Clinical evaluation of a direct RT-qPCR method for feline coronavirus detection in effusions using the PicoGene PCR1100 system

Abstract

Feline coronavirus (FCoV) infects both domestic and wild felids and has the potential to cause feline infectious peritonitis (FIP), a progressive and often fatal systemic disease. Although rapid diagnosis and treatment are crucial in cases of FIP, conventional reverse-transcription quantitative real-time PCR (RT-qPCR) requires RNA extraction and specialized equipment, limiting its use for timely testing in general veterinary practice. We evaluated the performance of a direct RT-qPCR method using the PicoGene PCR1100 system (GoFoton, Ibaraki, Japan), which omits the RNA extraction step and delivers results within ~40 min. Compared with FCoV culture supernatants and extracted RNA, we estimated the limit of detection of this direct RT-qPCR method to be 150 copies/reaction—a detection sensitivity equivalent to that of conventional RT-qPCR targeting the FCoV 3′-UTR. We observed no cross-reactivity with other feline viruses or SARS-CoV-2. We subsequently analyzed 28 pleural and abdominal effusions collected from cats suspected of having FIP to compare the direct RT-qPCR method with the conventional approach. The sensitivity of the direct RT-qPCR method was 95.5% (95% CI: [78.2, 99.2]) and the specificity was 100% (95% CI: [61.0, 100.0]), which supports the use of the PCR1100 system as a rapid and user-friendly point-of-care tool for the detection of FCoV RNA in effusion samples.

Keywords

direct RT-qPCR feline coronavirus feline infectious peritonitis point-of-care testing

Feline coronavirus (FCoV; family Coronaviridae, taxon species Alphacoronavirus suis; https://ictv.global/report/chapter/coronaviridae/coronaviridae/alphacoronavirus) is an enveloped, positive-sense, single-stranded RNA virus classified within the genus Alphacoronavirus, and was merged with canine coronavirus (CCoV) and transmissible gastroenteritis virus (TGEV). The viral genome encodes replicase polyprotein 1ab, 4 structural proteins (spike, envelope, membrane, nucleocapsid), and 2 nonstructural accessory proteins (3abc, 7ab), and includes a 5′–untranslated region (UTR), a 3′-UTR, and a poly(A) tail.³⁵

FCoV is widespread among both domestic and wild felids and exists as 2 serotypes, types I and II.^{3,11,13,17,24} Epidemiologic studies indicate that type I FCoV is the predominant serotype in natural infections. Although most FCoV infections remain subclinical or cause only mild, self-limiting enteritis, some infected cats develop feline infectious peritonitis (FIP), a life-threatening systemic disease.^15,25,37 In FIP, peritoneal or pleural effusion and granulomatous inflammation involve serosal surfaces and internal organs.^22,23 Historically regarded as uniformly fatal, FIP is now treatable with the nucleoside analogues GS-441524 and EIDD-2801 (Molnupiravir), which have improved clinical outcomes.^{9,20,26,27,31,32} However, delayed antiviral use has been associated with reduced therapeutic efficacy, underscoring the importance of early and accurate diagnosis.

The gold standard for the definitive diagnosis of FIP involves the immunohistochemical or immunocytochemical detection of the FCoV antigen localized within macrophages.^34,36 Although these methods are highly specific, their reliance on invasive procedures, such as tissue biopsy and the need for a specialized laboratory infrastructure, limit their feasibility in clinical practice. Consequently, a reverse-transcription quantitative real-time PCR (RT-qPCR) is widely used as a supportive testing tool in clinical practice.^14,27,28,32

RT-qPCR enables the highly sensitive and quantitative detection of FCoV RNA and may be applied to various sample types, including effusions, CSF, aqueous humor, and fine-needle aspirates.^5,8,30 Given the high sensitivity and specificity of RT-qPCR, this test is commonly employed in clinical testing of effusion samples in FIP cases with effusion.^5,18 However, conventional RT-qPCR requires RNA extraction, expensive equipment, and technical expertise, often necessitating referral to external laboratories, which may delay a diagnosis by several days and hinder the timely initiation of treatment.

In response to these challenges, point-of-care (POC) testing has been gaining in interest, offering veterinary clinics a rapid and simple approach to molecular testing. The PicoGene PCR1100 system (GoFoton, Ibaraki, Japan) is a compact real-time PCR device based on microfluidic technology. The PCR1100 system enables rapid nucleic acid amplification and fluorescence-based detection by using a dedicated reaction chip ( Fig. 1 ). The sensitivity and specificity of this system for detecting pathogens, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and foot-and-mouth disease virus (FMDV), are similar to those of conventional RT-qPCR.^19,33 Notably, this system supports direct RT-qPCR without RNA extraction, which should simplify the testing process and deliver results within 40 min.

We investigated the performance of the PCR1100 system and its dedicated direct RT-qPCR kit for detecting FCoV RNA in pleural and abdominal effusions collected from cats suspected of having FIP. We compared sensitivity, specificity, and limit of detection (LOD) with conventional RT-qPCR methods to examine the utility of this system as a rapid and user-friendly tool in veterinary clinical practice.

Materials and methods

Viruses

Type I FCoV strain KU-2 was isolated in our laboratory. Type II FCoV strain 79-1146 was provided by Dr. M. C. Horzinek (Utrecht University, the Netherlands). Type II CCoV strain 1-71 and TGEV strain TO-163 were obtained from the University of Tokyo and the National Institute of Animal Health, Japan, respectively. Felid alphaherpesvirus 1 (FeAHV1) strain C7301, feline calicivirus (FCV) strain F4, and feline panleukopenia virus (FPLV) strain TU-1 were also obtained from the University of Tokyo. Feline immunodeficiency virus (FIV) strain Petaluma was provided by Dr. J. K. Yamamoto (University of Florida). Feline leukemia virus (FeLV) strain KT-FeLVUCD-1 and the persistently infected cell line FL-74 (CRL-8012) were obtained from the American Type Culture Collection. SARS-CoV-2 strain JPN/Kanagawa/KUH003 was provided by Dr. Katayama (Kitasato University, Tokyo, Japan).

The accessions (GenBank or RefSeq) for the viral strains used are as follows: FCoV strain KU-2 (LC880185), FCoV strain 79-1146 (AY994055.1), CCoV strain 1-71 (AY796289.1), TGEV strain TO-163 (AB115401.1), FeAHV1 strain C7301 (LC880187), FCV strain F4 (D31836.2), FPLV strain TU-1 (LC880188), FIV strain Petaluma (NC_001482.1), FeLV strain KT-FeLV-UCD-1 (MT129531.1), and SARS-CoV-2 strain JPN/Kanagawa/KUH003 (LC630936).

DNA or RNA was extracted from the culture supernatants of cells infected with FeAHV1, FCV, FIV, FPLV, FeLV, CCoV, and TGEV. The genomes of FeAHV1, FCV, FIV, FPLV, and FeLV were confirmed using single-plex PCR or RT-PCR with the primer sets described elsewhere.³⁹ Regarding SARS-CoV-2, total RNA (1.61 × 10⁷ copies/0.2 mL) was extracted from the lung homogenate of an infected Syrian hamster as described.²¹ The genomes of CCoV strain 1-71 and TGEV strain TO-163 were confirmed using conventional RT-qPCR targeting the FCoV 3′-UTR, as described later.

Clinical samples

Pleural effusion and ascitic fluid specimens were collected from client-owned cats with suspected FIP that were presented with confirmed effusion to veterinary clinics in Japan. Samples were submitted to our laboratory for the detection of FCoV RNA by RT-PCR. Clinical data were limited to signalment (age, sex); detailed medical histories and other background information were not accessible and, thus, were not included in the analysis. Specimens leftover from FIP testing were submitted for research use with informed owner consent. All clinical fluid samples were stored at –80°C until further use.

FCoV RNA extraction

Total RNA was extracted from 200 μL of either a FCoV virus stock or clinical fluid samples (High Pure RNA isolation kit; Roche), following the manufacturer’s instructions. RNA was eluted in 50 μL of elution buffer and stored at –80°C until used.

Quantification of FCoV 3′-UTR by conventional RT-qPCR

The quantification of FCoV 3′-UTR RNA was performed using conventional RT-qPCR with primers targeting 3′-UTR, as described.^6,10 Briefly, reactions were conducted (RNA-direct realtime PCR master mix; TOYOBO) on a real-time PCR system (StepOne; ThermoFisher). Absolute quantification was performed using a standard curve generated from in vitro transcribed FCoV 3′-UTR RNA. These values were used to estimate RNA levels in samples tested with the PCR1100 system. The LOD was evaluated across a concentration range of 100–1,000 copies/reaction and ultimately was determined to be 100 copies/reaction.⁶ For each conventional RT-qPCR assay targeting the FCoV 3′-UTR, external positive and negative controls were included to validate assay performance. Internal controls were not incorporated in these assays.

FCoV RNA detection using the PCR1100 system

Direct RT-qPCR was performed using the PCR1100 system combined with a dedicated direct RT-qPCR kit (GoFoton). Equal volumes of lysis buffer and either FCoV culture supernatants or clinical fluid samples were mixed and incubated at 20°C for 5 min. A 4-μL aliquot of the mixture was added to the RT-qPCR reagent containing proprietary primers and probes targeting FCoV RNA and feline GAPDH (internal control). The total reaction volume (20 μL) was loaded onto the PCR1100 chip, and amplification was performed at 62°C for 10 s and 95°C for 10 s, followed by 50 cycles at 95°C for 5 s and 62°C for 30 s. FCoV RNA was automatically detected by the instrument. Additionally, the PCR1100 system outputs Ct values for each RT-qPCR reaction, enabling an evaluation of amplification performance.

Samples that tested positive for FCoV RNA were analyzed once. Samples that tested negative were reanalyzed in 2 additional RT-qPCR runs, and the final result was determined based on triplicate testing.

For analytical validation, extracted RNA was added directly to the RT-qPCR reagent without the lysis step. However, not all clinical samples were tested using extracted RNA. All other procedures were identical to those used in the direct RT-qPCR protocol.

Given that the naturally occurring FCoV infections in cats are attributed to type I FCoV,^1,12,16 we evaluated the ability of the RT-qPCR reagent provided by GoFoton to specifically detect type I FCoV. We performed RT-qPCR using the PCR1100 system with nucleic acids extracted from type I FCoV, common feline viruses, and SARS-CoV-2, a primarily human virus that has been reported to infect cats.²⁹ We also tested CCoV strain 1-71 and TGEV, which are both classified under the species Alphacoronavirus suis.

To evaluate the clinical applicability of the FCoV RT-qPCR assay using the PCR1100 system, we assessed its sensitivity and specificity using ascitic and pleural fluid samples collected from cats suspected of having FIP. As a reference method, conventional RT-qPCR targeting FCoV 3′-UTR was performed using RNA extracted from the same samples.

The optimized RT-qPCR reagent for FCoV RNA detection included in the direct RT-qPCR kit eliminates the need for RNA purification. To assess the impact of RNA extraction on RT-qPCR performance using the PCR1100 system, unprocessed culture supernatants and purified RNA extracted from FCoV strain 79-1146 (~1.5 × 10⁶ copies/reaction of FCoV 3′-UTR RNA) were both analyzed.

Concordance analysis

Concordance between the direct RT-qPCR and conventional RT-qPCR methods was evaluated using JMP Pro v.18.0.2 software (SAS Institute).

Results

Analytical performance of direct RT-qPCR using the PCR1100 system

FCoV RNA was successfully detected in both unprocessed culture supernatants and in purified RNA extracted from FCoV strain 79-1146 using the PCR1100 RT-qPCR system. Positive results were obtained in all replicates down to a 10^–4 dilution for both culture supernatants and extracted RNA ( Table 1 ). At a 10^–5 dilution (~15 copies/reaction), all samples tested negative in triplicate assays, indicating that the LOD for the assay was 150 copies/reaction.

Table 1.

Limit of detection for type II feline coronavirus (FCoV) 79–1146 RNA using RT-qPCR with the PCR1100 system.

Log₁₀ relative RNA concentration	Estimated FCoV 3′-UTR RNA levels (copies/reaction, PCR1100)	PCR1100 result
Log₁₀ relative RNA concentration	Estimated FCoV 3′-UTR RNA levels (copies/reaction, PCR1100)	Supernatant	Extracted RNA
–1	150,000	Pos	Pos
–2	15,000	Pos	Pos
–3	1,500	Pos	Pos
–4	150	Pos	Neg
–5	15	Neg*	Neg

Neg = negative; Pos = positive.

All negative results were validated by 3 independent replicates.

Based on Ct values from serial dilutions, PCR efficiency was slightly higher in direct RT-qPCR using culture supernatants (82.1%) than in conventional RT-qPCR using extracted RNA (78.9%; Fig. 2 ).

Ct values obtained from serial dilutions of both extracted RNA and culture supernatants using the PCR1100 system were compared. At each dilution (10^–1 to 10^–4), the Ct values for extracted RNA were consistently lower than those for direct testing: 30.5 vs. 31.9 (10^–1), 35.0 vs. 35.6 (10^–2), 38.9 vs. 39.8 (10^–3), and 42.4 vs. 43.3 (10^–4). Although the differences were relatively small, the largest gap (4.5 cycles) was observed between the 10^–1 and 10^–2 dilutions in extracted RNA, suggesting that dilution effects may influence amplification efficiency. The underlying cause of this discrepancy remains unclear.

Analytical specificity of the RT-qPCR assay using the PCR1100 system for Alphacoronavirus suis detection

A positive signal was detected for type I FCoV, CCoV 1-71, and TGEV, whereas FeAHV1, FPLV, FCV, FIV, FeLV, and SARS-CoV-2 yielded negative results in all 3 replicates ( Table 2 ). Hence, the direct RT-qPCR assay has high analytical specificity for viruses belonging to the species Alphacoronavirus suis, indicating detection capability beyond feline coronavirus. In addition, the detection of type II FCoV (strain 79-1146) was confirmed in the analytical sensitivity evaluation (Table 1; Fig. 2), suggesting that the assay detects both type I and type II FCoVs.

Table 2.

Cross-reactivity of the PCR1100 RT-qPCR assay with feline viruses and SARS-CoV-2.

Virus species	Virus strain	PCR1100 result
Alphacoronavirus suis	Type I FCoV KU-2	Pos
	Type II CCoV 1-71	Pos
	TGEV TO-163	Pos
Varicellovirus felidalpha1	FeAHV1 C7301	Neg*
Protoparvovirus carnivoran1	FPLV TU-1	Neg
Vesivirus felis	FCV F4	Neg
Lentivirus felimdef	FIV petaluma	Neg
Gammaretrovirus felleu	FeLV KT-FeLV-UCD-1	Neg
Betacoronavirus pandemicum	SARS-CoV-2 JPN/Kanagawa/KUH003	Neg

Neg = negative; Pos = positive.

All negative results were validated by 3 independent replicates.

Clinical validation of FCoV RT-qPCR using the PCR1100 system

Of the 28 clinical samples tested, conventional RT-qPCR detected FCoV RNA in 22 samples and yielded negative results in 6 samples ( Table 3 ). The direct RT-qPCR method using the PCR1100 system identified 21 samples as positive and 7 as negative. Internal control signals were successfully amplified in all clinical samples, regardless of FCoV RNA detection, confirming the validity and integrity of each RT-qPCR reaction. Based on these results, the sensitivity of the direct RT-qPCR method was 95.5% (21 of 22; 95% CI: [78.2, 99.2]) and its specificity was 100% (6 of 6; 95% CI: [61.0, 100.0]).

Table 3.

Characteristics and RT-qPCR results of clinical fluid samples tested using the PCR1100 system.

Sample	Sample type	PCR1100 (Ct)	FCoV RNA estimated by conventional RT-qPCR, log₁₀ copies/reaction
1	Ascites	Pos (43.3)	6.4
2	Pleural fluid	Pos (38.5)	5.5
3	Ascites	Pos (37.3)	5.2
4	Ascites	Pos (35.5)	5.1
5	Pleural fluid	Pos (34.4)	5.1
6	Ascites	Pos (30.4)	4.7
7	Ascites	Pos (38.6)	4.6
8	Ascites	Pos (34.1)	4.3
9	Ascites	Pos (41.4)	3.9
10	Ascites	Pos (40.2)	3.8
11	Pleural fluid	Pos (38.8)	3.6
12	Ascites	Pos (33.5)	3.4
13	Ascites	Neg*	3.4
14	Ascites	Pos (41.9)	3.1
15	Ascites	Pos (42.7)	3.0
16	Ascites	Pos (40.9)	2.9
17	Ascites	Pos (34.5)	2.9
18	Ascites	Pos (40.5)	2.8
19	Ascites	Pos (45.6)	2.1
20	Ascites	Pos (40.8)	1.8
21	Pleural fluid	Pos (40.9)	1.5
22	Pleural fluid	Pos (43.3)	1.3
23	Ascites	Neg	ND
24	Ascites	Neg	ND
25	Ascites	Neg	ND
26	Pleural fluid	Neg	ND
27	Pleural fluid	Neg	ND
28	Pleural fluid	Neg	ND

ND = not detected; Neg = negative; Pos = positive; Pos (Ct) = Ct values from PCR1100 system.

All negative results were validated by 3 independent replicates.

Among the 28 clinical samples, 27 results were concordant (21 positive, 6 negative); 1 sample was discrepant (positive by conventional RT-qPCR and negative by direct RT-qPCR). The discordant sample was retested using extracted RNA with the PCR1100 system, yielding a positive result with a Ct value of 31.1. The overall agreement rate was 96.4%. The Cohen kappa coefficient was 0.90 (SE = 0.098; 95% CI: [0.708, 1.000]), indicating almost perfect agreement. The result was statistically significant (p < 0.0001), supporting the reliability of the direct RT-qPCR method.

Discussion

Although the RNA extraction step was omitted, the direct RT-qPCR method using the PCR1100 system accurately identified FCoV RNA in 28 clinical samples, with a sensitivity of 95.5% and specificity of 100%. These values were comparable to those obtained using conventional RT-qPCR; however, given the limited sample size and absence of confirmed negative controls, further validation is necessary to substantiate the accuracy of this method.

Among the 28 clinical samples tested, only 1 was discrepant between the 2 methods, being positive by conventional RT-qPCR and negative by direct RT-qPCR. However, when RNA was extracted from the same sample and retested using the PCR1100 system, a positive result was obtained, which suggests that the false-negative result in direct RT-qPCR may have been caused by PCR inhibitors in the sample.

The PCR1100 system successfully detected FCoV RNA in both culture supernatants and extracted RNA samples down to a 10^–4 dilution. Based on these results, we estimated the LOD to be ~150 copies/reaction. This value reflects the combined performance of sample preparation, reagents, and the device, and is comparable to that of conventional RT-qPCR using extracted RNA and a benchtop RT-qPCR instrument (i.e., StepOne).^6,10 However, in clinical samples, carryover of PCR inhibitors into the RT-qPCR reaction may have caused a false-negative result. This observation implies that direct RT-qPCR using pleural or ascitic fluid with the PCR1100 system may have a higher LOD than conventional RT-qPCR using extracted RNA, although detection in the PCR1100 system may be limited by PCR inhibitors in clinical samples. Further investigation is needed to determine whether the PCR1100 system or direct testing offers a lower LOD under specific conditions. It should be noted that the 3′-UTR of FCoV is a sequence common to both genomic and subgenomic RNAs⁷; therefore, the copy number obtained by quantification does not directly reflect the number of viral particles. Additionally, the primer and probe sequences used in the direct RT-qPCR kit were not disclosed by the manufacturer, and the exact target region is unknown. Thus, direct comparison with the 3′-UTR used in conventional methods has inherent limitations.

Nevertheless, the direct RT-qPCR method demonstrated high detection performance comparable to conventional RT-qPCR in clinical samples. Furthermore, the kit was confirmed to detect both type I and type II FCoV. Cases of FIP caused by FCoV-23 have been reported, in which most of the spike gene of type I FCoV is thought to be replaced by that of pantropic CCoV (type IIb CCoV).^2,38 Considering that the direct RT-qPCR kit detected not only type I and type II FCoV but also CCoV and TGEV, all classified under the species Alphacoronavirus suis, it is reasonable to assume that FCoV-23 could also be detected. Although we did not evaluate the LOD for type I FCoV or directly assess the detectability of FCoV-23, our results suggest that differences in target regions may have limited impact on test sensitivity. Moreover, evaluation against Alphacoronavirus outside Alphacoronavirus suis, such as human coronavirus 229E, was not feasible because of limited access to viral stocks. However, such testing would further strengthen the inclusivity assessment of the assay.

Regarding PCR efficiency, the direct RT-qPCR method yielded a slightly lower maximum efficiency of 82.1%. This result likely reflects the influence of multiple factors, including the microfluidic architecture of the PCR1100 system, the presence of potential inhibitors in the reaction mixture, and the design of primers and probes.¹⁹ Notably, a study using FMDV RNA as the sample type reported a decrease in PCR efficiency from 90.1% to 76.1% when comparing a benchtop RT-qPCR instrument with the PCR1100 system, even when using the same primer set. Our findings are consistent with that report and further support the notion that PCR efficiency can be affected by the device-specific reaction environment.

Although the calculated PCR efficiency was slightly higher in direct RT-qPCR using culture supernatants (82.1%) than in conventional RT-qPCR using extracted RNA (78.9%), the Ct values obtained from serial dilutions were consistently lower for extracted RNA. This suggests that extracted RNA may have been amplified more efficiently. However, the largest Ct gap (4.5 cycles) was observed between the 10^–1 and 10^–2 dilutions, indicating that dilution effects may also contribute to the variation. Because both assays were performed using the same PCR1100 system, the discrepancy cannot be attributed to differences in instrumentation. The underlying cause remains unclear, and further investigation is needed to determine whether sample handling, RNA integrity, or other factors influence amplification efficiency in these conditions.

The calculated PCR efficiency of the direct RT-qPCR method was slightly lower than that of the conventional RT-qPCR method, but this did not affect its clinical applicability. The method reliably detected FCoV RNA down to a 10^–4 dilution and demonstrated strong concordance with conventional RT-qPCR results. However, the exact copy number of target RNA in effusion samples was unknown, and the influence of effusion-derived inhibitors carried over into the RT-qPCR reaction could not be evaluated. Therefore, we could not assess the quantification performance in clinical samples. FCoV RNA can be detected systemically even in apparently healthy cats, but the amount of FCoV RNA detected in cats with FIP is typically higher.⁴ Therefore, interpreting RT-qPCR results based on viral load is recommended in FIP diagnostic guidelines.^34,36 Nevertheless, previous studies have shown that RT-qPCR using effusion samples can achieve >85% sensitivity and 100% specificity even without considering quantification.^5,18 Based on these findings, we suggest that the PCR1100 system and the FCoV direct RT-qPCR method could be useful POC testing tools for diagnosing wet-type FIP using effusion samples. The elimination of the RNA extraction step offers practical benefits in clinical settings, including a short turnaround time, simplified workflow, and reduced risk of contamination. The ability to perform rapid on-site detections without outsourcing to external laboratories enables timely initiation of treatment for suspected FIP cases.

Considering that the validity of PCR1100 quantitative evaluation remains uncertain at this stage, interpretation of results should be approached with caution. In particular, for use in cases such as monitoring treatment efficacy rather than diagnosis, it is important to confirm the disappearance of viral RNA rather than relying solely on changes in Ct values.

Limitations of our study include the limited number of samples analyzed, highlighting the need for validation through multi-institutional research with large cohorts. Although we focused on pleural and abdominal effusions, not all FIP cases have effusions. In particular, the diagnosis of the “dry form” of FIP, which lacks characteristic fluid accumulation, remains more challenging. We evaluated only effusion samples, and not the diagnostic performance of the direct RT-qPCR method for dry-type FIP. For the diagnosis of dry-type FIP, alternative sample types, such as CSF, aqueous humor, tissue aspirates, and blood, should be tested. Fluid-based samples, such as CSF and aqueous humor, have been reported to allow highly specific detection of FCoV RNA,^5,8,30 suggesting potential applicability of the PCR1100 system. However, tissue aspirates often contain non-cellular tissue components and other debris that may interfere with nucleic acid extraction or hinder the flow path of the device, necessitating validation separate from effusion samples. Blood samples can be processed with the PCR1100 system, but the sensitivity of FCoV RNA detection in blood is known to be relatively low, which limits the testing value of blood. Given that direct RT-qPCR is more susceptible to the effects of sample-derived inhibitors, a detailed assessment of the impact of inhibitors on sensitivity is essential to improve diagnostic reliability. Continued validation and refinement of these issues are expected to enhance the practicality and reliability of the PCR1100 system.

Footnotes

Acknowledgements

We thank Dr. Ryohei Yashima, Director of Aozora Animal Hospital, for generously providing feline ascitic and pleural effusion samples. We are truly grateful to all those who contributed to the success of our study.

Declaration of conflicting interests

GoFoton provided the PicoGene PCR1100 system device on loan, as well as the dedicated RT-qPCR chips and direct RT-qPCR reagents used in our study. GoFoton did not provide any financial support, was not involved in the study design, data collection, analysis, interpretation, manuscript preparation, or the decision to submit the article for publication. The company also had no role in the procurement of other reagents or the collection of clinical samples. The authors affirm that the study was conducted independently and without influence from the manufacturer.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Tomomi Takano

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