019-nCoV Nucleic Acid Detection Kit which runs for 1 hour 51 min 37 sec according to the manufactures' procedure.     .807 From the results of the seven already tested positive samples, it was clear that 1%WK-30 had no effect in the extraction process and overall result.

Figure 2
Representative RT-qPCR results after extraction with 1% WK-30 (black curve) and 1% RNase free water (purple). From the results, 1% WK-30 had no effect on the extraction process and overall result.

Assay sensitivity and specificity
An in-silico probe and primer test using the basic local alignment search tool available online from NCBI resulted in 100% specificity of the in-house primers and probes in the detection of the novel coronavirus. The performance of the whole workflow including reagents was tested using 250 patient samples. The result of this test is summarized in Table 4. From the results, the novel coronavirus was detected in 116/250 (46.4%) hospital samples using the commercial kit. The in-house assay was a little more sensitive than the commercial assay as 119/250 (47.6%) hospital samples tested positive for the novel coronavirus using the in-house assay. Of note, the concordance between the two assays was high 247/250 (98.8%). However, three samples that tested negative by the commercial assay tested positive by the in-house assay resulting in a specificity of 97.76%. All the samples that tested positive by the commercial assay also tested positive by the in-house assay 116/166 (100% sensitivity) indicating that the inhouse assay was greatly comparable to the commercial assay with improved sensitivity.

Discussion
Since the discovery of the first case of COVID-19 in Wuhan, China [1][2][3], scientists have been working hard to diagnose the disease. Efforts geared towards the diagnosis of COVID-19 have been patient centered and applied in hospital set-ups. However, with the rising numbers of cases across the globe [11,28,29], more efforts should be explored especially in community diagnostics. There may be a number of cases being missed from patients in the community hence field work tests may prove beneficial in the treatment and diagnosis of COVID-19. A field based diagnostic approach has not yet been explored. In this article, we used locally available instruments and reagents to develop a rapid RT-qPCR diagnostic workflow that is not only suitable for POC but also field deployable. In the field setup, a rapid direct sample inactivation method that needs no additional instruments could be of great importance [27]. To develop such a method, we explored the biocidal effects of Rewocid WK 30 [30]. We tested if the surfactant had the potential of killing both bacteria and viruses. In both cases, 1% WK-30 was shown to be virucidal to SARS-CoV-2 and also bactericidal to different bacteria in vitro. The virucidal effects of 1% WK-30 also had no effect on the extraction process. So far, no literature has cited or tried to explore the biocidal capabilities of Rewocid WK 30. The ability of 1% WK-30 to inactivate and kill the novel coronavirus at room temperature within a minute of exposure makes it a suitable reagent for direct sample inactivation in the field set-up. However, when handling samples, one should also take care to observe biosafety and wear the appropriate personal protective equipment. Once the sample has been inactivated, the sample should proceed directly to extraction of the viral nucleic acid necessary for detection. A fast, portable automated extractor with minimal sample handling steps will be suitable for field extraction experiments. Automation with minimal sample handling steps ensures safety to the worker and also ensures sample integrity is kept intact throughout extraction in the field [27] where there are minimal or no specialized equipment e.g. biosafety cabinets. A portable instrument on the other hand meant that it could fit well in a mobile hospital. Of all the available extraction methods and instruments tested in this workflow, the Purifier™ Modesty instrument was the fastest and most portable. The instrument was also fitted with an ultraviolet light source to ensure the cleanliness of the extraction environment pre and post extraction. Up to 32 samples could be extracted in a minimal time of about 21 minutes. This was highly scalable as 32 patients could be served in a single run of extraction.
To make the workflow complete, we coupled the workflow with a fast detection protocol using a portable MyGo Pro real-time PCR instrument for detection. The instrument was highly compatible with the Purifier™ Modesty nucleic acid extractor as it could also process a maximum of 32 samples. The fast RT-qPCR protocol ensured samples were detected within 47 min 17 sec. This detection time was faster and results were highly comparable with that of the commercial assay. The MyGo Pro real-time PCR instrument could be run with a USB drive. This meant that multiple instruments could be used and run with only a single computer using multiple USB programmed drives. The multiple options for running the PCR instrument gave room for scaling. Portable instruments are important for onsite diagnosis and commutability [31]. All the instruments used in the optimization of the proposed workflow were highly portable ( Figure 3). This meant that the workflow instruments could easily be fitted and adapted in a mobile hospital before deployment to the community to detect and diagnose COVID-19. The complete workflow from sample inactivation to detection was approximated to be no more than 80 minutes using the field deployable workflow. This workflow was two times faster compared to that of the normal workflow in the laboratory setup (183 min 9 sec). Additionally, after the first detection of approximately 80 minutes, the proceeding samples can be extracted and detected within an hour. As the first samples are running, a new batch of samples may be loaded into the extractor which will run for 21 min, the extra 26 minutes before the RT-qPCR results are read can be used for reaction mix preparation and programming. Once the first batch finishes, the newly extracted samples can be detected immediately. This workflow is a first in exploring the potential of community diagnosis using a field deployable nucleic acid detection system for COVID-19. All the instruments highlighted to be used in the field set-up are highly portable and can fit well in a mobile hospital. The instruments can also be powered by a car battery and be used in remote locations where electricity is not accessible. Additionally, the workflow is flexible and can be modified depending on the available resources in different laboratories and countries. The workflow can also be set up in hospitals, clinics, and public health laboratories that cannot access electricity. We plan to actualize this workflow in our subsequent tests using a mobile hospital to help in the diagnosis of COVID-19. Actualization of this workflow ensures that patients are treated anywhere and at any time [25,31]. This will also help in reducing the large number of patients that visit hospitals. Patient treatment at community level decreases their risks of getting infected when visiting the hospitals. Different researchers and laboratories can use our workflow and modify it if needed to best fit their laboratory.

Conclusion
Portable instruments that can be used in the extraction and detection of nucleic acids already exist. We used locally available instruments and reagents to develop a new field-deployable workflow that can also be used at POC. We found that using Rewocid WK 30 as a biocidal agent could help in the inactivation of SARS-CoV-2 samples directly without the need of any extra equipment. This workflow was two times faster than that of the normal workflow in the laboratory set-up. We believe that the workflow described is easily adaptable and flexible. This workflow will help in the fight against the current SARS-CoV-2 pandemic and other future outbreaks. Lastly, the workflow may be useful in routine sample collection and detection in future fieldwork surveillance studies.