Development of a loop-mediated isothermal amplification assay for the rapid detection of six common respiratory viruses

Abstract

Due to the highly contagious and spreads quickly of respiratory infectious diseases (ADR), the availability of rapid, sensitive, and reliable diagnostic methods is essential for disease control. Here, we develop an approach based on loop-mediated isothermal amplification (LAMP) for the detection of influenza A virus (Flu A), Flu A subtypes H1N1and H3N2, influenza B virus (Flu B), respiratory syncytial virus (RSV) subtypes A and B, human adenovirus (HAdV), parainfluenza virus (PIV) subtypes 1 and 3, and human rhinovirus (HRV) simultaneously. We designed primers specific to detect respiratory viruses above, optimized the RT-LAMP assay and evaluated it for its sensitivity and specificity of detection using real-time monitoring based on SYBR Green I. We also evaluated the result of our RT-LAMP assay on 638 nasopharyngeal swab specimens with the commercial RT-PCR by Cohen's Kappa. The inconsistent results were verified by Sanger sequencing furtherly. The developed RT-LAMP assay displayed a detection limit of 1 × 10 2 copies/ml RNA close to that of RT-PCR; no cross-reactivity was observed in the 10 kinds of viruses studied. The results obtained with 638 clinical samples indicate that the developed method has high specificity (0.988-1) and sensitivity (0.863-1) for viruses studied, and the Kappa value of all viruses was more than 0.85 revealed an excellent agreement between the two methods. We developed an RT-LAMP-based method and optimized for the detection of common respiratory viruses. It may be a powerful tool for rapid and reliable clinical diagnosis of ADR in primary hospitals.

Development of a novel integrated isothermal amplification system for detection of bacteria-spiked blood samples

“…This system greatly simpli es the detection process of traditional RPA-LFD technique and effectively avoids the contamination caused by ampli cation products. At the same time In summary, the RPA-LFD assay is time-saving, more effective and sensitive than conventional identi cation methods, which has the potential to be applied in primary hospitals (Chen et al 2021;Yin et al 2017). Moreover, this novel integrated isothermal ampli cation system will become a powerful tool for the identi cation of bacteria or other pathogens, especially suitable for use in low-resource settings (Sadaow et al 2020).…”

Using Saliva and LAMP for Non-invasive Detection of Communicable Pathogens in Low Medical Access Regions Reham

“…As a result, LAMP has recently been implemented to detect many infectious diseases [4], [23]- [25]. Depending on the condition, the most common matrices collected in LAMP are from a venous blood draw or capillary fingerpick, nasopharyngeal swabs, urine or sputum collection [24] [26], [27]. 4) The detection using spectroscopy, colorimetry or turbidity can be performed.…”

“…The LAMP assay exhibited a limit of detection of 5 fg, while analysis with mPCR showed a detection limit of 5 x 10-5 ng (corresponding to 50 fg) of DNA, which makes the LAMP assay ten times more sensitive than mPCR [19]. Another recent study from 2019 performed by Shete et al [24] also assessed the diagnostic accuracy of TB-LAMP as an alternative to sputum smear microscopy [24]. The study demonstrated a higher sensitivity of the TB-LAMP assay compared to the smear microscopy but with similar specificity [24].…”

“…Another recent study from 2019 performed by Shete et al [24] also assessed the diagnostic accuracy of TB-LAMP as an alternative to sputum smear microscopy [24]. The study demonstrated a higher sensitivity of the TB-LAMP assay compared to the smear microscopy but with similar specificity [24]. One aspect of TB, however, is that the disease needs to be in its active form to be detected.…”

“…The group assessed the RT-LAMP for the diagnosis of COVID-19 disease as a comparison to the use of RT-PCR. Jiang et al [24], [62]concluded that the analytical specificity and sensitivity of the RT-LAMP assay was 100% with a determined LOD between 500-1000 copies/ml of the virus. Another study performed by Garneret et al [63] also demonstrated a LOD of 1 genome copy/ul (1000 copies/ml) when the performance of RT-LAMP was compared to RT-PCR (Table 2) [63].…”