Rubella, also known as German measles, is an infectious disease caused by rubella virus (RuV). It spreads through droplets in the respiratory secretions of infected individuals. The symptoms of individuals infected with rubella after birth are often mild, including slight fever, rash, lymphadenopathy, and arthralgia, and approximately 30% of cases are asymptomatic. However, infection of pregnant women, especially during the first trimester, sometimes leads to miscarriage, stillbirth, and birth defects such as hearing loss, cataracts, heart disease (patent ductus arteriosus, pulmonary stenosis, atrial heart septal defect), microcephaly, and developmental delays, termed congenital rubella syndrome (CRS) (Miller et al., 1982, Rendle-Short, 1964). Several safe and effective vaccines have been developed to prevent rubella and CRS worldwide (Huygelen and Peetermans, 1967, Huygelen et al., 1969). However, because of the lower vaccine coverage for rubella compared with measles as well as insufficient surveillance, rubella is thought to be underreported. The World Health Organization (WHO) advocates achieving and sustaining the regional measles and rubella elimination goals in the Immunization Agenda 2030 following the Global Vaccine Action Plan 2011-2020 (World Health Organization 2013a; 2020; 2021). The WHO Western Pacific Region (WPR) has identified the elimination of rubella infection as a regional goal, and zero cases of endemic rubella infection and domestically acquired CRS as a regional target (World Health Organization Regional Office for the Western Pacific 2022). One of the strategies to accomplish this regional target is to “strengthen the capacity to rapidly detect and to implement a coordinated, timely and effective response to rubella outbreaks”. Consequently, rapid and accurate diagnostic methods are becoming increasingly important.

RuV is a member of the genus Rubivirus in the family Matonaviridae and has a positive-sense single-stranded RNA genome of approximately 10 kb. RuV strains are classified into 13 genotypes (1a, 1B, 1 C, 1D, 1E, 1 F, 1 G, 1H, 1I, 1 J, 2 A, 2B, 2 C) according to the sequences within the E1-coding region (nucleotides 8731–9469) (World Health Organization 2013b). The genotypes currently circulating globally are 1a (as vaccine strains), 1E, 1 G, 1H, and 2B, with only genotypes 1E and 2B reported in the WPR (Brown et al., 2019, Mulders et al., 2016, World Health Organization Regional Office for the Western Pacific, 2022).

Diagnostic methods for rubella include the detection of RuV antigen-specific IgM in serum and detection of the RuV genome by nucleic acid amplification methods, such as real-time RT-PCR, in blood samples or throat swabs. Several IgM detection kits are commercially available and have benefits including simplicity, safety, and reproducibility of the detection. However, the positivity rate for IgM detection immediately after disease onset is not very high, because the IgM level begins to increase from the onset but does not reach a peak until late in the first week (Abernathy et al., 2009, Kurata et al., 2019, World Health Organization, 2008a, World Health Organization, 2008b). Virus excretion is highest at disease onset, and thus detection of the RuV genome by nucleic acid amplification methods is considered to have an advantage for the diagnosis of rubella. Several nucleic acid amplification methods have been developed in addition to the conventional RT-PCR and real-time RT-PCR assays (Guatelli et al., 1990, Ishiguro et al., 2003, Kouguchi et al., 2010). Loop-mediated isothermal amplification (LAMP) is a rapid assay system that does not require multiple handling and thermal steps or a sophisticated machine (Nagamine et al., 2002, Notomi et al., 2000). The early LAMP assays detected turbidity caused by precipitation of magnesium pyrophosphate that accumulated during nucleic acid amplification (Abo et al., 2014, Kurosaki et al., 2016). To improve the sensitivity, reduce nonspecific amplification, and monitor the nucleic acid amplification in real time, real-time fluorescent LAMP assays were developed using a quenching primer (Qprimer) or quenching probe (Qprobe) based on guanine base quenching technology (Crockett and Wittwer, 2001, Kurata et al., 2001, Torimura et al., 2001). This method has been applied to viral genome detection assays for several viruses, including SARS-CoV-2, because the results can be obtained within 30 min and nonspecific amplification is reduced (Nakauchi et al., 2019, Park et al., 2020, Shirato et al., 2018, Takayama et al., 2019). In the present study, a novel detection method for the RuV genome by real-time fluorescent RT-LAMP using Qprimer (Q-LAMP assay) was developed. The cytosine at the 5′ end of a portion of a Loop primer molecule (usually 1/20) was labeled with a BODIPY FL fluorescent dye (Qprimer). Hybridization of the Qprimer and the target nucleotide sequence causes the quenching of fluorescence by photo-induced electron transfer between the fluorescent dye and a guanine residue in the target. The Q-LAMP assay in this study detected fluorescent quenching. Because the Qprimer requires only one fluorophore and no quencher, hairpin, or complementary strand of a probe, it can be designed and produced less expensively and more easily than other real-time monitoring methods such as assimilating probes, molecular zippers, molecular beacons, and MD-probes. The positive percent agreement (PPA) of the new assay was compared with that of a real-time RT-PCR assay using synthesized RNA, viral RNA, and clinical specimens.