PHWR Public Health Weekly Report

The coronavirus disease 2019 (COVID-19) pandemic has underscored the importance of pathogen surveillance systems that are capable of early detection of emerging infectious agents and the need to advance genetic diagnostic technologies [1,2]. Major international public health organizations, including the World Health Organization (WHO) and the Coalition for Epidemic Preparedness Innovations, have emphasized the development and implementation of universal diagnostic methods for pathogens as the key components of strategies to prepare for potential future emerging infectious agents, referred to as “Pathogen X” [3]. The WHO has also established research and development priorities and released a list of pathogens based on factors such as pandemic potential and the feasibility of developing treatments. The organization recommends surveillance and pathogen testing technologies to detect the emergence of these listed pathogens, as well as the proactive development of vaccines and therapeutics [4]. In the Republic of Korea (ROK), the Korea Disease Control and Prevention Agency (KDCA) has designated and managed novel infectious disease syndromes as Class 1 infectious diseases, further emphasizing the need to develop and validate diagnostic methods for new pathogens that cannot be detected using existing diagnostic reagents.

Against this background, the development of diagnostic methods that can achieve both rapidity and broad applicability has emerged as an important task among various diagnostic technologies. Next-generation sequencing (NGS) enables the comprehensive identification of pathogen genomic information and plays a major role in diagnosis and research; however, its high cost, long analysis time, and equipment requirements limit its use for on-site testing or large-scale screening [5]. In contrast, polymerase chain reaction (PCR)-based assays (Table 1) remain key diagnostic tools in clinical and field settings thanks to their rapid turnaround time, high sensitivity and specificity, accessibility of equipment, and cost-effectiveness. In particular, pan-PCR and multiplex PCR, characterized by their universality and multiplexing capability, respectively, have drawn attention as suitable technologies for early infectious disease response and large-scale surveillance systems [6,7].

Table 1 Comparative analysis of PCR-based assays

Criterion	Conventional PCR	Pan-PCR	Multiplex PCR
Target pathogen range	Detection of single pathogen	Simultaneous detection of diverse pathogens within the same family	Simultaneous detection of specific genes from multiple individual pathogens
Primer design criterion	Single pathogen-specific primers	Primers targeting conserved common gene regions within the same family	Specific primers for each pathogen
Amplification method	Single target amplification	Common region amplification	Multiple target amplification
Application purpose	Diagnosis of single pathogen infection	Early discovery and screening of unknown pathogens	Simultaneous diagnosis of multiple pathogen infections

PCR=polymerase chain reaction..

Pan-PCR targets conserved sequences within a specific family or genus, providing broad applicability that enabled the detection of multiple subtypes, variants, recombinants, and even previously unknown or emerging pathogens [8]. This approach is particularly advantageous for the early identification of novel or variant pathogens, including “Pathogen X,” where high genetic diversity, recombination, or mutation may occur. Therefore, pan-PCR can serve as an effective diagnostic tool for the early detection of new and variant infectious diseases arising from such genetic changes. During the COVID-19 pandemic, the use of pan-PCR helped overcome the limitations of single-pathogen diagnostic methods, further underscoring the importance and utility of universal diagnostic technologies.

Multiplex PCR uses multiple sets of specific primers to simultaneously detect and differentiate various pathogens, making it effective for precise and rapid differential diagnosis. Along with infections caused by a single pathogen, the number of co-infection cases has recently been increasing [9]. Importantly, respiratory infectious diseases present limitations for conventional single-pathogen diagnostic methods because of the interactions among pathogens and their high mutation rates. Under these circumstances, multiplex PCR contributes to the simultaneous diagnosis of co-infections and the strengthening of surveillance systems and has become the most widely used platform among PCR-based multiplex diagnostic methods.

This study focuses on analyzing the strategic value and current status of pan-PCR and multiplex PCR technologies as molecular diagnostic platforms for responding to emerging infectious diseases. Although these technologies can be applied to a wide range of pathogens, this review primarily discusses viral pathogens that exhibit frequent mutations and a higher likelihood of emerging as new infectious agents rather than bacteria or fungi, which tend to be more genetically stable. The cases of application of multiplex PCR are examined mainly through widely known commercial panels whose clinical utility has been demonstrated. In the changing landscape of infectious diseases characterized by the emergence of new and variant pathogens and an increase in co-infections, these PCR technologies are becoming increasingly important as the key platforms that complement the limitations of single-pathogen diagnostic methods and enable early detection and response. By reviewing the global and domestic trends in the development of diagnostic technologies focused on pan-PCR and multiplex PCR, this study aims to present the roles, limitations, and future directions of these methods, thereby contributing to the establishment of effective and proactive response systems for emerging infectious diseases such as COVID-19.

To examine the domestic and international research trends and implementation status of pan-PCR and multiplex PCR assays, this study referred to pathogen-specific diagnostic methods utilizing pan-PCR and multiplex PCR as described in academic literature on PCR-based diagnostics. In addition, pandemic response reports and diagnostic case studies published by national and international public health organizations, including the KDCA, WHO, and United States Centers for Disease Control and Prevention (CDC), were reviewed. Based on these cases, this study presents insights into the future directions and prospects for the development of these diagnostic technologies.

1. Introduction to the Pan-PCR Assay

1) Technical principles and limitations of the pan-PCR assay

Pan-PCR is a technique that designs diagnostic primers based on the conserved gene sequences shared among pathogens within the same taxonomic lineage. The process begins by collecting genomic data from the target pathogen group and performing multiple sequence alignment to identify the gene regions with a high degree of conservation. Primers are then designed within these conserved regions, considering both specificity and amplification efficiency. The designed primers enable amplification even in pathogens that differ at the variant or subtype level. Although pan-PCR is useful for the early detection and phylogenetic identification of unknown pathogens, further analyses such as sequencing of the amplified products are required for precise species-level identification. This additional step increases the total diagnostic turnaround time and may incur costs associated with NGS linkage, which can be a limiting factor for clinical application [10]. In bacterial diagnostics, conserved genes such as 16S ribosomal RNA have been widely used as targets, allowing broad detection capability. However, in the case of viruses, universal detection is more challenging because of high genomic diversity, rapid mutation rates, and absence of common genes. Therefore, Pan-PCR has been developed as a detection strategy focused on pathogens within the same family, and major public health organizations worldwide are actively incorporating this technology into infectious disease surveillance and response systems.

2) Application of the pan-PCR assay

(1) Coronaviruses

The pan-coronavirus PCR assay developed by the Rega Institute for Medical Research at KU Leuven in Belgium targets a highly conserved region within the RNA-dependent RNA polymerase (RdRp) gene, enabling the comprehensive detection of various coronavirus lineages, including multiple genetic variants. In fact, this technique has demonstrated high detection rates for viruses of SARS, MERS, and diseases caused by other coronaviruses and has been reported to be minimally affected by genetic variations in the pathogen [11].

(2) Paramyxoviridae and pneumoviridae

The pan-paramyxoviridae PCR assay developed at the Erasmus Medical Center in Rotterdam, the Netherlands, uses a single primer set that targets a conserved region within the RdRp gene of viruses belonging to the paramyxoviridae and pneumoviridae families, allowing simultaneous detection with high universality. This method has also been successfully applied to wild bird samples, enabling the detection of various pathogens, including avian metapneumovirus [12].

(3) Filoviridae

The pan-Filoviridae PCR assay enables the broad detection of mammalian filoviruses, primarily Ebola virus and Marburg virus. SYBR green–based quantitative real-time PCR (qPCR) developed by the National Institute for Communicable Diseases in South Africa and the high-throughput real-time PCR (RT-PCR) system developed by the University of Washington School of Medicine in the United States have both been applied, offering advantages in real-time surveillance and efficient processing of large-scale or low-concentration samples. In previous studies of wild bats, this assay has been used for the early detection of potential pathogens and has demonstrated reliable performance. Moreover, its value has been emphasized as a proactive public health tool for preventing community transmission of high-risk viruses such as Ebola and Marburg [13,14].

(4) Other families and genera

The Norwegian Institute of Public Health developed a diagnostic assay capable of comprehensively detecting major human-infecting Caliciviruses, including norovirus and sapovirus, which cause outbreaks through gastrointestinal infection. The study reported that the assay demonstrated superior speed and accuracy compared with existing diagnostic techniques [15].

At the Center for Biological Safety in Berlin, Germany, a pan-Flaviviridae PCR assay was developed targeting a highly conserved region of the NS5 gene, enabling the broad detection of multiple Flavivirus pathogens such as yellow fever virus, Dengue virus, and Zika virus. This assay is effective as a surveillance tool in regions where two or more of these viruses are co-circulating (Table 2) [11-16].

Table 2 Application of pan-PCR assays by pathogen family

Pathogen family	Country or institution	Target gene	Features	Reference
Coronaviridae	University of Leuven, Belgium	RdRp	Broad detection of various coronavirus lineages including genetic variants; less affected by pathogen gene mutations	[11]
Paramyxoviridae & pneumoviridae	Erasmus Medical Center, Netherlands	RdRp	Simultaneous detection of paramyxoviridae and pneumoviridae; high versatility in wildlife surveillance (e.g., wild birds)	[12]
Filoviridae	NICD, South Africa; University of Washington, USA	Nucleoprotein	Broad detection of high-risk filoviruses (Ebola, Marburg); efficient for large-scale, low-concentration sample processing and real-time surveillance	[13,14]
Caliciviridae	Norwegian Institute of Public Health	RdRp	Comprehensive detection of major human caliciviruses, including norovirus and sapovirus, responsible for outbreaks	[15]
Flaviviridae	Berlin Centre for Biological Safety, Germany	Non-structural protein	Comprehensive detection of multiple flaviviruses (Yellow fever, Dengue, Zika); useful for surveillance in co-circulating regions	[16]

PCR=Polymerase chain reaction; RdRp=RNA-dependent RNA polymerase; NICD=National Institute for Communicable Diseases..

3) Global and domestic trends in the application of pan-PCR technology

Pan-PCR is an advanced molecular diagnostic technology capable of simultaneously detecting a wide range of viral pathogens. It has recently emerged as a key strategy to enhance the early detection and response capacity for infections of unknown origin.

The KDCA is promoting the standardization and pilot implementation of pan-PCR assays focusing on 13 viral families considered highly likely to be introduced into the country as part of its strategic preparedness for “Pathogen X.” The selection criteria were based on four factors: the level of attention from major international public health organizations, potential public health impact due to respiratory transmissibility, zoonotic risk, and the likelihood of domestic introduction and occurrence. Through this initiative, the KDCA aims to contribute to the advancement of the national infectious disease response system. The WHO is also promoting the development of a pan-Filovirus PCR assay that can simultaneously detect multiple hemorrhagic fever pathogens of unknown origin. In addition to establishing laboratory-based surveillance systems, the WHO is expanding its support for strengthening the capacity of healthcare personnel, setting up isolation facilities, and developing comprehensive and sustainable epidemic response frameworks [17].

Furthermore, the WHO provides a universal RT-PCR protocol targeting the matrix gene common to influenza A viruses, facilitating the early detection and surveillance of influenza A, including highly pathogenic avian influenza subtypes such as H5 and H7 [18]. This represents one of the essential diagnostic strategies for proactively addressing the potential risks of zoonotic infectious diseases. Similarly, the CDC has enhanced its infectious disease response capabilities through the detection of pathogens using pan-enterovirus and pan-parechovirus PCR assays combined with a sequencing analysis [19].

2. Introduction to the Multiplex PCR Assay

1) Technical principles and limitations of multiplex PCR

Recently, studies have been conducted on primer design using an in silico analysis, evaluation of dimers (undesired primer–primer binding), and optimization methods for multi-gene diagnostics. A 2022 article in Nature Communications introduced the Simulated Annealing Design using Dimer Likelihood Estimation (SADDLE) method—an algorithmic approach for designing multiplex PCR primer sets that minimizes primer dimer formation while optimizing target amplification efficiency and uniformity. This method was also reported to be applicable to qPCR panel design and applications combined with Sanger sequencing [20]. However, the detection sensitivity of multiplex PCR assays may decline because of continuous viral mutations. This reduction is attributed not only to the decreased amplification efficiency caused by interactions among multiple primer sets but also to single-nucleotide variations occurring in primer-binding regions, which can substantially impair assay performance [21].

2) Application of the multiplex PCR assay

Multiplex PCR technology has attracted global attention as a key tool for the early diagnosis of infectious diseases and simultaneous detection of multiple pathogens. Research, development, and commercialization efforts are actively underway in several countries. Countries are applying this technology in public surveillance systems and clinical settings to improve the speed and accuracy of infectious disease response (Table 3).

Table 3 Global application of multiplex PCR assays

Country	Application	Target pathogens	Features
Germany	FTD Respiratory Pathogens Assay	Total of 16 types including Influenza A/B, parainfluenza, adenovirus, coronavirus (etc.)	Simultaneous detection of 16 respiratory viruses using a multi-tube format
USA	FilmArray Respiratory	Total of 22 respiratory pathogens/8 viruses, 18 bacteria, 7 antimicrobial resistance genes	BioFire Diagnostics FilmArray multiplex panel utilized globally (e.g., Taiwan, Japan)
China	Multiplex RT-PCR+MassARRAY	Total of 27 types including viruses and bacteria	Quantitative analysis of amplification products via MassARRAY after RT-PCR; capable of co-infection diagnosis
Republic of Korea	PowerChek SARS-CoV-2, Influenza A & B multiplex real-time PCR Kit	SARS-CoV-2 and Influenza A & B	Strengthens domestic diagnostic capacity with performance comparable to international standards (over 97% agreement)

PCR=polymerase chain reaction; FTD=fast track diagnostics; RT-PCR=real-time PCR; SARS-CoV-2=severe acute respiratory syndrome coronavirus 2..

In Germany, the Fast Track Diagnostics Respiratory Pathogens Assay has been commercialized and is being used for respiratory infectious disease surveillance. This assay is designed to detect a total of 16 respiratory viruses, including influenza A and B, parainfluenza virus, adenovirus, and coronavirus (e.g., NL63, OC43), using a multi-tube format [22]. The FilmArray Respiratory Panel, developed and commercialized by BioFire Diagnostics in the United States, enables the simultaneous detection of 22 respiratory pathogens, including both viruses and bacteria. This panel is currently used in clinical settings and surveillance systems in Taiwan and Japan [23]. The Taiwan Centers for Disease Control conducted a comparative analysis by simultaneously performing multiplex PCR assays using the FilmArray Respiratory Panel and the conventional PCR assays previously in use. The study concluded that multiplex PCR assays demonstrated a significantly higher positive detection rate than conventional assays and effectively identified cases of multiple co-infections. These results confirmed the utility of multiplex PCR for both respiratory pathogen surveillance and clinical diagnosis [24]. In Japan, Nagasaki University Hospital conducted diagnostic testing using the FilmArray Respiratory Panel in patients with acute respiratory infections. The study confirmed that this method could detect a total of 17 viral and 3 bacterial respiratory pathogens with high specificity and sensitivity [25]. In addition, the FilmArray Pneumonia Panel, a system specialized for pneumonia diagnosis, can simultaneously detect 8 viral species, 18 bacterial species, and 7 antibiotic resistance genes from lower respiratory tract specimens within approximately 1 hour. Notably, it provides quantitative results for 15 clinically significant bacterial pathogens associated with pneumonia, enabling the rapid diagnosis of bacterial–viral co-infections and serving as a key reference for guiding treatment decisions [26]. In China, a diagnostic method combining multiplex RT-PCR with MassARRAY, a mass spectrometry–based technology, has been developed to enable the simultaneous detection of a broader range of pathogens. This assay is designed to detect a total of 27 respiratory pathogens, including both viruses and bacteria, within a single panel. Compared with conventional PCR-based diagnostic methods, the assay demonstrates higher sensitivity and specificity and allows for the accurate identification of multiple infections, even in cases of complex co-infection, in local health centers and hospitals across China [27]. In the ROK, following the COVID-19 pandemic, multiplex RT-PCR diagnostic reagents capable of simultaneously detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/B viruses have been successfully developed and commercialized. A comparative study evaluating the performance of this assay against the BioFire Respiratory Panel demonstrated a concordance rate exceeding 97%, confirming equivalent diagnostic performance [28].

3) Global and domestic trends in the application of multiplex PCR

Multiplex PCR is utilized both domestically and internationally to strengthen infectious disease response capabilities through the diagnosis of infections with unknown etiologies. In particular, the WHO and CDC are actively incorporating this technology into public health surveillance and diagnostic systems.

The KDCA is currently developing panels for syndrome-based multiplex testing across five categories—respiratory, hemorrhagic, exanthematous, neurologic, and diarrheal—to strengthen proactive laboratory response capabilities for infections of unknown origin. To enhance national surveillance and early outbreak response, the agency has designed a system that enables the rapid screening and subtype differentiation of four avian influenza viruses frequently detected in domestic poultry (H5N1, H5N6, H5N8, and H9N2) within a single reaction [29]. In addition, a diagnostic assay has been developed that can simultaneously detect the pandemic influenza A (H1N1)pdm09 virus and five seasonal influenza subtypes within a single reaction using specific primers and probes [30].

Such multidimensional efforts to develop and preemptively secure diagnostic technologies represent a key strategy for proactively addressing the potential risks of future infectious diseases. The WHO strongly encourages the development of diagnostic methods capable of the simultaneous detection of multiple pathogens and provides a multiplex RT-PCR protocol for the concurrent detection of influenza viruses and SARS-CoV-2, particularly for the early diagnosis and surveillance of respiratory viruses [31]. The CDC has also developed the Flu SC2 Multiplex Assay, which enables the simultaneous detection of influenza A/B and SARS-CoV-2 with high accuracy from both upper and lower respiratory specimens [32].

This study analyzed the strategic value and current status of pan-PCR and multiplex PCR technologies as molecular diagnostic platforms for responding to emerging infectious diseases. Focusing on viral pathogens with high mutation rates and potential for novel emergence, this review examined the research, development, and practical applications of widely known commercial panels whose utility has been demonstrated.

Pan-PCR serves as a universal detection tool targeting conserved genetic regions, thereby enabling the identification of unknown and emerging pathogens; however, it requires additional analyses for precise species-level identification. Meanwhile, multiplex PCR has demonstrated excellent efficiency in the simultaneous differentiation of multiple pathogens and the diagnosis of co-infections through technical advancements such as in silico optimization. In the ROK, the clinical utility of multiplex PCR has been successfully validated through applications involving the simultaneous detection of SARS-CoV-2 and other pathogens. These technologies can ultimately be integrated with NGS-based genomic analysis to establish an efficient diagnostic workflow encompassing screening, confirmation, and characterization. Together, pan-PCR and multiplex PCR represent specific directions for technological advancement aimed at simultaneously meeting the demands for rapidity, universality, and specificity in response to future infectious disease threats. Moving forward, pan-PCR should evolve beyond simple positive/negative detection toward the direct integration of amplified products with genomic analysis. The technological goal should be to establish a system that rapidly determines the phylogeny of “Pathogen X” in the field by quickly linking the amplification data from conserved gene regions to NGS or Sanger sequencing. For multiplex PCR, which remains the most widely used method for the simultaneous detection of multiple pathogens, enhancing the reliability of primer design is critical. By applying in silico optimization algorithms to minimize unnecessary primer–primer interactions, diagnostic panels can be expanded according to the priority of domestic prevalence and potential introduction, while maintaining high sensitivity and specificity.

As such, the efforts by international public health organizations such as the WHO and CDC to strengthen infectious disease response capabilities based on pan-PCR and multiplex PCR assays indicate the global recognition of these technologies as key public health tools for the early detection and surveillance of unknown pathogens. In addition, the KDCA is focusing on pan-PCR and multiplex PCR as key diagnostic tools to proactively respond to future infectious disease threats and enhance domestic surveillance capacity. Since 2023, as part of the Pathogen X response strategy, the agency has been developing pan-PCR assays for 13 viral families prioritized based on their high risk of domestic introduction and pandemic potential, while simultaneously developing syndrome-based multiplex test panels utilizing multiplex PCR technology. Pan-PCR provides breadth and rapidity for the detection of novel or unknown pathogens and for the primary screening of high-risk groups with potential for domestic introduction. In contrast, multiplex PCR is used in clinical settings that require specificity and efficiency, such as the simultaneous differential diagnosis of multiple pathogens and the rapid identification of co-infections. Ultimately, the KDCA aims to maximize diagnostic efficiency through the flexible integration and complementary use of these two technologies (Figure 1), thereby establishing a diagnostic system that enables rapid patient management and effective public health response. The advancement of molecular diagnostic technologies based on pan-PCR and multiplex PCR is expected to play a pivotal role in public health diagnostics in case of future pandemics.

Figure 1. Diagnostic system for emerging and unknown pathogens
PCR=polymerase chain reaction; NGS=next-generation sequencing.

Ethics Statement: Not applicable.

Funding Source: None.

Acknowledgments: None.

Conflict of Interest: The authors have no conflicts of interest to declare.

Author Contributions: Conceptualization: YJA, CWJ. Data curation: YJA. Formal analysis: YJA. Investigation: YJA, CWJ, JER, EJK. Methodology: YJA. Project administration: JER, EJK. Resources: YJA, CWJ, JER, EJK. Supervision: JER, EJK. Validation: JER, EJK. Visualization: YJA, CWJ. Writing – original draft: YJA, CWJ. Writing – review & editing: JER, EJK.