Automated multiplex nucleic acid tests for rapid detection of SARS-CoV-2, influenza A and B infection with direct reverse-transcription quantitative PCR (dirRT-qPCR) assay in a centrifugal microfluidic platform

The coronavirus disease 2019 (COVID-19) pandemic, caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, has posed a threat to public health worldwide. Also, influenza virus has caused a large number of deaths annually. Since co-infection of SARS-CoV-2 and influenza virus, which share similar symptoms, hampers current treatment efficiency, multiple simultaneous detection of these viruses is needed to provide the right treatment for patients. We developed a microfluidic disc-direct RT-qPCR (dirRT-qPCR) assay for rapid multiplex detection of SARS-CoV-2, influenza A and B viral infection in pharyngeal swab samples in an automated manner. Choices of the DNA polymerase, concentrations of dTPs and MgCl2 were characterized to optimize the assay. A detection limit of 2 × 101 copies per reaction was found in all three viral RNAs with as little as 2 μL of swab samples. The accuracy of our assay was evaluated with 2127 clinical swab samples of infection with these three viruses and healthy controls, and it possessed a consistency rate of 100, 99.54 and 99.25% in SARS-CoV-2, influenza A and B detection in comparison to standard RT-qPCR. The reported scheme of our assay is capable of screening other viral infections for up to 16 targets simultaneously. The whole diagnosis could be completed in 1.5 hours after simple sample loading by a non-technical expert. This constitutes an enabling strategy for large-scale point-of-care screening of multiple viral infections, which ultimately lead to a pathway for resolving the critical issue of early diagnosis for the prevention and control of viral outbreaks.


Introduction
3][4] The annual inuenza peak causes around 670 000 deaths worldwide.The World Health Organization (WHO) stressed the urgent need for tools, such as affordable point-of-care testing, to detect inuenza for better prevention and control in a country-level approach and to serve in pandemic preparedness. 5These casualties can be prevented or minimized by early diagnosis prior to treatment, such as administration of anti-viral drugs, instead of relying on human immunity and passive monitoring.Yet, co-infection of SARS-CoV-2 and inuenza virus hampers treatment efficiency in the worst-case scenario.Therefore, reliable multiple detection of both SARS-CoV-2 and inuenza virus is of great signicance in timely diagnosis to provide correct treatment of patients. 6,7ucleic acid amplication tests (NAAT) are the primary method for sensitive detection of SARS-CoV-2 and inuenza viral RNA.However, this detection involves multiple steps, from swab sampling to nucleic acid extraction and amplication, which are both time-consuming and labour-intensive even This journal is © The Royal Society of Chemistry 2020

RSC Advances
][10][11][12] Crosscontamination and environmental pollution due to human errors could lead to false-positive or negative results in clinical samples and create unnecessary public panic. 13Also, the nucleic acid extraction is inefficient in samples with low viral titer. 14irect polymerase chain reaction (PCR) was proposed as a rapidly emerging nucleic acid amplication detection. 15,16imple sample pretreatment, instead of complicated nucleic acid extraction, such as freezing-thawing of samples, treating samples with formamide and using lysates, amplied the DNA directly from the clinical samples, e.g.blood.8][19] Therefore, recent optimization of sample treatment and nucleic acid amplication in the presence of inhibitors inside crude clinical samples has supported rapid viral detection. 20It also prevents clinical sample loss, which is critical for detecting samples of low viral titer, in each step.
However, manual operation is still required for sample mixing and reagent loading.Automatic qPCR test as a nearpoint-of-care or near-patient laboratory test has been described as a less time-consuming method for COVID-19 test, but such test machines are usually stationed in centralized laboratories due to its bulkiness.Inappropriate specimen shipping to laboratories is another cause of false-negative in COVID-19 test. 21,22Microuidic platform (MP), also called the microuidic chip laboratory or lab-on-a-chip (LoC), is a bioassay approach that performs uidic manipulation in a micrometer scale.It has become a promising method for an automatic qPCR test because it miniaturizes conventional equipment for uid actuation.Moreover, a micron-scale structure and a reaction chamber in the microuidic chip, meaning a large surface area to volume ratio, enable faster heat transfer to shorten the thermocycling time in the RT-qPCR assays.Compared to conventional experimental techniques, the microuidic approach offers better prospects for incorporation of integrated electronic microcontrollers, faster reaction speed and smaller sample consumption.Meanwhile, the centrifugal microuidic platform employs a centrifugal force, instead of an external pumping system, for easy control of multiple and simultaneous uid actuation.Also, spinning action could actuate uid to multiple chambers under the same centrifugal force to support a highly accurate sample aliquoting.Consequently, the MP approach incurs less pollution and lower cost, making it more suitable for the high throughput and multiplex detection of pathogenic microorganisms. 23Various functions of conventional chemical or biological experiments have been demonstrated with the microuidic network formed by microchannels in an automated manner. 246][27][28] Precise temperature control is critical for an effective nucleic acid amplication and it could be achieved in centrifugal microuidics during the spinning mode. 29In contact-heating method, the centrifugal microuidic disc is attached to the heating source for heat conduction, and it is desirable for isothermal DNA amplication than PCR as thermocycling speed is slow. 25Double-sha turntable disc for bidirectional ow of uid supports the actuation of the PCR reaction mixture to various temperature zones repeatedly for rapid thermocycling, and multiple of PCR discs can be placed in the main turntable disc to increase throughput. 30Non-contact sensing and heating for temperature regulation has simplied the embedment of the microuidic disc onto spinning platform to support robust thermocycling without the need of complicated uidic actuation or heating-cooling electronic components. 31his study primarily aims to provide an immediate solution to tackle both the current pandemic and a possible outbreak of inuenza in near future.To ensure a high degree of automation and lower risk of cross-contamination, we have designed and fabricated a microuidic platform and established the direct reverse-transcription quantitative PCR (dirRT-qPCR) assay on the microuidic platform for multiplex detection of SARS-CoV-2, inuenza A and B viruses in pharyngeal swab samples.Our optimized dirRT-qPCR assay, integrated with centrifugal microuidics, provided a reliable direct sample detection.Compared to the conventional RNA-extraction based RT-qPCR method, the microuidics disc-dirRT-qPCR assay has achieved high sensitivity and automation to detect SARS-CoV-2, inuenza A and B viral RNA simultaneously in swab samples.Accuracy on direct detection in clinical swab samples has also been validated.

Collection and processing of clinical samples
Clinical specimens, including 29 SARS-CoV-2, 169 inuenza A and 356 inuenza B positive samples, and 1572 negative samples from healthy individuals or infected with other respiratory diseases, were collected from August 2019 to May 2020 in the health and quarantine laboratory of Shenzhen International Travel Health Care Center according to the standard clinical sample collection protocol, with ethical approval and written consent from the patients and the volunteers.The samples were additionally treated with Minimal Essential Medium Eagles and Earle's Balanced Salts (MEM/EBSS) liquid broth and stored at À80 C prior to the experiment.Standard RNA of SARS-CoV-2, inuenza A and B were extracted from patient samples with the conventional RNA extraction kit as described previously. 20

Design of the primer pair and probes
The primer pair and the probe were synthesized by UNIM-EDICA.The design of the primer pair and the probe was based on the alignment of N gene of SARS-CoV-2 (Gene ID: 43740575), conserved matrix (M1) gene of inuenza A virus (Gene ID: 956527), and variant hemagglutinin (NP) gene of inuenza B virus (Gene ID: 26824002) published in GenBank.The primers and the probe were designed with Primer Premier 5.0 and Primer Express 3.0.1 to generate an amplicon size of 99, 106 and 84 bp of SARS-CoV-2, inuenza A and B virus respectively.The sequence of the primer pair and the hydrolysis probe, along with the sizes of the expected amplicons, were listed in Table 1.For the fabrication of microuidic disc, injection molding technology with a molding angle of 3 on transparent PMMA was used to mass-produce the base microuidic disc layer, which provides the microuidic network and the reaction vessel for the uid actuation.Fig. 2A shows the design of the disc with a diameter of 120 mm and thickness of 3 mm.The microuidic system is divided into four independent reaction units and each unit accounts for a quarter of the disk area.Each unit includes a sample-loading hole and a sample-loading tank for mixing of sample and dirRT-qPCR reaction mixture, a siphon sample inlet channel, metering chambers, a series of capillary tension valves, PCR reaction tanks, a waste liquid tank.The designed micro-uidic disc in our work can simultaneously detect four clinical samples, and 16 targets of each sample can be evaluated at the same time.Aer pre-loading of primer pairs, probes and dirRT-qPCR reaction mixture, the base layer is sealed with a transparent lm on top, for the ease of observation of uidic ow, and stored at 4 C until use.

Microuidic disc-dirRT-qPCR assay
Four DNA polymerases, PrimeDirect (Takara), OmniTaq (DNA Polymerase Technology Inc), Alpha Taq (VitaNavi) and TTX (TOYOBO), which are mutants of DNA polymerase resistant to the inhibitory effect of blood, were selected to evaluate the efficiency of our microuidic disc-dirRT-qPCR assay.The initial PCR conditions were established according to the melting temperature (T m ) value of the primer pairs and the probe and the recommended reaction temperature of the DNA polymerases.We selected the reaction mixture containing DNA polymerase, a primer pair, a probe, and a sample template with optimized concentrations of MgCl 2 and dNTPs.The micro-uidic disc-dirRT-qPCR reactions were performed in our microuidic machine with a volume of 20 mL each and capable for, at maximum, 16 simultaneous target detection.Each experiment was repeated in triplicate.Positive control and no template control (NTC) were included in each experiment.Pri-meDirect polymerase was selected for the downstream PCR assay with the corresponding reaction procedure shown as follows: reverse transcription was performed at 90 C for 3 min and 60 C for 5 min for RT reaction, then 95 C for 5 s, followed by 60 C for 30 s, and repeated for 40 cycles.To validate the accuracy of the nucleic acid amplication, agarose gel electrophoresis was performed on the samples aer 30 and 40 cycles of reaction.The gels were imaged to evaluate the amplicon size and number of the bands.To validate the practical performance, sensitivity and accuracy of this assay were evaluated using the standard viral samples and clinical swab samples.

Statistical analysis
Statistical analyses were conducted using OriginPro (version 2018) and GraphPad Prism (version 5).The sensitivity and reliability of the dirRT-qPCR assay were determined by analyzing the mean cycle threshold (Ct) values and standard deviations (SD) of Ct values with that in conventional RT-qPCR assay.

Results
Validation of the sample-to-answer multiplex assays on the centrifugal microuidic platform Fluidic actuation inside the disc was rst characterized before integrating the dirRT-qPCR assay into it for thermocycling reaction and uorescence detection.We adjusted the rotating speed based on the design of uidic channels on the micro-uidic disc to ensure the uidic ow is highly consistent.Microuidic-disc dirRT-qPCR experiment was performed on the disc with the automated spinning prole in Fig. 2B.The schematic diagram (upper) and photos (lower) of each uidic actuation step in our centrifugal microuidic are shown in Fig. 2C.The unpinned disc was rst increased to 800 rpm for 50 s to draw the loaded samples, mixed with dirRT-PCR reaction cocktails, from the sample-loading tank to the metering chamber through a siphon valve.It was increased to 1600 rpm for 30 s to pass all the excess samples aer metering to the waste chamber.It was then increased to 2600 rpm for 60 s to overcome the resistance of the capillary tension valve and ow to the dirRT-qPCR reaction tanks, pre-loaded with primer pairs and probes, for the target multiplex viral detection.It was increased to 3000 rpm for 6 s to ensure all the metered uid owed into the reaction tanks.The centrifugal speed was set as 400 rpm to hold the uids inside the reaction tanks for the subsequent dirRT-qPCR reactions.The total centrifugation time for sample mixing, metering and loading to multiplex reaction sites was within 180 s.

Sensitivity of the microuidic-disc multiplex dirRT-qPCR assay
Aer characterization of uid actuation in our centrifugal microuidic disc, dirRT-qPCR was integrated into the reaction on our microuidic platform and its performance was evaluated.Four commercially available polymerases (PrimeDirect, This journal is © The Royal Society of Chemistry 2020

RSC Advances Paper
OmniTaq, Alpha Taq, and TTX) that claimed to work on clinical samples, such as blood, for direct PCR were included in our assay.Amplication efficiency was evaluated by both the amplication curves and the Ct value of these four reaction mixtures with the standard viral RNAs-sparked swab samples (Fig. 3).All of them worked in our dirRT-qPCR assay, showing positive signals with a mean Ct below 30.Also, amplication efficiency of PrimeDirect polymerase was the highest, with its lower Ct value and higher overall uorescence signals with time, out of the four polymerases (Fig. 3A).Further statistical analysis (Fig. 3B) shows that the use of PrimeDirect polymerase outweighed the other polymerases and provided faster detection.Amplication efficiency was further characterized using endpoint dirRT-PCR followed with gel electrophoresis, which could provide not only evidence on amplication efficiency, determined by the intensity of the bands, but also its accuracy on amplifying the correct amplicon size.Fig. 3C shows the gel electrophoresis of the dirRT-qPCR assay with four polymerases at both cycle 30 and 40.All polymerases could amplify the correct amplicon size without amplifying other regions or potential primer-dimer regions.PrimeDirect polymerase showed a brighter band compared to other polymerases at both cycle 30 and 40.Therefore, PrimeDirect polymerase was selected for the downstream experiments.Apart from the evaluation of the polymerases to select the best one for our dirRT-qPCR assay, we have characterized the dirRT-qPCR reagent used, including the essential cofactor magnesium ions (Mg 2+ ) and substrate dNTP for polymerase to construct the amplicon, on both amplication efficiency and accuracy.In addition, the optimized concentration of these components is critical to reducing the false negative by dNTPs, with a chelating effect to interact with magnesium ions, at nonoptimal high concentrations to inhibit dirRT-qPCR reaction.To determine the optimal concentrations of dNTPs and MgCl 2 , the dirRT-qPCR was performed with PrimeDirect polymerase and the reaction mixture and a combination of various concentrations of MgCl 2 (2.5-6.5 mM) and dNTPs (0.4-1.0 mM).To evaluate the amplication efficiency, the Ct values were summarized in three-dimensional bar graphs in Fig. 3D.The optimal concentrations of dNTPs and MgCl 2 were determined by the lowest Ct value, indicating the highest amplication efficiency.The increase in the concentration of MgCl 2 showed an insignicant improvement in amplication efficiency, while a high concentration of dNTPs inhibited the amplication at  Aer the optimal condition was established, we evaluated its sensitivity in detecting standard concentrations of three viral RNAs sparked in swab samples.It was rst evaluated using simulated clinical samples with standard viral RNAs ranging from 2 Â 10 0 to 2 Â 10 6 copies per reaction.There were amplication curves in all the concentrations when compared to negative (black curve) in SARS-CoV-2, inuenza A and B screening (Fig. 4A-C), suggesting that this dirRT-qPCR on the microuidic platform could be performed with direct sample addition without sample pretreatment.The high linearity (R 2 ¼ 0.99) of all three standard curves (Fig. 4D) supports the use of dirRT-qPCR assay for quantication of viral RNA copies number ranging from 2 Â 10 1 to 2 Â 10 6 copies per reaction in clinical This journal is © The Royal Society of Chemistry 2020

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Paper infection samples.The detection limits were 2 Â 10 1 copies per reaction in all three viruses (Table 2).

Clinical evaluation of the microuidic disc-dirRT-qPCR assay on clinical samples
Aer understanding its sensitivity and dynamic range of our microuidic disc-dirRT-qPCR assay, we employed it for screening samples with clinical infection of these three viruses.The accuracy was evaluated by comparing our established microuidic disc-dirRT-qPCR assay and the current standard RNA-extraction based RT-qPCR on clinical specimens of these viral infections.Fig. 5A-C shows typical amplication curves of our established microuidic-disc-dirRT-qPCR (solid lines) and standard RNA-extraction based RT-qPCR (dash lines) from two patients (red and blue lines) infected with the indicated disease.
The overlapping curves of solid and dash lines of each patient suggest the amplication rate is similar between our micro-uidic disc-dirRT-qPCR and conventional RT-qPCR and thus resulted in similar Ct values for disease screening.Fig. 5D-F show a statistically signicant difference in the Ct value between 29 positive samples of the three types of virus infection compared to the negative controls.It therefore supports the use of our platform as a potential standard method for effective screening of positive/negative disease infection.40 cycles with sample pre-processing in our microuidic platform only requires as short as 90 min.The mean Ct value were 27.71, 28.20 and 29.80 in SARS-CoV-2, inuenza A and B respectively.It suggested that positive results can be conrmed in 57 min aer sample loading in our assay.
Furthermore, the evaluation of the accuracy, an important criterion for effective biosensing, has been done with 2127 clinical swab samples of infection with the three viruses and healthy controls in comparison to the current gold standard, i.e. conventional RNA-extraction based RT-qPCR detection to support the reliability of our methods in practical use.Table 3 shows the summary of larger-scale sample analysis in screening patients infected with SARS-CoV-2, inuenza A and B with a total of 2127 clinical samples (29 SARS-CoV-2-positive samples, 171 inuence A-positive samples, 355 inuence Bpositive samples), and 1572 negative samples from healthy individuals or infected with other respiratory diseases were used in a larger-scale sample analysis, and it showed 100%, 99.54% and 99.25% accuracy on the diagnosis of SARS-CoV-2, inuenza A and B respectively.Positive predictive value (PPV) of detecting SARS-CoV-2, inuenza A and B are 100%, 97.11% and 97.75% and negative predictive value (NPV) of detecting them are 100%, 99.81%, and 99.71% respectively.To show the consistency of these methods on clinical detection, Fig. 5G-I shows the relationship of Ct between our microuidic-disc-dirRT-qPCR (x-axis) and conventional RT-qPCR (y-axis) in each positive (blue) or negative (orange) sample for SARS-CoV-2, inuenza A and B screening, respectively.The positive sample (blue dots) tends to shi in upper le region (65.5%, 61.4% and 63.4% for SARS-CoV-2, inuenza A and B screening respectively) indicated smaller Ct values were found in our microuidic-disc-dirRT-qPCR.Therefore, our nucleic amplication assay of this paper is a less time-consuming method compared to standard RNA-extraction based RT-qPCR.3][34][35][36] The overall reduction in time and reagent loading and downstream processes support the use of our method over standard RNA-extraction based RT-qPCR.

Discussion
The novelty of this work lies in the implementation of our automated centrifugal microuidic platform and optimized dirRT-qPCR reaction for sample-to-answer diagnosis of multiple viral infections from unpuried clinical pharyngeal swab sample.It offers a simple operating process, i.e. loading swab samples onto the disc, as well as high sensitivity and accuracy to demonstrate a simultaneous screening of clinical SARS-CoV-2, inuenza A and B virus infection.The entire detection process can be completed within 1.5 hours, and positive signals can be detected in 57 min, making it at least three times faster with the conventional RT-qPCR approach. 37 [32][33][34]36 This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 34088-34098 | 34095 Paper RSC Advances The test can be conducted with a very small sample volume, thus greatly reducing the risk of human contact during sample collection.Moreover, the use of small sample volume also facilitates its application in situations where the availability of sample is relatively scarce.The reported centrifugal micro-uidic platforms could provide the simple processes in an automated manner, similar to other reported centrifugal microuidic systems, but our integration with dirRT-qPCR assay with conditions optimized could further simplify the design of microuidic disc and reduce the processes to be executed during disc spinning, thus increasing the robustness and accuracy for clinical use.
In recent years, microuidic-based assays and direct PCR amplication have gained popularity, since they reduce laborious procedures and time needed for the extraction and puri-cation of nucleic acid and sample aliquoting for multiple detection.9][40] This reported work is mainly focused on swab as it has been considered as a better minimally invasive method than blood for sampling.Pharyngeal swabs, the most common sample acquired, are believed to contain fewer PCR inhibitors and are relatively simple to collect than blood or plasma.Non-invasive saliva collection is also considered as an alternative to replacing invasive venipuncture for virus diagnosis, as a recent study showed that saliva is a more reliable tool for the detection of SARS-CoV-2. 41It was also observed that positive detection of inuenza viral genetic markers in the pharyngeal swab samples was only possible within two days aer the onset of the disease, while detection in saliva was possible for at least four days on average.The range of RNA viral load in saliva is 10 4.1 to 10 7.4 copies per mL. 42Therefore, NAAT has shown both good sensitivity and specicity for inuenza screening, where nearly both 100% PPV and NPV could be achieved using different combinations of primers, probes and RT-qPCR cocktails. 43,44Although our primers targeting SARS-CoV-2 viral sequence was established recently, a higher specicity lies in a more comprehensive genome analysis of the virus.Unfortunately, the high mutation rate of RNA-based virus is one of the reasons of false negative in NAAT, where the primer or the probe-binding site does not match the mutated viral sequence.Addition of COVID-19-RdRp/Hel was evaluated lately to provide a higher speci-city. 45It can be incorporated into our multiplex detection system without affecting the workow.In our platform, the high dynamic range, from 2 Â 10 1 to 2 Â 10 6 copies per reaction, supports diagnosis of viral infection in different stages.Viral load in patients with SARS-CoV-2 infection ranged from 10 2 to 10 6 copies per sample in different phases of infection and sample types.High viral load was found in samples of asymptomatic patients. 46Therefore, our platform could provide not only screening in suspected cases for early diagnosis, but largescale screening to nd disease carriers.It is worth noting that viral shedding may start before the onset of symptoms and therefore creates difficulties in accurate detection.Complete SARS-CoV-2 viral shedding may occur aer a median of 20 days, with Ct > 40 that leads to false negative in RT-qPCR assay. 47he detection time of other methods that omit RNA extraction can be ranging from 0.5-3.5 hours, for example, the SHERLOCK method takes 2 hours, 48 while the heat treatment approach takes 3.5 hours. 49Other nucleic acid amplication strategies, such as isothermal DNA amplication, instead of PCR, can also be used to further increase its sensitivity, speci-city and shorten its reaction time. 50Although LAMP-based isothermal amplication on SARS-CoV-2 can be completed within 0.5 hours, it is not suitable for quantication, which is important to evaluate the severity of the infection and potential transmission rate of the patient for disease control. 51Moreover, our platform can be translated to detecting other biomarkers rather than viral RNA.One example is human microRNAs, which are upregulated or downregulated in different disease infections, as alternative biomarkers to tackle the problem of false-negative in targeting RNA virus with high mutation issue. 52,53Apart from detecting nucleic acid for disease diagnosis, our centrifugal microuidic platform could be used in protein-based immunoassays to provide additional information of infection status by screening viral surface protein antigens, or seroconversion, i.e. the extent and duration of immunity to the virus, by screening human antibodies regenerated aer infection, where the latter is particularly useful for longitudinal serological studies in post-pandemic monitoring. 54The multiplex immunoassay based on multiple lateral ow paper strip assays has been demonstrated to extend its rapid, automated and simultaneous detection of multiple kinds of pathogen surface antigen with the use of centrifugal microuidics. 55,56ith the ongoing COVID-19 pandemic, the WHO expected that the COVID-19 may become an endemic disease and constant outbreaks may be common in the future.At the same time, the uncontrolled spread of inuenza A and B viruses in high-population areas has been an emerging issue.Inuenza season in the fall-winter period contributes to a signicant amount of disease-caused death, and as its symptoms are similar to COVID-19 it may create public panic.In addition, since lockdown measures of different countries will be loosened in the coming months, reliable continuous monitoring of the virus is needed among cross-border individuals, especially children, who had a high incidence of other common coronavirus infections and were more likely to be infected and transmit viruses across the border, and the elderly, who in general have a weaker immune system. 57Our microuidic disc-dirRT-qPCR with its platform could achieve a more straightforward and convenient detection as an alternative to the current standard RNA-based RT-qPCR method.Our platform enables the high-throughput sample-to-answer multiplex detection of SARS-CoV-2, inuenza A and B viruses and other emerging infectious diseases, supporting point-of-care diagnosis for on-site screening.It ultimately realizes the goal of large-scale screening of viral infection for early diagnosis and the prevention and control of viral outbreaks for post-pandemic prevention and monitoring.
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 34088-34098 | 34089 Paper RSC Advances Centrifugal microuidic platform Our centrifugal microuidic platform consists of the following compartments: a microuidic disc layer, an optical detection unit, a temperature control unit, and a mechanical control unit for both nucleic acid amplication and real-time uorescence detection.Fig. 1A shows the image of the machine layout and a magnied view of the microuidic layer.Fig. 1B is the schematic diagram showing the light path in an optical detection unit, regulated heat ow in the temperature control unit, and a motorized stage for disc spinning in the mechanical control unit.Air ow for heating and cooling was employed to allow a rapid temperature thermocycling to shorten the time of RT-qPCR.Incident LED light excitation of 494 and 538 nm wavelength and a photomultiplier tube (PMT) light detector with a bandpass lter for 518 and 554 nm were used for collection of the uorescence emission light in this experiment.

Fig. 1 (
Fig. 1 (A) Image of the constructed centrifugal microfluidic equipment and magnified view of the microfluidic cassette.The diameter of the disk is 125 mm, and the reaction tank is located on the periphery.(B) Schematic diagram showing the spinning, thermocycling and optical detection in the microfluidic equipment.

Fig. 2
Fig. 2 Microfluidic disc configuration and spinning profile in the microfluidic platform.(A) Schematics of the microfluidic disc showing four independent units in a single disc, with all of the features and the chambers labelled.(B) Configuration and (C) characterization of the spinning profile of the designed disc in our microfluidic platform.Schematic diagram (upper) and photos (lower) show the sequential step of the fluid actuation under spinning program.

Fig. 3
Fig. 3 Optimization of the reaction mixture in microfluidic disc-dirRT-PCR to achieve effective detection in the swab samples.(A) Amplification curves and (B) statistical Ct analysis shows the amplification efficiency of four selected DNA polymerases, i.e.PrimeDirect, OmniTaq, Alpha Taq, and TTX on the standard viral RNAs-sparked swab samples.(C) Image of gel electrophoresis of end-point dirRT-PCR using four polymerases at both cycle 30 and 40.(D) Summary of Ct analysis using PrimeDirect polymerase and varying MgCl 2 (2.5-6.5 mM) and dNTPs (0.4-1.0 mM) concentrations in the reaction mixture.(E) Image of gel electrophoresis of end-point dirRT-PCR followed with gel electrophoresis on four selected conditions (unoptimized concentrations of dNTPs and MgCl 2 , lowest concentrations of dNTPs and MgCl 2 , highest concentrations of dNTPs and MgCl 2 , optimized concentrations of dNTPs and MgCl 2 ) at both cycle 30 and 40.

Fig. 4
Fig. 4 The sensitivity of the microfluidic disc-dirRT-qPCR assay in multiplex detection of SARS-CoV-2, influenza A and B. (A-C) Amplification curves using standard viral RNAs of (A) SARS-CoV-2, (B) influenza A and (C) influenza B sparked swab samples.(D) Standard curve of mean Ct with standard viral RNAs concentrations of the three viruses.

Fig. 5
Fig. 5 Accuracy of clinical detection with our microfluidic dirRT-qPCR compared to standard RNA-extraction based RT-qPCR for multiplex detection of SARS-CoV-2, influenza A and B viruses in clinical infectious swab samples.Typical amplification curves of our microfluidic-disc-dirRT-qPCR (solid lines) and standard RNA-extraction based RT-qPCR (dash lines) from two patients (red and blue) and Ct analysis on effective screening of patients infected with (A and D) SARS-CoV-2, (B and E) influenza A, (C and F) influenza B as well as healthy individuals as the negative control.(G-I) Relationship of Ct between our microfluidic-disc-dirRT-qPCR (x-axis) and conventional RT-qPCR (y-axis) of each positive (blue) or negative (orange) sample for (G) SARS-CoV-2, (H) influenza A and (I) influenza B screening.Percentages of the positive sample (blue dots) shifted in upper left and lower right regions were reported.

Table 1
Sequences of primer pairs and hydrolysis probes used in this study

Table 2
Summary of sensitivity of microfluidic-disc dirRT-qPCR assay

Table 3
Summary of evaluation on the accuracy of clinical detection with our microfluidic disc-dirRT-qPCR compared to standard RNAextraction based RT-qPCR

Table 4
Summary of operational processes with our microfluidic disc-dirRT-qPCR compared to standard RNA-extraction based RT-qPCR