An HFman Probe-Based Multiplex Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Simultaneous Detection of Hantaan and Seoul Viruses

Abstract

Hantaviruses are zoonotic pathogens that are widely distributed worldwide. Hantaan virus (HTNV) and Seoul virus (SEOV) are two most common hantaviruses that infect humans and cause hemorrhagic fever with renal syndrome (HFRS). Rapid and sensitive detection of HTNV and SEOV are crucial for surveillance, clinical treatment and management of HFRS. This study aimed to develop a rapid HFman probe-based mulstiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to simultaneously detect HTNV and SEOV. A novel multiplex RT-LAMP assay was developed, and 46 serum samples obtained from clinically suspected patients were used for evaluation. The novel RT-LAMP assay can detect as low as 3 copies/reaction of hantaviruses with a detection limit of 41 and 73 copies per reaction for HTNV and SEOV, respectively. A clinical evaluation showed that the consistencies of the multiplex RT-LAMP with RT-qPCR assay were 100% and 97.8% for HTNV and SEOV, respectively. In view of the high prevalence of HTNV and SEOV in rural areas with high rodent density, a colorimetric visual determination method was also developed for point-of-care testing (POCT) for the diagnosis of the two viruses. The novel multiplex RT-LAMP assay is a sensitive, specific, and efficient method for simultaneously detecting HTNV and SEOV.

Keywords: HFman probe; Hantaan virus; LAMP; POCT; Seoul virus.

Figure 1

Genomic location and screening strategy of RT-LAMP primers for HTNV and SEOV. The location of each primer set is detailed in Table S1.

Figure 2

The genomic location of the RT-LAMP primers in the S gene of HTNV and SEOV. The primers include F3, B3 (outer primer), F2, F1c, B2, and B1c (inner primers), and LB (loop primer).

Figure 3

Sensitivity and specificity of the multiplex RT-LAMP assay. (A) Sensitivity. The RT-LAMP reaction was carried out with serial dilutions of 3 × 10³–3 × 10⁰ copies/μL of HTNV and SEOV standard RNA. (B) Specificity. Tested viruses included influenza A, HCoV-229E, parainfluenza virus 3, human metapneumovirus, human rhinovirus, HCoV-HKU-1, and bocavirus. (C) Colorimetric reaction of the RT-LAMP assay for HTNV and SEOV. The color change from burgundy to orange or yellow was considered as positive. NTC, non-template control.