Evaluation of a Sample-to-Result POCKIT Central SARS-CoV-2 PCR System

The emergence of COVID-19 has caused unprecedented impacts on global public health and many other aspects. Meanwhile, many types of methods have been developed to detect the causative agent, SARS-CoV-2; this has greatly advanced the technologies in the diagnostic field. Here, we describe the development and validation of a sample-in-result-out POCKIT Central SARS-CoV-2 PCR system for detecting SARS-CoV-2 in comparison with a commercial reference real-time RT-PCR assay (TaqPath COVID-19 Combo Kit). Both assays were specific and did not cross-react with non-SARS-CoV-2 agents. Both assays were able to detect various SARS-CoV-2 strains including some variants. Based on testing serial dilutions of SARS-CoV-2 USA-WA1/2020 isolate, the limit of detection was 0.8 TCID50/mL (1.87 × 103 genomic copies/mL) for POCKIT Central SARS-CoV-2 PCR and 0.16 TCID50/mL (3.75 × 102 genomic copies/mL) for the reference PCR. Subsequently, 183 clinical samples were tested by both assays and the diagnostic sensitivity, specificity, and agreement of the POCKIT Central SARS-CoV-2 PCR were 91.7%, 100%, and 94.0%, respectively, when compared to the reference PCR. The compact sample-to-result POCKIT Central SARS-CoV-2 PCR system is a simplified and efficient point-of-care tool for SARS-CoV-2 detection. In addition, this platform can be readily adapted to detect other human and animal viruses.

Coronaviruses are well known for high mutation and recombination rates which drive their genetic diversification [6,7]. In the period of over three years (late 2019 to early 2023), the SARS-CoV-2 virus evolved while rapidly spreading in the human population, resulting in the emergence of various virus variants with different characteristics compared to the ancestral/original strains [8]. Different nomenclature systems have been proposed to describe genetically diverse SARS-CoV-2 strains, and some most notable classification systems include GISAID (www.gisaid.org), Nextstrain (clade.nextstrain.org), and Pango lineages [9,10]. The original SARS-CoV-2 strains in early outbreaks included two Pango lineages A and B [9]. However, with the rapid evolution of SARS-CoV-2, a few thousand Pango lineages have been described (cov-lineages.org). For easy and efficient communications, from May 2021, WHO began to use the Greek alphabet to label the key SARS-CoV-2 variants such as the Alpha (B. 1 Rapid, sensitive, and specific methods to detect SARS-CoV-2 and identify the infected individuals are critical to better monitor the infection and limit the spread of COVID-19 [11]. Many advances have been made in laboratory testing for SARS-CoV-2 in the past few years. Molecular diagnostic tools (e.g., real-time PCR, droplet-digital PCR [ddPCR], loopmediated isothermal amplification [LAMP] assays, and genome sequencing approaches, etc.), rapid antigen tests, antibody tests, and various methods in other formats (e.g., microfluidic and/or biosensor methods) have been developed for detecting or confirming SARS-CoV-2 infection [11][12][13][14][15][16][17][18][19]. Nucleic acid extraction followed by real-time RT-PCR conducted in the central laboratory is considered the gold-standard method for SARS-CoV-2 detection. However, the accuracy of this assay relies on trained personnel and it also requires time-consuming laboratory processes with complex and expensive equipment [11,20]. For these reasons, point-of-care (POC) testing is needed in some resource-limited areas and/or in nursing homes or long-term care facilities [20,21].
Insulated isothermal PCR (iiPCR) is a fluorescent hydrolysis probe-based technology that provides isothermal heating at the bottom of special capillary tubes to induce thermal convection and temperature gradient in an insulated environment to drive PCR reaction within a relatively short amount of time [22,23]. The POCKIT Nucleic Acid Analyzer series, which is a user-friendly iiPCR system (GeneReach Biotech), provide various iiPCR assays for detecting different human pathogens, including malaria, Zika virus, Middle East respiratory syndrome coronavirus (MERS-CoV), and dengue virus [24][25][26][27][28]. The newgeneration POCKIT Central system automatically completes both nucleic acid extraction and iiPCR amplification and eventually generates a qualitative result. Simply, it is a sample-in-answer-out equipment that is easy to operate and could be a tool for point-ofcare testing.
In 2020, due to the urgent need for SARS-CoV-2 PCR testing, the POCKIT Central SARS-CoV-2 PCR was clinically evaluated on 100 oropharyngeal swab samples [29] in order to apply for emergency use authorization from various agencies. However, in Chang et al. paper [29], the POCKIT Central SARS-CoV-2 PCR was not validated for analytical sensitivity (by testing serial dilutions of a SARS-CoV-2 isolate with known concentration) and specificity (by testing against other non-SARS-CoV-2 pathogens) and was not tested for its capability to detect various SARS-CoV-2 variants. In this study, we thoroughly evaluated the accuracy of the POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR assay in comparison with a commercial TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific).

POCKIT Central SARS-CoV-2 Orf1ab RT-iiPCR System
The POCKIT Central SARS-CoV-2 RT-iiPCR system (GeneReach Biotech, Taichung, Taiwan) used in this study included nucleic acid extraction reagents, SARS-CoV-2 (orf1ab) Premix reagent, and POCKIT Central instrument that allowed to test 8 samples in one run (Figure 1). The PCR assay was designed to target the conserved orf1ab genomic region of SARS-CoV-2 together with a specific Internal Control. The Internal Control template is a plasmid containing a fragment of nucleotides that was artificially designed and synthesized and is not present in any analyzed pathogens or host species. The Internal Control template and the corresponding primers and probe were included in the Premix to monitor the performance of the PCR system. The SARS-CoV-2 primer and probe sequences are: forward primer (ORF1ab-F) 5 -CCCTGTGGGTTTTACACTTAA-3 , reverse primer (ORF1ab-R) 5 -ACGATTGTGCATCAGCTGA-3 , probe (ORF1ab-P) 5 -FAM-CCGTCTGCGGTATGTGGAAAGGTTATGG-BHQ1-3 . The amplicon size is 119 base pairs with the location at nucleotides 13,331-13,449 according to the isolate SARS-CoV-2/human/USA/CA-IGI-0045/2020. Premix for each sample. Both the nucleic acid extraction and PCR amplification were conducted in one POCKIT Central instrument following the manufacturer's protocol. Briefly, the Premix vials were snapped into the Transfer Cartridges and placed in the designated slots of the POCKIT Central instrument. Samples (200 µ L for each) were loaded into the Extraction Cartridges which were placed in the designated slots of the POCKIT Central instrument. After that, a button was pressed to start the run and it took ~85 min to obtain the results. POCKIT Central SARS-CoV-2 (orf1ab) P(+) Control Reagent (GeneReach Biotech, Taichung, Taiwan) was used as an external positive control and a provided Negative Control Reagent was included.
POCKIT Central system automatically completed nucleic acid extraction, iiPCR amplification, and qualitative result interpretation sequentially. Fluorescent signals at 520 nm and 550 nm were generated, respectively, when the targeted SARS-CoV-2 sequences and the internal control sequences were amplified. For results to be valid, the negative control and the SARS-CoV-2 positive control included in the kit must be correct and all of the samples must be Internal Control positive regardless of the SARS-CoV-2 status in the sample.  There was one set of Extraction Cartridges, Transfer Cartridges, and SARS-CoV-2 Premix for each sample. Both the nucleic acid extraction and PCR amplification were conducted in one POCKIT Central instrument following the manufacturer's protocol. Briefly, the Premix vials were snapped into the Transfer Cartridges and placed in the designated slots of the POCKIT Central instrument. Samples (200 µL for each) were loaded into the Extraction Cartridges which were placed in the designated slots of the POCKIT Central instrument. After that, a button was pressed to start the run and it took 85 min to obtain the results. POCKIT Central SARS-CoV-2 (orf1ab) P(+) Control Reagent (GeneReach Biotech, Taichung, Taiwan) was used as an external positive control and a provided Negative Control Reagent was included.
POCKIT Central system automatically completed nucleic acid extraction, iiPCR amplification, and qualitative result interpretation sequentially. Fluorescent signals at 520 nm and 550 nm were generated, respectively, when the targeted SARS-CoV-2 sequences and the internal control sequences were amplified. For results to be valid, the negative control and the SARS-CoV-2 positive control included in the kit must be correct and all of the samples must be Internal Control positive regardless of the SARS-CoV-2 status in the sample.

The Reference TaqPath COVID-19 Real-Time RT-PCR System
In the reference TaqPath COVID-19 real-time RT-PCR system, nucleic acid extraction and PCR reaction were conducted in two separate steps ( Figure 1).
A MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, USA) and a Kingfisher Flex instrument (Thermo Fisher Scientific, Waltham, MA, USA) were used to extract nucleic acids from viral pathogens and clinical samples. First, 500 µL of Wash Buffer for Wash 1 Plate, 1000 µL of 80% Ethanol for Wash 2 Plate, and 50 µL of Elution Solution for Elution Plate were prepared per well. Then the Binding Bead Mix with 265 µL of Binding Solution and 10 µL of Total Nucleic Acid Magnetic Beads per reaction was prepared. Next, the sample plate was prepared with 5 µL of Proteinase K, 200 µL of sample, and 275 µL of the Binding Bead Mix per well. Subsequently, 5 µL of MS2 Phage Control (Thermo Fisher Scientific) was added to each sample well and the Negative Control well. Eventually, all of the prepared plates were loaded into a Kingfisher Flex instrument and the automated program was run to extract nucleic acids. The extracted nucleic acids were immediately used for TaqPath COVID-19 PCR testing. The remaining nucleic acids were saved at −80 • C freezer.
TaqPath COVID-19 Combo Kit (Thermo Fisher Scientific) was included as the reference PCR assay for evaluation and comparison in this study. For PCR reaction setup, 6.25 µL of TaqPath 1-Step Multiplex Master Mix (No ROX) (4×), 1.25 µL of COVID-19 Real-Time PCR Assay Multiplex, 7.50 µL of nuclease-free water and 10.0 µL of the RNA template were included in a 25 µL reaction on the 96-well reaction plate. The Positive Control of this assay was TaqPath COVID-19 Control (1 × 10 4 copies/µL) diluted to a working stock of 25 copies/µL provided by the manufacturer. The Negative Control was included for each RT-PCR reaction plate. PCR reactions were performed on the ABI 7500 Fast instrument (Thermo Fisher Scientific) with the following conditions: one cycle of 25 • C for 2 min, one cycle of 53 • C for 10 min, one cycle of 95 • C for 2 min, and 40 cycles of 95 • C for 3 s and 60 • C for 30 s. COVID-19 Interpretive Software 1.5 version was used for analysis. The reference TaqPath COVID-19 assay included primers and probes targeting three SARS-CoV-2 genes (ORF1ab, N, and S) and one internal positive control MS2. For each target, Ct < 37 was considered Positive and Ct ≥ 37 was considered Negative. For the result interpretation, if ≥2 SARS-CoV-2 targets were positive and MS2 was either positive or negative, the sample was considered Positive; if one SARS-CoV-2 target gene was positive and MS2 was either positive or negative, the sample was considered Inconclusive and would be retested; if 3 SARS-CoV-2 targets were all negative and MS2 was positive, the sample was considered Not Detected for SARS-CoV-2; if 3 SARS-CoV-2 targets and MS2 were all negative, the result was Invalid and retest was required.

Viral and Bacterial Pathogens
To evaluate the cross-reactivity (analytical specificity) of the POCKIT Central SARS-CoV-2 PCR assay and the reference TaqPath COVID-19 assay, a variety of human viral and bacterial pathogens were included for testing (Table 1). To evaluate the inclusivity of the assays, different SARS-CoV-2 original strains, and variants were tested. These 22 SARS-CoV-2 original strains and variants were obtained from BEI Resources in the format of either genomic RNA or heat-inactivated virus ( Table 2). For the heat-inactivated virus strains, 200 µL of the virus was used in the sample-in-result-out POCKIT Central SARS-CoV-2 PCR system; 200 µL of the virus was used for nucleic acid extraction followed by PCR in the TaqPath COVID-19 PCR system. For SARS-CoV genomic RNA obtained from BEI Resources, 10 µL of the genomic RNA was diluted into 190 µL of nuclease-free water and then used in the POCKIT Central SARS-CoV-2 PCR system while 10 µL of the genomic RNA was directly used for TaqPath COVID-19 PCR reaction without going through nucleic acid extraction.
The whole genome sequences of these 22 SARS-CoV-2 strains were obtained from GenBank or GISAID. Then, the 22 whole genome sequences were used as the query sequences at the websites clades.nextstrain.org and gisaid.org to determine the Pango lineage, Nextstrain clade, and GISAID clade information of these strains. In addition, the ORF1ab sequences of 23 SARS-CoV-2 strains (22 strains included in this study and the primary strain Wuhan/WIV04/2019) were aligned and compared using BioEdit software (version 7.2.5) to determine the conservation of POCKIT Central SARS-CoV-2 PCR primer and probe sequences.

Clinical Samples
To evaluate the diagnostic accuracy of the POCKIT Central SARS-CoV-2 PCR in comparison with the reference TaqPath COVID-19 PCR, a total of 183 human nasopharyngeal swab samples collected and submitted to the State Hygienic Laboratory of University of Iowa or the Iowa State University Public Health Testing Services in 2020 and 2021 were tested. Among the 183 samples, 100 of them were collected in 2020 and the remaining 83 samples were collected in 2021; all of the samples were collected in the USA.

Limit of Detection of POCKIT Central SARS-CoV-2 PCR Assay and the Reference TaqPath COVID-19 PCR Assay
The heat-inactivated SARS-CoV-2 USA-WA1/2020 isolate (BEI Resources Cat#: NR-52286) with a titer of 1.6 × 10 5 TCID 50 /mL before heat-inactivation and 3.75 × 10 8 genomic copies (GC)/mL determined by digital PCR at BEI Resources were serially diluted and used to determine the limit of detection (LOD) of the POCKIT Central SARS-CoV-2 PCR and the reference PCR with 5 replicates at high concentrations and 20 replicates at low concentrations. For the reference PCR, the serially diluted isolate was first extracted with 200 µL input for each dilution and eluted into 50 µL, and then 10 µL of the extracted RNA was used for PCR setup. For the POCKIT Central SARS-CoV-2 PCR, 200 µL of the sample at each dilution was directly used in the system in which nucleic acid extraction and PCR reaction were combined in one instrument.

Analytical Specificity of POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR Assay and the Reference TaqPath COVID-19 PCR Assay
As shown in Table 1, both POCKIT Central SARS-CoV-2 PCR and the reference PCR had great analytical specificity and did not cross-react with any of the tested non-SARS-CoV-2 viral or bacterial pathogens.

Inclusivity of POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR Assay and the Reference TaqPath COVID-19 PCR Assay
A total of 22 different SARS-CoV-2 isolates obtained from BEI Resources were tested by both PCR assays. These 22 isolates include the original SARS-CoV-2 strains detected in various countries and regions as well as the SARS-CoV-2 Alpha, Beta, Delta, and Omicron variants. We also determined the Pango lineage, Nextstrain clade, and GISAID clade information of these SARS-CoV-2 isolates with the data summarized in Table 2. All of the 22 isolates tested positive by both PCR assays ( Table 2), suggesting that both PCR assays had good coverage to detect genetically diverse SARS-CoV-2 strains. Sequence alignment indicated that the ORF1ab genomic region targeted by POCKIT Central SARS-CoV-2 PCR primers and probe was conserved among all of the evaluated SARS-CoV-2 strains except one SARS-CoV-2 isolate USA/MD-HP05285/2021 which had one nucleotide substitution compared to other SARS-CoV-2 isolates ( Figure 2). However, this nucleotide substitution did not affect the detection of USA/MD-HP05285/2021 isolate by POCKIT Central SARS-CoV-2 PCR.

Limit of Detection of POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR Assay and the Reference TaqPath COVID-19 PCR Assay
Different dilutions of the SARS-CoV-2 isolate USA-WA1/2020 were tested by both the POCKIT Central SARS-CoV-2 PCR and the reference PCR, with 5 replicates at high concentrations and 20 replicates at low concentrations. As shown in Table 3, the limit of

Limit of Detection of POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR Assay and the Reference TaqPath COVID-19 PCR Assay
Different dilutions of the SARS-CoV-2 isolate USA-WA1/2020 were tested by both the POCKIT Central SARS-CoV-2 PCR and the reference PCR, with 5 replicates at high concentrations and 20 replicates at low concentrations. As shown in Table 3, the limit of detection (at least 95% of reactions were positive) of the POCKIT Central SARS-CoV-2 PCR was 1.87 × 10 3 genomic copies/mL (corresponding to 0.8 TCID 50 /mL) while the limit of detection of the reference PCR was 3.75 × 10 2 genomic copies/mL (corresponding to 0.16 TCID 50 /mL) under the conditions of this study.

Diagnostic Accuracy of POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR Assay
The diagnostic accuracy of the POCKIT Central SARS-CoV-2 PCR was evaluated by testing 183 clinical samples in comparison with the reference TaqPath COVID-19 PCR. The detailed results are shown in Supplemental Table S1. For all 183 samples, the Internal Control of the POCKIT Central SARS-CoV-2 PCR and the internal positive control (MS2 gene) of the reference PCR were all positive; hence, all of the SARS-CoV-2 results of the two PCR assays were valid.
Regarding the reference PCR, 51 samples were negative (Ct ≥ 37) by all of the three SARS-CoV-2 target genes ORF1ab, N, and S, 94 samples were positive (Ct < 37) by all of the three target genes, and 38 samples were positive by the ORF1ab and N target genes but negative by the S target gene (Table S1). According to the interpretation criteria from the kit manufacturer, it was concluded that 51 samples were negative and 132 samples were positive for SARS-CoV-2 by the reference PCR. For the 132 samples positive by SARS-CoV-2 ORF1ab and N target genes of the reference PCR, the Ct values ranged from 9.46-33.86 (ORF1ab target gene) and 8.12-36.97 (N target gene). For the 38 samples that were positive by the ORF1ab and N target genes but negative by the S target gene, their Ct values ranged from 10.04-33.86 (ORF1ab target gene) and 8.81-34.20 (N target gene).
Regarding the POCKIT Central SARS-CoV-2 PCR, 62 samples were negative and 121 samples were positive for SARS-CoV-2. Compared with the reference PCR, the diagnostic sensitivity, specificity, and agreement of the POCKIT Central SARS-CoV-2 PCR were 91.7%, 100%, and 94.0%, respectively, with all of the 183 samples being accounted for (Table 4). There were 11 discrepant results among the 183 clinical samples between the two PCR assays and all of these 11 samples were negative by the POCKIT Central SARS-CoV-2 PCR but positive by the reference PCR. These 11 samples had relatively high Ct values of 29.10-33.86 (ORF1ab target gene) and 29.19-36.97 (N target gene) according to the reference PCR. As shown in Table S1,

Discussion
The COVID-19 pandemic has prompted an unprecedented global effort to develop diagnostic methods, vaccines, antiviral drugs, and bioinformatics tools to track virus evolution and spread; all of these have greatly contributed to controlling SARS-CoV-2 infection worldwide. In addition, the knowledge and experience learned from these efforts are invaluable for fighting against other infectious diseases. The speedy development and application of effective mRNA vaccines against COVID-19 is a stellar example of new technology. Similarly, significant advances have been made in the SARS-CoV-2 diagnostic field. According to the 360D× that tracks the commercially available SARS-CoV-2 tests for diagnostic and clinical use (https://www.360dx.com/coronavirus-test-tracker-launched-COVID-19-tests), up to 15 March 2023, there have been 403 different types of PCR tests, 36 isothermal amplification tests, 102 antigen tests, 155 antibody tests, and a few biosensor tests for SARS-CoV-2 worldwide.
Various real-time RT-PCRs targeting ORF1ab, N, S, and E genomic regions have been developed and widely used for detecting SARS-CoV-2, and are considered the gold standard for SARS-CoV-2 testing [11][12][13]19]. However, at the early stage of the COVID-19 outbreak, human medicine diagnostic laboratories did not have sufficient capacity (shortage of supplies, reagents, and instruments for nucleic acid extraction and real-time RT-PCR) to meet the exploding SARS-CoV-2 PCR testing requests. Many veterinary diagnostic laboratories (including our lab at Iowa State University College of Veterinary Medicine) were brought in to test human samples for SARS-CoV-2 by real-time RT-PCR in a highthroughput format. However, the instrument for such high-throughput SARS-CoV-2 real-time RT-PCR is not field deployable and is unsuitable for point-of-care use.
POCKIT Central insulated isothermal PCR system combines nucleic acid extraction and PCR reaction in one instrument and automates the sample-to-answer process withiñ 85 min; this system can be a useful POC tool. In this study, we evaluated POCKIT Central SARS-CoV-2 PCR system in comparison with a commercial TaqPath COVID-19 real-time RT-PCR which was used as a reference assay. The POCKIT Central SARS-CoV-2 PCR assay showed great analytical specificity and inclusivity. The primers and probe of POCKIT Central SARS-CoV-2 PCR target the conserved ORF1ab genomic region, enabling this assay to successfully detect all of the 22 SARS-CoV-2 original strains and various variants evaluated in this study ( Table 2).
The POCKIT Central SARS-CoV-2 PCR (LOD 1.87 × 10 3 genomic copies/mL) showed lower analytical sensitivity than the reference TaqPath PCR (LOD 3.75 × 10 2 genomic copies/mL) based on testing the serial dilutions of heat-inactivated SARS-CoV-2 isolate USA-WA1/2020 (Table 3). For the POCKIT Central SARS-CoV-2 PCR, the equipment automatically processed 200 µL of samples from the beginning to the result interpretation. As for the reference PCR, 200 µL of the samples were first extracted using the Mag-MAX Viral/Pathogen Nucleic Acid Isolation Kit on a Kingfisher Flex instrument to obtain 50 µL of viral nucleic acids, then 10 µL of the nucleic acid extracts were used to set up the PCR reaction. In order to reasonably compare the LODs of these two PCR assays, we calculated the LODs based on the genomic copies of the SARS-CoV-2 isolate before nucleic acid extraction. We did not calculate the LODs based on the concentration of viral RNA used for the PCR reaction because we could not measure the viral RNA concentration in the POCKIT Central SARS-CoV-2 PCR system due to its automated sample-to-result process. Therefore, it is noteworthy that the LOD of the reference TaqPath COVID-19 PCR determined in this study may be different from the LOD based on the quantitation of viral RNA used for PCR. For example, in this study, if we calculated the LOD in another way, the LOD of the reference TaqPath COVID-19 PCR would be 15 genomic copies per reaction (3.75 × 10 2 genomic copies/mL × 0.2 mL × 10 µL/50 µL).
In this study, among the 183 clinical samples, 38 samples were positive by the reference TaqPath COVID-19 PCR ORF1ab and N target genes but negative by the reference PCR S target gene (Table S1). But, this is not surprising because the S gene is known to be highly prone to mutation. This also emphasizes that when developing SARS-CoV-2 screening PCR, the S gene should not be the single target for primers and probes. When all of the 183 clinical samples were used to evaluate the diagnostic accuracy, the POCKIT Central SARS-CoV-2 PCR system had 91.7% sensitivity (121/132) and 100% specificity (51/51) compared to the reference TaqPath COVID-19 PCR system (  (Table S1). In 2020, the POCKIT Central SARS-CoV-2 PCR was clinically evaluated on 100 oropharyngeal swab samples and it had 96.8% diagnostic sensitivity compared to a SARS-CoV-2 real-time RT-PCR assay [29]. Such preliminary data were used to apply for emergency use authorization from various agencies. Specifically, in 2020-2022, POCKIT Central SARS-CoV-2 PCR was successfully registered under Food and Drug Administration for emergency use authorization in Taiwan, under Agência Nacional de Vigilância Sanitária (ANVISA) in Brazil, and under the In Vitro-Diagnostic Medical Devices Directive 98/79/EC (IVDD) in Europe. In our current study, the POCKIT Central SARS-CoV-2 PCR was thoroughly validated and the study can be considered a combination of technical and clinical validations. The data from this study confirms the validity of the authorization to use this test in an emergency setting based on previous incomplete data. This work demonstrates the diagnostic reliability of the test and the data presented in the current study can be used for additional marketing registrations if needed.
According to the literature, various other PCR-based POC devices have also been developed and validated for SARS-CoV-2 testing [19]. For example, when compared with a standard real-time RT-PCR, the Accula SARS-CoV-2 POC PCR had 68% overall positive percent agreement (34/50) but had 100% positive percent agreement (27/27) for samples with Ct < 30 [30]. Similarly, when compared with a standard real-time RT-PCR, the Visby Medical COVID-19 POC PCR had 95% overall positive percent agreement (58/61) but had 100% positive percent agreement (49/49) for samples with Ct < 31 [31]. Many rapid antigen tests have also been developed for SARS-CoV-2 POC testing. In one review study [32], the authors included 93 studies (reported in 88 publications) that evaluated 36 rapid SARS-CoV-2 antigen tests in 104,961 participants and the rapid antigen tests had an overall sensitivity of 75%. In one study, the SARS-CoV-2 antigen test had 78.9% sensitivity among all symptomatic participants but 96.3% sensitivity in the symptomatic participants who had PCR Ct values of <29 [33]. Similarly, other studies showed that SARS-CoV-2 antigen tests had better sensitivity in samples with PCR Ct values of <25 and the sensitivity decreased a lot in samples with Ct values of >30 [17,34]. Hence, if the sensitivities of various assays are simply compared relative to the real-time RT-PCR Ct values, it appears that the POCKIT Central SARS-CoV-2 PCR had at least comparable sensitivity when compared to other point-of-care PCR and antigen tests.

Conclusions
In this study, we described a novel, automated sample-to-result POCKIT Central SARS-CoV-2 orf1ab RT-iiPCR system that combined nucleic acid extraction and PCR reaction in one instrument but still had comparable diagnostic accuracy to the reference real-time RT-PCR, especially for samples with Ct < 30. The compact POCKIT Central SARS-CoV-2 PCR system can be easily set up and implemented in local clinics, health centers, nursing homes, naval ships, cruises, and remote areas when high-throughput testing of a large number of samples is not needed. This system will allow timely (~85 min from loading samples to obtaining the results) on-site detection without transporting samples to a central diagnostic laboratory. In addition, this platform can be readily adapted to detect other human and animal viruses.