Comparing clinical utility of STANDARD M10 rapid rtRT-PCR assay with pooled testing for SARS-CoV-2 screening

Abstract

Pre-admission screening for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) was essential during the COVID-19 pandemic in Korea. The pooled real-time reverse transcription (rtRT)-PCR test, recommended for optimizing testing capacity, had long turnaround times, limiting its use for same-day hospitalizations. This study evaluated the performance and clinical utility of a rapid rtRT-PCR test as an alternative screening tool for same-day admissions, compared to pooled testing with standard rtRT-PCR. Between July 2022 and January 2023, nasopharyngeal and oropharyngeal swabs were collected twice from each of 3931 patients. Samples were tested using the STANDARD M10 SARS-CoV-2 rapid rtRT-PCR assay and the pooled Allplex SARS-CoV-2 assay. The SARS-CoV-2 positivity rates were 2.4% (n = 96) for the STANDARD M10 and 2.6% (n = 104) for pooled testing, with an overall agreement of 97.3%. The mean turnaround time for the STANDARD M10 was 2.1 h; with 90% of results reported within 2.9 h, compared to 10.7 and 17.1 h for pooled screening-negative and individual test cases, respectively. The STANDARD M10 assay demonstrated high concordance with pooled testing, offering a simple procedure and rapid reporting, making it suitable for same-day admission screening. Rapid rtRT-PCR was crucial for healthcare system resilience during COVID-19 pandemic and will be essential for future preparedness.

Introduction

During the coronavirus disease 2019 (COVID-19) pandemic, all patients requiring hospitalization in Korea were screened for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to ensure adherence to a rigorous disease control strategy implemented by the Korea Disease Control and Prevention Agency (KDCA)^1,2. To meet the demand for large-scale screening, conserve medical resources, and manage national healthcare expenditures, pooled testing with standard real-time reverse transcription PCR (rtRT-PCR) was introduced nationwide³. However, pooled testing lacked standardization and had notable limitations, including longer turnaround times and the need for two-step confirmation of positive results⁴. As a result, pooled testing was mainly used for pre-scheduled hospitalization screenings, as the turnaround time was unsuitable for same-day hospital admissions, which required more rapid SARS-CoV-2 screening.

Rapid rtRT-PCR methods, which deliver results in under an hour, are essential for minimizing treatment delays and preventing the spread of infectious agents^5–8. However, at the onset of the pandemic, only a few global companies had developed rapid rtRT-PCR assays, leading to a supply–demand imbalance in many countries. In Korea, emergency use authorization was granted for two rapid rtRT-PCR assays: the Xpert Xpress SARS-CoV-2 assay (Cepheid, Sunnyvale, California, USA) and the FilmArray respiratory panel assay (BioFire, bioMérieux, Marcy l’Etoile, France). The government allocated daily quotas for imported rapid rtRT-PCR reagents and restricted their use to critically ill patients in emergency departments. After a Korean company (SD Biosensor, Seoul, Korea) developed the STANDARD M10 SARS-CoV-2 rapid rtRT-PCR assay, the system was rapidly and widely adopted during the Omicron surge^9,10.

Although the STANDARD M10 assay was introduced urgently without sufficient initial performance evaluation, subsequent studies have demonstrated its reliable clinical performance compared with established rtRT-PCR tests. Clinical data accumulated during the pandemic showed over 99.0% overall agreement in patients visiting emergency departments⁹ and in those with acute respiratory symptoms¹¹. However, to the best of our knowledge, no study has yet compared the clinical performance of the STANDARD M10 assay with pooled testing as a screening tool for patients with low clinical suspicion for COVID-19. At the study institution, the STANDARD M10 assay was implemented as a pre-admission screening tool, allowing the resumption of same-day hospital admissions, which had previously been suspended in many hospitals due to a lack of isolation rooms for patients awaiting pooled test results. Building on this experience, the present study compares the STANDARD M10 assay with pooled testing using standard rtRT-PCR to evaluate its clinical performance and utility as a screening tool for same-day admission.

Methods

Patients and specimens

Patients who met all of the following criteria underwent both the STANDARD M10 assay and pooled testing prior to admission and were consecutively enrolled in this study:

Same-day admission: Patients who presented to the emergency department or outpatient clinics of Hanyang University Guri Hospital between July 1, 2022, and January 31, 2023, and for whom same-day admission was determined at the discretion of the attending physician.

Low clinical suspicion of COVID-19: Patients for whom the attending physician assessed a low clinical suspicion of SARS-CoV-2 infection. For example, patients presenting with symptoms such as fever that were attributed to an underlying disease rather than to COVID-19 were included in the study.

Two combined specimens—each consisting of nasopharyngeal and oropharyngeal swabs soaked in a single viral transport medium (VTM) kit—were collected from each patient by trained medical staff. One specimen, placed in a VTM kit manufactured by RM Life Science (Seoul, Korea), was transported to the laboratory at Hanyang University Guri Hospital, where the STANDARD M10 SARS-CoV-2 assay was performed immediately. The second specimen, placed in a GeneTM gene transport medium kit (SG Medical, Seoul, Korea), was stored at 4 °C and transported to a central laboratory (Seegene Medical Foundation, Seoul, Korea) for pooled SARS-CoV-2 testing.

After clinical testing with the STANDARD M10 assay, residual specimens were stored at 4 °C for up to 48 h for result verification using the Allplex SARS-CoV-2 rtRT-PCR assay (Seegene, Seoul, Korea) in cases with discordant results relative to pooled testing. These residual samples were subsequently stored at − 70 °C for long-term preservation until testing with the Xpert Xpress SARS-CoV-2 assay.

This study was conducted in accordance with the principles of the Declaration of Helsinki. The Institutional Review Board of Hanyang University Guri Hospital approved the study protocol (2023–07-038) and waived the need for informed consent given the study’s retrospective nature.

STANDARD M10 SARS-CoV-2 assay

The STANDARD M10 assay was performed on individual specimens. Laboratory staff loaded 600 μL of each specimen into the sample chamber of a STANDARD M10 SARS-CoV-2 cartridge, which was then inserted into the STANDARD M10 instrument. The system automatically performed the complete rtRT-PCR process—including nucleic acid extraction—within the cartridge and displayed the final result within 1 h⁹. Four STANDARD M10 instrument modules were used during the study period. The assay targets the envelope (E) and open reading frame 1ab (ORF1ab) genes, with a cycle threshold (Ct) cutoff of 40.0. Results were interpreted as follows: detection of both target genes indicated a positive result; detection of only one target gene indicated an inconclusive result; and detection of neither target gene indicated a negative result. An exogenous control gene (bacteriophage MS2) was used as an internal control.

Pooled test for SARS-CoV-2

A two-stage method was used for pooled testing:

1st stage (pooled screening test):

The VTM from five specimens (300 μL each) were manually combined into one tube. Nucleic acids were extracted from 300 μL of the pooled sample using the Maelstrom™ 9600 system (Taiwan Advanced Nanotech Inc., Taoyuan, Taiwan). An exogenous internal control was added during the extraction step. rtRT-PCR was then performed using Allplex SARS-CoV-2 reagents (Seegene, Seoul, Korea) on a CFX96 thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer’s protocol⁹.

The Allplex assay targets the E gene, nucleocapsid (N) gene, RNA-dependent RNA polymerase (RdRP) region of the ORF1ab gene, and the spike (S) gene¹². Signals from RdRP and S are detected on the same fluorescence channel. Results were interpreted using Seegene Viewer software (versions 3.28.000 and 3.29.000). A pooled test was considered positive if Ct values for all three fluorescence channels (E, N, and RdRP/S) were within the 40.0-cycle cutoff. Inconclusive results were defined as amplification in more than one fluorescence channel across the full 45-cycle run, accounting for dilution effects due to pooling¹³. A negative result was defined as the absence of amplification in all channels across 45 cycles and was reported as pooled screening–negative without proceeding to individual testing.

2nd stage (individual test):

If a non-negative result (inconclusive or positive) was obtained in the pooled screening test, the procedure advanced to the second stage for individual testing. Nucleic acids were extracted from 300 μL of each unpooled specimen, and rtRT-PCR was performed using the same Allplex reagents and CFX96 thermal cycler. Individual test results were interpreted as follows:

• Positive Ct values within 40.0 cycles for all three fluorescence channels

• Inconclusive Ct values within 40.0 cycles for one or two fluorescence channels

• Negative No Ct value detected within 40.0 cycles in any channel

Supplemental rtRT-PCR for discordant results

Supplemental rtRT-PCR was performed for specimens with discordant results between the STANDARD M10 assay and the pooled test. The specimen used for supplemental testing was the same sample previously tested with the STANDARD M10 assay. Two supplemental rtRT-PCR assays were employed: the Allplex SARS-CoV-2 assay and the Xpert Xpress SARS-CoV-2 assay. The Allplex assay was conducted within 48 h of sample collection for all cases with discrepant results between the STANDARD M10 assay and pooled testing. If the results of the STANDARD M10 assay and Allplex assays were also discordant, the Xpert assay was subsequently used. The Xpert assay targets the E gene and N2 region of the N gene (N2), with a Ct cutoff of 45.0 cycles¹⁴. According to the KDCA guidelines, confirmation of SARS-CoV-2 infection requires detection of all tested genes³; thus, positive SARS-CoV-2 samples were defined as those yielding positive results for the E and N2 genes.

Turnaround time

Turnaround time was defined as the interval between patient sample collection and the reporting of results in the electronic medical records. Specimens for the STANDARD M10 assay were delivered to the laboratory hourly, with testing and result reporting conducted on an individual basis. In contrast, specimens for pooled testing were transported to the central laboratory three times daily—at 12:30, 17:30, and 21:30 on regular working days— and twice daily on holidays, at 12:30 and 21:30. Results from the central laboratory were electronically transmitted and recorded in the hospital’s medical records system.

Statistical analyses

The overall percentage agreement of the STANDARD M10 was calculated using the pooled test results as the reference, with MedCalc software (version 22.016; MedCalc, Ostend, Belgium). For statistical comparison between the STANDARD M10 assay and the pooled test, inconclusive results were classified as positive.

Results

Patient characteristics

A total of 3931 patients were enrolled in this study (Table 1), with a higher proportion of women (57.9%) than men (42.1%). The median age was 59 years (range: 0–101), and 145 patients (3.7%) were under 18. Physicians in the emergency department (49.9%) or outpatient department (50.1%) determined the need for admission and SARS-CoV-2 screening in patients not suspected of having COVID-19. Among patients visiting the outpatient department, the reasons for admission included treatment for medical conditions in internal medicine (32.9%), surgical departments (41.5%), and other specialties such as neurology and pediatrics (25.6%). The overall study flow is illustrated in Fig. 1.

Table 1.

Demographic characteristics of 3931 patients without COVID-19-associated symptoms who underwent testing using the STANDARD M10 assay and pooled test for SARS-CoV‐2.

Characteristics	Values
Total patients, n	3931
Sex, n (%)
Men	1654 (42.1)
Women	2277 (57.9)
Median age, years (range)	59 (0–101)
Age, years, n (%)
0 ≤ age < 18	145 (3.7)
18 ≤ age < 40	515 (13.1)
40 ≤ age < 60	1383 (35.2)
60 ≤ age < 80	1506 (38.3)
80 ≤ age	382 (9.7)
Medical department
Emergency department	1962 (49.9)
Outpatient department	1969 (50.1)
Internal medicine	648 (32.9)
Surgical departments	817 (41.5)
Other	504 (25.6)

Abbreviations: SARS-CoV‐2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019.

Fig. 1.

Flow chart of the study. A total of 3931 patients simultaneously underwent pooled testing (specimen 1) and STANDARD M10 testing (specimen 2). Samples with inconclusive or positive pooled screening results proceeded to individual testing. Discrepant results between the pooled test and STANDARD M10 assay were further evaluated using a supplemental Allplex assay (n = 124). If discordance persisted and sufficient sample volume was available, the Xpert assay was subsequently performed (n = 72).

Comparison of STANDARD M10 and pooled tests

Among the 3931 specimens tested by both pooled testing and the STANDARD M10 assay, 3300 (83.9%) were reported as negative for SARS-CoV-2 at the pooled screening stage and classified as pooled screening-negative (Table 2). For the remaining 631 specimens (16.1%), classified as pooled screening-non-negative, individual tests were performed. The positivity rates for SARS-CoV-2 were 2.4% (n = 96) for the STANDARD M10 assay and 2.6% (n = 104) for the pooled test. For statistical analysis, inconclusive results were treated as positives. The overall percentage agreement between the two assays was 97.3% (95% confidence interval: 96.8–97.8%).

Table 2.

Comparison of the STANDARD M10 assay and pooled test.

		Total number	Pooled test
			Pooled screening–negative (n = 3300)	Individual test (n = 631)a
				Negative	Inconclusive	Positive
STANDARD M10
Negative		3756	3239	493	8	16
Inconclusive		79	48	12	3	16
Positive		96	13	8	3	72
Total number		3931	3300	513	14	104
Overall percentage agreement (95% CI)b		97.3% (96.8–97.8%)

^aPooled screening-non-negative specimens were tested individually at 2nd stage of the pooled test.

^bInconclusive results were considered positive results in the calculation of agreement.

Abbreviations: CI, confidence interval.

The distribution of turnaround times for the STANDARD M10 and pooled tests is shown in Fig. 2. The mean turnaround time for the STANDARD M10 assay was 2.1 ± 0.7 h, with 90% of results reported within 2.9 h. In comparison, the mean turnaround times for pooled tests were 10.7 ± 4.1 h for pooled screening-negative cases and 17.1 ± 4.1 h for pooled screening-non-negative cases requiring individual testing. Additionally, results were reported within 18.0 and 23.8 h for 90% of cases in the pooled screening-negative and individual test groups, respectively.

Fig. 2.

Turnaround times for the STANDARD M10 assay and pooled tests. The STANDARD M10 assay demonstrated a significantly shorter turnaround time, with a mean of 2.1 ± 0.7 h (a). In contrast, pooled tests exhibited longer turnaround times, with mean values of 10.7 ± 4.1 h for pooled screening-negative cases (b) and 17.1 ± 4.1 h for individual tests (c). Solid downward-pointing triangles indicate the time points by which 90% of results were reported: 2.9 h, 18.0, and 23.8 h for (a), (b), and (c), respectively. The delayed minor histogram peaks observed in graphs (b) and (c) reflect longer delivery intervals during nighttime and holidays.

Discordant results and supplemental Allplex assay

A total of 124 discordant results were identified between the STANDARD M10 assay and the pooled test: 61 occurred in pooled screening-negative cases, and 63 in pooled screening–non-negative cases undergoing individual testing (Table 3). Supplemental rtRT-PCR using the Allplex assay was performed on the specimens originally tested with the STANDARD M10 assay. Among the 61 pooled screening-negative cases, four (6.6%) showed concordant results with the STANDARD M10 assay on the Allplex assay (three inconclusive and one positive). Among the 63 cases discordant individual tests, 35 (55.6%) yielded concordant results between the STANDARD M10 and Allplex assays (17 negative, 11 inconclusive, and 7 positive). In total, the Allplex assay produced concordant results in 39 of the 124 discordant cases. However, the discordant findings in the remaining 85 cases could not be resolved through supplemental rtRT-PCR testing with the Allplex assay.

Table 3.

Results of the supplemental Allplex SARS-CoV-2 assay for 124 specimens with discordant results between the STANDARD M10 assay and pooled testing.

	Pooled test				Allplex SARS-CoV-2
	Pooled screening-negative				Negative	Inconclusive	Positive
STANDARD M10
Inconclusive	48				43	3	2
Positive	13				9	3	1
Discordance number	61				57

		Individual test			Allplex SARS-CoV-2
		Negative	Inconclusive	Positive
STANDARD M10
Negative		–	8	–	7	1	0
		–	–	16	10	4	2
Inconclusive		12	–	–	6	6	0
		–	–	16	6	5	5
Positive		8	–	–	1	1	6
		–	3	–	1	1	1
Discordance number		63			28
Total final discordance number		85

Concordant results between the STANDARD M10 and Allplex SARS-CoV-2 assays are underlined.

Supplemental Xpert assay

Supplemental rtRT-PCR using the Xpert assay was performed on 72 of the 85 cases, with discordant results between the STANDARD M10 and Allplex assays (Table 4). Thirteen specimens were excluded due to insufficient sample volume. For nearly all discordant cases, the Ct values were greater than 30.0 in the STANDARD M10 assay and greater than 33.0 in the Allplex assay across all target genes, except for one case (case 6), which showed Ct values of 29.58 and 30.35 for the E gene in the STANDARD M10 and Allplex assays, respectively. Among the 14 Xpert-positive cases (cases 1–14), five were concordant with the STANDARD M10 assay (cases 1–5), and five were concordant with the Allplex assay (cases 6–9, 13). The Xpert assay yielded 12 inconclusive results (cases 15–26), characterized by Ct values detected only for the N2 target. Of these, one was positive according to the STANDARD M10 assay (case 15) and three were positive according to the Allplex assay (cases 16–18). The Xpert assay produced 46 negative results (cases 27–72). Among these, eight had previously tested positive with the STANDARD M10 assay (cases 27–54), while none were positive according to the Allplex assay.

Table 4.

Comparison of cycle threshold (Ct) values across rtRT-PCR assays for SARS-CoV-2 detection.

Case number	rtRT-PCR resultsa	Ct values of rtRT-PCR
		Xpert		STANDARD M10		Allplex
		E	N2	E	orf1ab	E	RdRP/S	N
Concordant results
1–80	-PPb	nt		13.53–35.77	14.14–35.77	13.57–38.62	15.18–39.11	12.31–39.13
Discordant results
1	PPI	33.8	35.1	32.65	32.96		36.35	36.57
2	PPI	35.0	36.1	31.69	32.81	36.26		36.84
3	PPI	35.9	38.9	34.16	34.96	37.22	37.55
4	PPI	36.9	40.0	35.64	35.46	38.09
5	PPI	42.5	43.6	34.96	35.49	36.41
6	PIP	30.7	33.3	29.58		30.35	33.78	31.85
7	PIP	34.0	36.5	33.47		33.97	35.88	35.17
8	PIP	35.8	37.4	34.95		33.65	35.79	33.89
9	PIP	42.0	41.0	36.13		36.58	37.40	35.80
10	PIN	35.9	39.1	31.83
11	PIN	41.8	40.8	36.24
12	PIN	44.1	41.9	36.11
13	PNP	36.5	37.1			35.47	38.0	36.14
14	PNI	37.8	41.1			36.56	39.12
15	IPN		40.1	36.43	34.75
16–18	IIP		36.1–40.1	32.14–36.43		37.49–38.18	37.81–39.80	37.88–38.64
19–23	IIN		39.7–43.2	34.46–34.18
24	IIN		41.9		34.41
25, 26	INI		40.1–41.2			38.20		39.05
27–34	NPN			30.84–36.24	31.28–35.44
35–65	NIN			34.55–36.68
66–71	NIN				35.09–36.7
72	NNI					38.28
73–74	-PN	nt		35.24–35.81	35.22–35.32
75–82	-IN	nt		33.01–36.33
83	-IN	nt			35.11
84	-NP	nt				36.64	39.31	35.90
85	-NI	nt						37.90

^aOrder of the rtRT-PCR results is as follows: Xpert, STANDARD M10, and Allplex assays.

^bThe category consisted of 72 concordant results between the STANDARD M10 assay and the pooled test and 8 concordant results between the STANDARD M10 assay and the supplemental Allplex assay.

Abbreviations: rtRT-PCR, real-time reverse transcription polymerase chain reaction; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; Ct, cycle threshold; P, positive; N, negative; I, inconclusive; nt, not tested.

Discussion

The clinical performance of the STANDARD M10 rapid rtRT-PCR assay for SARS-CoV-2 was compared with that of pooled testing in patients with a low clinical suspicion of COVID-19. The assay demonstrated high concordance with pooled testing, achieving an overall agreement of 97.3%. Furthermore, its ability to report results within 2.9 h for 90% of cases enables same-day admissions, contributing to the normalization of healthcare operations during the pandemic.

Pooled testing significantly reduces the number of tests and associated costs when disease prevalence is low¹⁵, making it an effective tool for screening infectious diseases, particularly in settings such as blood donation. During the early phase of the COVID-19 pandemic, pooled testing for SARS-CoV-2 was rapidly implemented without prior validation, based on guidelines in Korea that recommended limiting the number of specimens per pool to five or six^3,13,16. Initially, low disease prevalence enabled cost-effective large-scale screening via pooled tests. However, with the emergence of new variants and increasing prevalence, the demand for individual testing rose, resulting in higher labor requirements, greater potential for human error, and increased reagent consumption¹⁷. Consequently, many hospitals discontinued in-house pooled testing and shifted to outsourcing via central laboratories, transferring the testing burden while underutilizing their own laboratory facilities and trained personnel.

Since the introduction of the STANDARD M10 assay, rapid rtRT-PCR testing has expanded from emergency departments to pre-admission screening for same-day hospitalizations. This allowed the study institution to resume same-day admissions, which had been suspended for 2 years, thereby demonstrating the clinical utility of this testing policy. During the study period, four STANDARD M10 modules were in operation. In instances where more than four samples were delivered at once, delays occurred due to limited module availability, extending overall turnaround times. Specifically, 4.4% of deliveries (94 out of 2162) involved more than four specimens, with a maximum of 12 specimens in a single batch. Overall, 221 specimens (5.6% of all specimens, 221/3931) were delayed due to instrument saturation: 197 were delayed by 1 h, 23 by 2 h, and one by 3 h. Although the STANDARD M10 has a run time of 1 h compared to 45 min for the Xpert and FilmArray assays, all three rapid rtRT-PCR platforms operate on a single-test-per-module basis. Therefore, performing multiple tests concurrently requires additional modules. To minimize delays in rapid rtRT-PCR testing, institutions must optimize the number of instrument modules, sample volume, and transport workflows based on their specific clinical demands.

Compared to previous studies^9,11, which reported > 99.0% overall agreement between the STANDARD M10 and Allplex assays, this study found a lower agreement of 97.3%, likely attributable to differences in analytical methods. Prior studies evaluated assay performance by directly comparing results from the Allplex assay retested using the same specimens as the STANDARD M10 assay. Additionally, those studies focused on diagnostic performance in symptomatic patients, under the assumption that SARS-CoV-2-related symptoms correlated with high viral loads¹⁸, while low viral load cases (inconclusive results) were considered clinically less relevant and grouped with negative results. In contrast, the present study aimed to compare the STANDARD M10 assay with pooled testing for screening purposes. Therefore, retested Allplex assay results of individual specimens were not included in the clinical performance analysis. Furthermore, inconclusive results with low viral loads were classified as positive in this study, based on the rationale that they may represent asymptomatic or presymptomatic infections^18–20.

One case (case 6) showed an inconclusive result with the STANDARD M10 assay, characterized by a relatively low Ct value of 29.8 for the E gene and no detection of the ORF1ab gene. This type of discordance may occur in rapid rtRT-PCR assays, including the Xpert assay, due to genetic mutations or suboptimal sample quality^21,22. Upon retesting with a diluted specimen, the result was positive, suggesting the presence of a PCR inhibitor in the original sample. The proportion of inconclusive results observed in this study with the STANDARD M10 assay (2.0%) was higher than that reported in a prior study conducted during the Omicron variant surge (1.2%)⁹. Another study performed during the same period as the present one reported a similar proportion of inconclusive results (2.4%)¹¹. As both studies took place shortly after the Omicron surge, it is likely that a substantial portion of the inconclusive results reflected prolonged shedding of incomplete viral genetic material in patients recovering from prior Omicron infection²³.

Among the 32 cases that initially tested positive only in the pooled test, 7 were confirmed positive, 16 were negative, and 9 were inconclusive when retested with the Allplex assay. This notable discrepancy is likely attributable to a high proportion of specimens with low viral loads, as reflected by high Ct values, given that the study population comprised patients not suspected of having COVID-19. Among pooled test–positive or –inconclusive cases, 26.3% had Ct values > 35.0—a proportion higher than that reported in previous studies. For instance, one study including 27.8% symptomatic patients reported 12.0% of specimens with Ct values > 35.0⁹, while another study focusing exclusively on symptomatic individuals reported only 2.5% with Ct values > 35.0 based on Allplex assay results¹¹. When viral loads approach the limit of detection, results can vary between samples from the same patient or upon repeat testing. Additionally, procedural errors such as sample contamination during pooled testing could contribute to discrepancies.

Pooled testing involves a five-fold dilution of individual specimens, which theoretically reduces assay sensitivity. To address this limitation, pooled testing guidelines recommend proceeding with individual testing when amplification is suspected at any point during the full PCR cycle—regardless of whether the signal crosses a predefined threshold—thus mitigating the effects of dilution¹³. In this study, individual testing was not performed for all pooled specimens. However, because the analysis was based on real-world clinical data rather than controlled experimental conditions, it effectively reflects the feasibility of replacing pooled testing with the STANDARD M10 assay in actual clinical practice. Notably, a same-day admission system was successfully maintained throughout the study period without any nosocomial outbreaks, which may support the clinical applicability of this approach.

Compared to pooled testing, rapid rtRT-PCR has disadvantages, including higher reagent costs and potential limitations related to instrument and cartridge supply²⁴. However, the findings from this study suggest that widespread development of validated rapid rtRT-PCR assays across multiple countries could help address supply chain issues. Moreover, increased competition among manufacturers may lower reagent costs over time, and healthcare expenditures could be managed by adjusting individual patient cost burdens. Although extensive efforts have been made to improve the accuracy and reporting speed of pooled testing, standardization remains lacking^25–29. Therefore, pooled testing is best suited for low-prevalence settings, where automation and optimization of workflows are in place. When laboratory resources and trained personnel are sufficient, revising testing policies to incorporate rapid rtRT-PCR or individual rtRT-PCR testing could improve the clinical efficiency of human and material resource utilization.

This study had several limitations. First, the results were not validated using alternative molecular methods, such as sequencing or digital PCR³⁰. However, a previous study by our research group involving droplet digital PCR experiments provides comparative data supporting the performance of the STANDARD M10 assay⁹. Second, the specific SARS-CoV-2 variants responsible for infection in this study were not identified. According to national variant surveillance reports, the Omicron sublineage BA.5 was predominant during the study period³¹. Nonetheless, SARS-CoV-2 continues to evolve rapidly, and the emergence of new variants may affect assay detection sensitivity³², underscoring the importance of ongoing variant identification.

In conclusion, the STANDARD M10 assay demonstrated a high concordance rate of 97.3% compared with simultaneously performed pooled tests. The procedure was simple, with an average turnaround time of 2.1 h, and 90% of results were reported within 2.9 h—supporting its utility as a screening test for same-day admissions during the pandemic. The diversification of rapid rtRT-PCR methods helped address supply chain constraints and contributed to the stabilization of healthcare systems during COVID-19. This testing approach is expected to play a significant role in enhancing preparedness for future pandemics.

Author contributions

Jinyeong Kim contributed to resource collection and drafting the original manuscript. Investigation, material preparation, and initial drafting were performed by Eunhee Han. Jieun Kim contributed to resource collection. Young Jin Kim was involved in conceptualization, formal analysis, and manuscript writing, review, and editing. Mi Hyun Bae contributed to conceptualization, resource collection, formal analysis, investigation, methodology, and manuscript writing, review, and editing. All authors read and approved the final version of the manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics approval

This study was conducted in accordance with the principles of the Declaration of Helsinki and was approved by the Institutional Review Board of Hanyang University Guri Hospital (2023–07-038). Due to the retrospective nature of the study, Institutional Review Board of Hanyang University Guri Hospital waived the need of obtaining informed consent.

Consent to participate

The need for informed consent was waived.