Respiratory syncytial virus (RSV) is a leading cause of acute lower respiratory tract infection worldwide, with a high clinical burden in infants, young children, older adults, and immunocompromised individuals. RSV exists as two antigenic subtypes, RSV A and RSV B, which co-circulate and show genetic variability, particularly in the attachment (G) glycoprotein gene. Subtype-resolved detection supports molecular epidemiology, outbreak monitoring, and evaluation of preventive interventions including monoclonal antibodies and vaccines.
Although reverse transcription quantitative PCR (RT-qPCR) is the reference method for RSV detection due to high analytical sensitivity and specificity, its dependence on centralized instrumentation and trained personnel limits use in decentralized or resource-limited settings. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides rapid amplification at a constant temperature and has lower equipment requirements, making it attractive for near‑patient or point-of-care testing. Prior RT-LAMP approaches for RSV have often relied on non–sequence-specific readouts (intercalating dyes, turbidity, colorimetric changes) that are vulnerable to non-specific amplification and are insufficient for reliable subtype discrimination in multiplex formats.
The authors developed a multiplex RT-LAMP assay that simultaneously detects and discriminates RSV A and RSV B and includes an internal control (IC) within a single isothermal reaction. Primer sets were designed to conserved regions: the RSV A primer set targets a conserved region of the matrix (M) gene, while the RSV B primer set targets a conserved region of the RNA-dependent RNA polymerase (L) gene. The internal control targets the human ACTB gene.
To achieve sequence-specific fluorescence readout in multiplex conditions, a dual-probe strategy was implemented: an assimilating probe for RSV A detection was chosen to generate a robust signal in multiplex reactions, while hybridization-based TaqMan-style (HyTaq) probes were applied for RSV B and for the ACTB internal control to enhance specificity. These probe-based detection approaches aim to reduce non-specific LAMP signals and permit reliable multiplex fluorescence discrimination between subtypes and the internal control.
A retrospective specimen set of 308 nasopharyngeal (NP) swabs collected during routine care at Korea University Guro Hospital between January 2018 and July 2019 was used for clinical validation. The set comprised 91 RSV A–positive, 97 RSV B–positive, and 120 RSV-negative samples as determined by prior routine diagnostic testing using the Allplex Respiratory Panel 1 assay. Extracted nucleic acids were prepared from 200 µL of each NP sample using an automated extraction system (PowerEXP™ 32) and stored at −20 °C until testing.
The study used anonymized residual clinical specimens and was approved by the institutional review board (Korea University Guro Hospital, approval number 2019GR0055) with an exemption from review and waiver of informed consent.
Analytical performance was assessed using serial dilutions of RSV-positive clinical specimens and plasmid standards to characterize assay sensitivity. Probe-based detection was employed to limit non-specific fluorescence associated with LAMP chemistry. The multiplex RT-LAMP assay was tested for cross-reactivity against a panel of common respiratory viruses; the authors report no cross-reactivity in those experiments.
Clinical performance was evaluated with the 308 retrospective NP swabs, using the previous Allplex Respiratory Panel 1 results as the reference standard. The RSV A RT-LAMP assay demonstrated a clinical sensitivity of 92.31%, while the RSV B RT-LAMP assay showed a sensitivity of 98.97%. Specificity was 100% for both RSV A and RSV B targets relative to the prior diagnostic classification of the specimens.
For comparative purposes, the commercial Allplex SARS-CoV-2/FluA/FluB/RSV Assay (Seegene) was run on the same specimen set. The RT-LAMP assay was positioned as a rapid, isothermal alternative to RT-PCR with sequence-specific probe readouts designed to permit subtype discrimination within a single reaction. The source reports performance metrics for the RT-LAMP assays but does not provide head-to-head sensitivity and specificity numbers for the commercial RT-PCR within the same manuscript text beyond noting its use for comparison.
The probe-based multiplex RT-LAMP assay described provides subtype-specific detection of RSV A and RSV B with an internal control in a single isothermal reaction. The dual-probe approach—assimilating probe for RSV A and HyTaq probes for RSV B and ACTB—is intended to overcome non-specific signal generation typical of dye- or turbidity-based LAMP readouts and to enable reliable multiplex fluorescence discrimination.
Clinically, the assay demonstrated high specificity (100% for both subtypes) and strong sensitivity (92.31% for RSV A and 98.97% for RSV B) in a retrospective panel of nasopharyngeal specimens. No cross-reactivity with other common respiratory viruses was observed in analytical testing. These features suggest potential applicability in decentralized or near‑patient testing environments where rapid subtype-resolved results could aid surveillance or clinical decision making.
The authors note that further validation is required before broad clinical deployment. Specific elements that would require additional description or study—such as limit of detection in standardized units, operational characteristics in point-of-care workflows, and broader external validation across diverse specimen sets and geographic regions—were not detailed in the provided source text.
Key methods reported include primer design using PrimerExplorer V5 for conserved regions of RSV A (M gene) and RSV B (L gene), nucleic acid extraction from 200 µL of NP swab specimen using the PowerEXP™ 32 automated system, and testing of the multiplex RT-LAMP assay against serial dilutions, plasmid standards, and a respiratory virus panel for cross-reactivity. The retrospective clinical specimen set (n = 308) and prior diagnostic classifications served as the reference for clinical performance evaluation.
All relevant data are reported to be within the manuscript and its supporting information files as stated by the authors. Ethical approval and a waiver of consent were obtained under IRB approval number 2019GR0055.