Toxoplasmosis, a parasitic disease resulting from Toxoplasma gondii infection, remains prevalent worldwide, and causes great harm to immunodepressed patients, pregnant women and newborns. Although various molecular approaches to detect T. gondii infection are available, they are either costly or technically complex. This study aimed at developing a rapid visual detection assay using recombinase-aided amplification (RAA) and lateral flow dipstick (LFD) coupled with CRISPR-Cas13a fluorescence (RAA-Cas13a-LFD) to detect T. gondii. The RAA-Cas13a-LFD assay was performed in an incubator block at 37 °C within 2 h, and the amplification results were visualized and determined through LFD by the naked eye. The detection limit was 1 × 10−6 ng/μL by our developed RAA-Cas13a-LFD protocol, 100-fold higher than that by qPCR assay (1 × 10−8 ng/μL). No cross-reaction occurred either with the DNA of human blood or Ascaris lumbricoides, Digramma interrupta, Entamoeba coli, Fasciola gigantica, Plasmodium vivax, Schistosoma japonicum, Taenia solium, and Trichinella spiralis, and the positive rate by RAA-Cas13a-LFD assay was identical to that by qPCR assay (1.50% vs. 1.50%) in detecting T. gondii infection in the unknown blood samples obtained from clinical settings. Our findings demonstrate that this RAA-Cas13a-LFD assay is not only rapid, sensitive, and specific and allows direct visualization by the naked eye, but also eliminates sophisticated and costly equipment. More importantly, this technique can be applied to on-site surveillance of T. gondii.

Coronavirus Disease 2019 (COVID-19) caused by SARS-CoV-2 poses a significant threat to global public health. Early detection with reliable, fast, and simple assays is crucial to contain the spread of SARS-CoV-2. The real-time reverse transcription-polymerase chain reaction (RT-PCR) assay is currently the gold standard for SARS-CoV-2 detection; however, the reverse transcription loop-mediated isothermal amplification method (RT-LAMP) assay may allow for faster, simpler and cheaper screening of SARS-CoV-2. In this study, the triple-target RT-LAMP assay was first established to simultaneously detect three different target regions (ORF1ab, N and E genes) of SARS-CoV-2. The results revealed that the developed triplex RT-LAMP assay was able to detect down to 11 copies of SARS-CoV-2 RNA per 25 µL reaction, with greater sensitivity than singleplex or duplex RT-LAMP assays. Moreover, two different indicators, hydroxy naphthol blue (HNB) and cresol red, were studied in the colorimetric RT-LAMP assay; our results suggest that both indicators are suitable for RT-LAMP reactions with an obvious color change. In conclusion, our developed triplex colorimetric RT-LAMP assay may be useful for the screening of COVID-19 cases in limited-resource areas.

No abstract