Loop-mediated isothermal amplification assay (LAMP) was originally designed for genetic testing and applied to viruses and other pathogens [11–13]. Using six primers that target eight distinct gene sequences, the reaction is initiated only if all the primers match with the targets sequence correctly. Thus, these assays offer high levels of specificity, selectivity, sensitivity, and efficiency. In particular, reverse transcription and amplification reactions of RNA viruses can be completed in the meantime under isothermal environments, saving time and reducing experimental error.
The most conserved segment within the N gene (GenBank EF081360) was selected as the target. The entire testing process under isothermal conditions of 65°C takes only 1.5 h (the DNA template must be pre-denatured at 95°C prior to the isothermal reaction). Additionally, results from the LAMP can be readily observed by the white precipitate of magnesium pyrophosphate generated during the reaction. Just because no laboratory equipment is needed for the determination of results, RT-LAMP can easily be used in field-testing. Moreover, A real-time monitoring turbidimeter was developed to monitor LAMP reactions . Through continuous exploration, LAMP has been combined with a fluorescent dye for quantification in real-time reactions [15, 16]. For example, a metal ion indicator, hydroxynaphthol blue (HNB), was added to LAMP assay [17–19]. The HNB dye-based assay has a remarkable advantage compared with other color-based assays in that HNB is mixed prior to amplification. Thereby, the reaction tubes need not to be opened, which reduce the risk of cross-contamination.
LAMP technology has been applied to a variety of nucleic acid detection assays for pathogens, such as H5N1-HPAIV, HBV, HIV-1, MTB, O157, trypanosomes, and plasmodium [15, 16, 20–23]. This study established an RT-LAMP method with HNB dye to detect the hMPV N gene, which has significantly higher sensitivity (10 copies) levels than the conventional RT-PCR method and did not cross-react with RSV, HRV, or A/PR/8/34 (H1N1) viral nucleic acids. Restriction enzyme and sequence analyses also validated its specificity.
In the testing of clinical respiratory samples, 18 hMPV positive samples were detected by the traditional RT-PCR method, but 23 hMPV positive samples by RT-LAMP method due to the higher sensitivity. The epidemiological analysis indicated that children with HMPV varied from 20 days to 146 months of age, and the majority of patients (82.6%, 18/23) were aged at under ≤5 years. The main clinical diagnoses included bronchopneumonia (52.2%, 12/23), acute respiratory tract infection (26.1%, 6/23), acute asthmatic bronchopneumonia (17.4%, 4/23) and pneumonia (4.3%, 1/23). In short, RT-LAMP with HNB dye was shown to be a sensitive and easy assay for detection of hMPV. Given these benefits, RT-LAMP is considered a useful and promising technique for the less equipped primary sector. It can also play an important role in the early diagnosis and control of diseases during outbreaks in remote areas or under field conditions.