Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus

Objective A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Methods Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. Results By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 μmol/L and 0.2 μmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 μmol/L; the final concentrations of FluA probe and primer were 0.2 μmol/L and 0.3 μmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 μmol/L and 0.25 μmol/L, respectively. Conclusion A multiplex real-time quantitative fluorescence RT-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes.


Background
Te coronavirus disease 2019 (COVID- 19) is an emerging acute respiratory infectious disease that has now evolved into a major global public health event.It has been demonstrated that early detection, reporting, and isolation of the disease can efectively contain the spread and dissemination of coronavirus [1][2][3].During winter, however, it becomes more difcult to diagnose COVID-19 with increasing transmission of other respiratory viruses with similar symptoms.
COVID-19 and infuenza are both respiratory infectious diseases, which can be transmitted through droplets and contact, and the early symptoms of both are similar and difcult to distinguish, such as fever, dry cough, and sore throat [4][5][6].
COVID-19 is caused by a type B coronavirus (SARS-CoV-2) with a genome of around 30 kb [7][8][9], which is a single-stranded RNA (+) virus belonging to betacoronavirus and is capable of infecting both the avian and human species.Infuenza is caused by RNA viruses of the Orthomyxoviridae family with genomes of about 14 kb, including infuenza A (FluA), infuenza B (FluB), and infuenza C (FluC) viruses [9,10].Infuenza A and B viruses may cause regional or even large-scale epidemics.Infuenza viruses and SARS-CoV-2 can invade the epithelial cells of the upper respiratory tract, and virions are spread by large droplets produced by infected individuals when they cough and sneeze, which leads to the invasion of the epithelial cells of the upper respiratory tract [11,12].
Te Diagnosis and Treatment Protocol of COVID-19 (Trial Version 8) requires that the suspected COVID-19 cases should be diagnosed by methods including rapid antigen testing and multiplex PCR nucleic acid testing to distinguish from infuenza virus infection.In addition, the combined assay for SARS-CoV-2, FluA, and FluB is able to detect coinfections.A study involving 93 cases found that 50% of SARS-CoV-2 infections were coinfected with FluA/B, which may lead to earlier organ damage in patients with critical conditions [2].Concurrent tests for COVID-19, FluA, and FluB not only reduce the number of tests required for patients but also allow a timely clinical treatment plan for coinfected patients.
Diferential diagnosis of SARS-CoV-2 and infuenza viruses will be helpful in establishing appropriate strategies for public health and patient management, especially in the diagnosis of suspected cases, critical cases, and in the identifcation of potential outbreak risks.Adding infuenza detection to COVID-19 assays can efectively shorten the test time and improve the efciency of available equipment, personnel, and reagents, which is cost-efective for the containment of the COVID-19 pandemic.A rapid test for SARS-CoV-2, FluA, and FluB is needed during the prevalent seasons of respiratory viruses to control the pandemic and allow timely diagnosis and treatment for patients.
Te most cost-efective preventive and control measure in the face of various emerging infectious diseases is to establish rapid and accurate nucleic acid molecular diagnostic methods, which are based on fully automated and integrated molecular diagnostic systems.Te fully automated and integrated molecular diagnostic system can automatically complete the entire process of testing, including sample lysis, nucleic acid extraction, nucleic acid rinsing, nucleic acid elution, gene amplifcation (PCR), and real-time fuorescence quantitative detection, and can realize the "samples-in, result-out" [13].
Te integrated molecular diagnostic system has several advantages over the common PCR method: it does not require a nucleic acid extractor or PCR instrument to be used in conjunction with the test, but only one integrated instrument.Compared with the ordinary PCR method, the integrated molecular diagnostic system has multiple advantages: it is not necessary to match the equipment such as nucleic acid extraction instruments, PCR instruments, and other equipment, and only one integrated instrument can be detected; the operator simply needs to add the sample to the kit and insert it into the instrument for testing, without requiring any additional steps during the testing process, thereby optimizing time and efort efciency; fully closed automated experimental process can avoid sample crosscontamination and environmental pollution to maximize the protection of the operator's safety.Nowadays, many organizations are actively carrying out the development and research of fully automated and integrated molecular diagnostic systems [14][15][16][17][18][19].

Design of Primers and Probes.
Te conserved sequences of the SARS-CoV-2 N gene, ORF1ab gene, FluA M gene, and FluB NS gene were selected as amplifcation targets, and specifc primers and fuorescent probes were designed to detect the sample RNA through the change of fuorescent signals (Table 1).

Establishment of PCR System.
A conventional PCR procedure and a rapid amplifcation procedure were set up in the experiment for the amplifcation of the SARS-CoV-2 N gene and FluA M gene to evaluate the efects of enzymes from diferent manufacturers on the amplifcation results and to select the best Taq DNA polymerase for the rapid amplifcation system.
2 International Journal of Analytical Chemistry (3) Te amplifcation reagents were prepared and tested separately on the prototype Fully Automated Nucleic Acid Amplifcation Testing System (PFANAT-1), with the number of parallel tests being N � 3 for each condition before calculating the average Ct values and selecting the best rapid amplifcation system.Te amplifcation procedure was confgured according to Table 3. Amplifcation procedure 1 is the best solution after considering the amplifcation conditions of the four manufacturers, which is defned as the conventional amplifcation procedure.Amplifcation procedure 2 is a fast amplifcation program optimized for amplifcation time, which is defned as the rapid amplifcation procedure.

Amplifcation System Optimization
(1) Ten diferent Mg 2+ concentrations (fnal concentrations of 0 mmol/L, 1 mmol/L, 1.5 mmol/L, 2 mmol/L, 2.5 mmol/L, 3 mmol/L, 3.  International Journal of Analytical Chemistry the standard curve.Ten, serial dilution samples were detected and a 90% positive detection rate was calibrated by the four-parameter ftting algorithm to determine the lowest detection limit, and the clinical samples were diluted to 200 copies/mL using a sample preservation solution to confrm the lowest detection limit.Te tests were repeated 20 times under the optimized conditions, and the positive detection rate was obtained based on the tests of three samples of diferent sources of each virus tested with three diferent batches of kits.Among them, FluA included fve subtypes, H1N1, H3N2, H7N9, H5N1, and H1N1 (2009), and FluB included two subtypes, Victoria and Yamagata.

Efects of Enzymes on SARS-CoV-2 and FluA Tests.
Te results are shown in Figure 1, which indicates that F enzyme is unable to achieve rapid amplifcation, E enzyme Ct is delayed, and A enzyme and N enzyme can lead to rapid amplifcation, with N enzyme working the best.

Results of FluA Tests at Diferent Mg 2+ Concentrations.
Te Ct value of FluA decreased with the rise of Mg 2+ concentration, and there was no signifcant diference when the concentration of Mg 2+ was at 3 mM or above.Terefore, 3 mM was determined as the optimal concentration of Mg 2+ considering that excessive concentration would lead to nonspecifc amplifcation (Figure 2).

Test Results of Diferent Concentrations of Primer Probes.
Te Ct value of SARS-CoV-2 N decreases when both primer and probe concentrations are increased, so the fnal concentration of N probe is set at 0.15 μM and the fnal concentration of primer can be 0.2 μM since the efect of primer is close at the concentrations ranging from 0.05 μM to 0.4 μM (Table 4); the Ct value of SARS-CoV-2 ORF increases when both primer and probe concentrations are increased, so the fnal concentration of ORF probe is set at 0.15 μM and the fnal concentration of primer can be 0.15 μM since the efect of primer is desirable at the concentrations ranging from 0.15 μM to 0.25 μM (Table 4).Te Ct value of FluA decreases when both primer and probe concentrations are increased, so the fnal concentration of FluA probe is set at 0.2 μM and the fnal concentration of primer can be 0.2 μM since the efect of primer is desirable at the concentrations ranging from 0.15 μM to 0.4 μM (Table 4); the Ct value of FluB decreases when both primer and probe concentrations are increased, and therefore the fnal concentration of FluB probe is set at 0.15 μM and the fnal concentration of primer can be 0.25 μM since the efect of primer is desirable at the concentrations ranging from 0.15 μM to 0.4 μM (Table 4).

Comparison of Results between Multiplex Systems and Single Systems.
In order to optimize the amplifcation system, amplifcation tests were performed using the synthetic RNA of SARS-CoV-2, FluA, and FluB, with Psrp RNA (fragment sequences derived from Arabidopsis genomes) as the internal control.Every assay had parallel tests of 3 samples, and the results showed no signifcant diference in amplifcations between the multiplex system and the singleweight system (Table 5).
As revealed by the results of the three batches of diferent detection reagents, 200 copies/mL for the clinical samples or viral cultures of SARS-CoV-2, FluA, and FluB all met the  International Journal of Analytical Chemistry 5 requirements of 95%-100% positive detection rate, as shown in Table 7 and Supplementary Table 3.So, the minimum detection limits of SARS-CoV-2, FluA, and FluB can be set to 200 copies/mL.

Discussion
In response to respiratory infectious diseases, early and rapid diagnosis can control the development of the disease as early as possible and reduce the number of critical patients.However, the premise of rapid diagnosis is to ensure the sensitivity and accuracy of the detection.Terefore, designing a reaction system to ensure rapid and efective amplifcation of nucleic acid is the core of this study.Finally, a multiplex fuorescence RT-PCR assay was designed in this study to establish and optimize a multiplex amplifcation system for COVID-19, infuenza A, and infuenza B to achieve the diferential diagnosis of COVID-19 and infuenza in 30 minutes.Tis study proved that the enzymes from diferent manufacturers could afect the amplifcation results, and N enzymes could achieve rapid amplifcation through experiments.Te detection principle is that the DNA polymerase with 5′∼3′ DNA exonuclease activity will degrade the probe when it meets the fuorescence-labeled probe bound to the template strand during the PCR extension, resulting in the release of fuorescence to be detected by the real-time quantitative PCR instrument [20].On Taq DNA polymerase, the polymerase active region and the 5′∼3′ DNA exonuclease active region are found in diferent structural domains [21], and these two active regions work together to initiate the synthesis of new DNA strands while cleaving the fuorescent probe and releasing the signal.Among the reactions of polymerization and exocytosis, the less efcient reaction directly determines the efciency of DNA amplifcation and the release of fuorescent signals.As reported, the Taq polymerase does not degrade the entire probe, and the degraded part is about 5-12 bp from the 5′ ends of the probe; the undegraded probe may participate in the subsequent PCR cycles, so as to inhibit the release of the fuorescent signal; the degradation of the probe is closely associated with the intensity of the Taq enzyme's exonuclease activity [22,23].In the case of the limited amount of enzyme (0.38, 0.19 U/reaction), once the Taq polymerase with the same polymerase activity is added to the reaction, the enzyme with higher exonuclease activity shows higher amplifcation efciency; when Taq polymerase with the same exonuclease activity is added, the amplifcation efciency is basically similar even though the polymerase activity is diferent.Te abovementioned results demonstrate that the exonuclease reaction is a key step for rate control, and the rate is crucial to the efciency of DNA amplifcation.Due to the dependence of enzyme activity on Mg 2+ concentration in the PCR reaction system, the absence of Mg 2+ will lead to enzyme inactivation, and the enzyme activity will be inhibited when the concentration of Mg2+ is high.In the study of optimizing the isothermal amplifcation reaction system, Mg 2+ concentration in the system has a signifcant efect on the amplifcation efciency [24].In addition, the divalent cations also afect the dissociation temperature and annealing temperature of the primer and template hybrid.Terefore, the concentration of Mg 2+ in the system will afect the amplifcation efciency.In this study, it was found that the amplifcation efciency was the highest and the expansion speed was the fastest when the Mg 2+ concentration was 3 mmol/L in the multiple amplifcation system.Te fnal concentrations of primers and probes for the three respiratory virus assays were determined through the optimization of concentrations of primer and probe in the amplifcation system.Te fnal concentrations of SARS-CoV-2 NP probe and primer were 0.15 μmol/L and 0.2 μmol/L, respectively; the fnal concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 μmol/L; the fnal concentrations of FluA probe and primer were 0.2 μmol/L and 0.3 μmol/L, respectively; the fnal concentrations of FluB probe and primer were 0.15 μmol/L and 0.25 μmol/L, respectively.Te detection of the three viruses using the multiplex assay system constructed in this study did not show a diference from those of the single system amplifcation, and the minimum detection limits of the present study for SARS-CoV-2, FluA, and FluB could all reach 200 copies/mL.Relevant research results show that the minimum detection limit of single nucleic acid detection of respiratory pathogens (such as SARS-CoV-2) is mostly in the range of 250-1000 copies/ mL, and the amplifcation time is mostly more than 1 h.Although some studies claim that the minimum detection limit of the method can reach 200 copies/mL or lower, the amplifcation time is close to 2 h [25][26][27][28][29].
In summary, by optimizing the amplifcation conditions and using a self-made device that integrates sample lysis, nucleic acid extraction, nucleic acid purifcation, multiplex fuorescent PCR, and result analysis, this study achieves the purpose of 30 min rapid detection without additional operation and the minimum detection limit of 200 copies/mL.International Journal of Analytical Chemistry Te assay can be used for clinical rapid diferential diagnosis, contributing to the combat against the SARS-CoV-2 pandemic as well as the seasonal infuenza.

Figure 1 :Figure 2 :
Figure 1: (a) Comparison of the amplifcation efciency of the SARS-CoV-2 N gene by diferent enzymes in the conventional amplifcation procedure and the rapid amplifcation procedure.(b) Comparison of the amplifcation efciency of FluA by diferent enzymes in the conventional amplifcation procedure and the rapid amplifcation procedure.

Table 4 :
Ct values under diferent concentrations of primers and probes.

Table 5 :
Ct values of the multiplex PCR system and single PCR system.

Table 6 :
Te concentrations of the clinical samples or viral cultures of SARS-CoV-2, FluA, and FluB.

Table 7 :
Positive detection rate for viral culture tests of 200 copies/mL for SARS-CoV-2, FluA, and FluB.