Evaluation of a new point‐of‐care quantitative reverse transcription polymerase chain test for detecting severe acute respiratory syndrome coronavirus 2

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection is rapidly spreading worldwide, and the resultant disease, coronavirus disease (COVID‐19), has become a global pandemic. Although there are multiple methods for detecting SARS‐CoV‐2, there are some issues with such tests, including long processing time, expense, low sensitivity, complexity, risk of contamination, and user friendly. This study evaluated the reproducibility and usability of a new point‐of‐care test (POCT) using real‐time quantitative reverse transcription polymerase chain reaction (qRT‐PCR) for detecting SARS‐CoV‐2. Methods Samples from 96 patients with suspected SARS‐CoV‐2 infection were assessed using the real‐time qRT‐PCR‐based POCT and the conventional real‐time qRT‐PCR method based on the Japanese National Institute of Infectious Diseases guidelines (registration number: jRCT1032200025). Results The real‐time qRT‐PCR‐based POCT had a positive agreement rate of 90.0% (18/20), a negative agreement rate of 100% (76/76), and a total agreement rate of 97.9% (94/96), and the significantly high score of questionnaire survey (total score p < 0.0001). In the two cases in which real‐time qRT‐PCR‐based POCT results did not match conventional real‐time qRT‐PCR test results, the SARS‐CoV‐2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real‐time qRT‐PCR. All patients could be triaged within 1 day using the real‐time qRT‐PCR‐based POCT without invalid reports. Conclusions The real‐time qRT‐PCR‐based POCT not only had high reproducibility and useability but also allowed rapid patient triage. Therefore, it may be helpful in clinical settings.


| INTRODUC TI ON
Coronavirus disease (COVID-19) is an acute respiratory infection caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 1 It was first reported as "pneumonia of unknown cause" in Wuhan, China, in December 2019, and the Chinese Centers for Disease Control and Prevention (CDC) officially announced on January 7, 2020, that COVID-19 was caused by the novel coronavirus.
There are some molecular diagnostic tools for detecting SARS-CoV-2, such as the standard quantitative reverse transcription polymerase chain reaction (qRT-PCR) test issued by the CDC in the United States, 2 the standard qRT-PCR test issued by the National Institute of Infectious Diseases (NIID) in Japan, 3 the high-throughput qRT-PCR kit test, the reverse transcription loop-mediated isothermal amplification test, 4 the viral antigen test, 5 the quantitative viral antigen test, and the viral antibody test. 6,7 There are also multiple sample collection methods, such as collection using nasopharyngeal swabs or saliva. 8 As all of these methods have both strengths and limitations, it is necessary to choose among them based on clinical requirements. Although the standard method is a qRT-PCR test from a nasopharyngeal swab, it has some limitations in clinical use, because of including long processing time, expense, low positive agreement rate, complexity, user friendly, and a level of personal protective equipment (PPE).
In this study, a point-of-care test (POCT) using a real-time one-step qRT-PCR method based on the existing Japanese test method "Manual for the Detection of Pathogen 2019-nCoV" was performed using nasopharyngeal swab specimens collected from patients with suspected COVID-19. 3 We compared our results with those of the existing method to assess its performance. In addition, we assessed the correlation between the RNA copy number using conventional real-time qRT-PCR and the cycle threshold in the real-time qRT-PCR-based POCT to determine the positive agreement rate of the evaluation kit in detecting small amounts of SARS-CoV-2. who had a history of close contact with an infected person; or who were cured from SARS-CoV-2 infection. Two nasopharyngeal swab specimens were collected from each patient: one was suspended in 1 ml of liquid culture media (UTM), and the other was suspended in the extraction reagent solution for the evaluation kit. 9 Questionnaire survey was conducted for all 6 laboratory staffs in charge involved in the analysis. There are 5 kinds of questions (1. Operability, 2.

| Patients
Contribution to reducing infection risk, 3. Ease of result judgment, 4.
Error handling, and 5. Total satisfaction level) and was 5 grades for each question.

| Conventional real-time qRT-PCR
RNA purification and real-time one-step qRT-PCR (conventional real-time qRT-PCR) were performed for specimens collected in UTM using a method based on the "Manual for the Detection of Pathogen 2019-nCoV" Ver.2.9.1 issued by the NIID. 3  Figure 1C). Then, the filter is tightened ( Figure 1D), and the vial is shaken several times to thoroughly mix the sample ( Figure 1E). Four drops of this sample are placed onto the sample spot of the test cartridge ( Figure 1F). After the sample drop is absorbed, the test cartridge is promptly inserted into the device, which begins the measurement.

| Real-time qRT-PCR-based POCT test using new evaluation kit for rapid gene detection
In general, a complicated purification step is required when amplifying RNA because impurities present in the sample, such as human genomic DNA, inhibit the reaction. In this method, DNA binds to silica particles in the extraction reagent solution, and these particles are removed by filtration. Thus, the target RNA is extracted into the droplet solution and easily measured ( Figure 1G).

| Measurement of SARS-CoV-2 RNA copy number
SARS-CoV-2 RNA was extracted from the residual sample of the evaluation kit using the QIAamp DNA Ⅿini Kit (Qiagen; Hilden), as in the control method, and the extracted RNA was used to quantitatively measure the SARS-CoV-2 RNA copy number from the calibration curve of N2 primers as previously described. 3

| Statistical analysis
Clinical characteristics and symptoms were counted and calculated statistical analysis each male and female using the unpaired t test GraphPad Software). No adjustment of multiple comparisons was made. All reported p-values were two-sided, and a p < 0.05 was considered significant.

| Clinical samples
In total, 96 patients who had a history of close contact with an infected person; experienced fever, cough, or pneumonia; or were cured from infection were included in our study. There were no significant differences in age or body temperature of individuals examined as outpatients based on gender; however, the body temperatures of male patients who were hospitalized were significantly higher than female patients (p = 0.0002). Patient details, including age, sex, patient background, and clinical symptoms, are shown in

| Correlation of results between the conventional and the evaluation of real-time qRT-PCR test
We The relationship between the number of SARS-CoV-2 RNA copies and the cycle number when quenching for the 18 positive samples is summarized in Figure 4A. There was a strong correlation (r = 0.91), and the limit of detection was estimated to be 5-10 copies per test ( Figure 4B). In the two cases in which real-time qRT-PCR-based POCT results did not match conventional real-time qRT-PCR test results, the SARS-CoV-2 RNA copy numbers were 8.0 copies per test in one case and below the detection limit in the other case when quantified using conventional real-time qRT-PCR.
Moreover, all patients successfully received medical triage from clinical doctors because they could undergo a test on the same day using the real-time qRT-PCR-based POCT (minimum, less than 1 h).

| Validation of the nucleotide variance of primer binding sites in SARS-CoV-2 mutant strains
To validate nucleotide variance of these CDC-based and NIIDbased primer binding sites in already reported SARS-CoV-2 mutant strains, we found some single nucleotide polymorphism

| Level of importance question in your satisfaction survey
Questionnaire survey was conducted for all 6 laboratory staffs in charge involved in the analysis. Real-time qRT-PCR-based POCT is not only significantly higher than conventional real-time qRT-PCR test in total score, but also every 5 questions ( Figure 4C).

| DISCUSS ION
We successfully evaluated the reproducibility and usability of a new point-of-care test (POCT) using real-time qRT-PCR for detecting SARS-CoV-2 (Real-time qRT-PCR-based POCT). Our real-time qRT-PCR-based POCT method was found to be as high accurate (high sensitivity, specificity, and reproducibility) as conventional real-time qRT-PCR without invalid reports in these cases. Moreover, we found real-time qRT-PCR-based POCT has the potential of high usability as a POCT by questionnaire survey. The usefulness of this device in clinical use (bedside) is to be high.
In this study, a point-of-care test (POCT) using a real-time one-step qRT-PCR method based on the existing Japanese test method "Manual for the Detection of Pathogen 2019-nCoV" was performed using nasopharyngeal swab specimens collected from patients with suspected COVID-19. 3 We compared our results with those of the existing method to assess its performance. In addition, we assessed the correlation between the RNA copy num-  Welfare in Japan. These testing systems are being expanded to take advantage of the characteristics of each method. However, these testing systems also have inadequacies that limit their broad use.
Immunochromatographic qRT-PCR-based POCT (minimum, less than 1 h). Therefore, we believe this new POCT using real-time qRT-PCR is the best method for clinical use.

CO N FLI C T O F I NTE R E S T
The authors state that they have no conflict of interest.

A PPROVA L CO D E I SS U E D BY TH E I N S TITUTI O N A L R E V I E W B OA R D (I R B)
The study was prospective observation study, carried out by the opt-in method of each institution, and approved by the ethics com- observational study) (https://jrct.niph.go.jp/re/repor ts/detai l/7882) and are available to share and download the files to all person who is interested (https://jrct.niph.go.jp/re/repor ts/detai l/7882), includes pdf format raw data, study protocol, statistical analysis plan, informed consent form, and clinical study report (from August 1 2021).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that provided the evidence for the study are available from the corresponding author upon reasonable request.