Porcine epidemic diarrhea virus (PEDV), porcine bocavirus (PBoV), and porcine rotavirus (PoRV) are associated with porcine viral diarrhea. In this study, triplex loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) was established for the simultaneous detection of PEDV, PoRV, and PBoV. The PEDV-gp6, PoRV-vp6, and PBoV-vp1 genes were selected to design LAMP primers. The amplification could be carried out at 64 °C using a miniature metal bath within 30 min. The triplex LAMP–LFD assay exhibited no cross-reactions with other porcine pathogens. The limits of detection (LODs) of PEDV, PoRV, and PBoV were 2.40 × 10¹ copies/μL, 2.89 × 10¹ copies/μL, and 2.52 × 10¹ copies/μL, respectively. The consistency between rt-qPCR and the triplex LAMP–LFD was over 99% in field samples testing. In general, the triplex LAMP–LFD assay was suitable for the rapid and simultaneous detection of the three viruses in the field.

Nipah virus (NiV) is a zoonotic pathogen that poses a significant threat because of its wide host range, multiple transmission modes, high transmissibility, and high mortality rates, affecting both human health and animal husbandry. In this study, we developed a one-step reverse transcription droplet digital PCR (RT-ddPCR) assay that targets the N gene of NiV. Our RT-ddPCR assay exhibited remarkable sensitivity, with a lower limit of detection of 6.91 copies/reaction. Importantly, it displayed no cross-reactivity with the other 13 common viruses and consistently delivered reliable results with a coefficient of variation below 10% across different concentrations. To validate the effectiveness of our RT-ddPCR assay, we detected 75 NiV armored RNA virus samples, mimicking real-world conditions, and negative control samples, and the RT-ddPCR results perfectly matched the simulated results. Furthermore, compared with a standard quantitative real-time PCR (qPCR) assay, our RT-ddPCR assay demonstrated greater stability when handling complex matrices with low viral loads. These findings show that our NiV RT-ddPCR assay is exceptionally sensitive and provides a robust tool for quantitatively detecting NiV, particularly in stimulated field samples with low viral loads or complex matrices. This advancement has significant implications for early NiV monitoring, safeguarding human health and safety, and advancing animal husbandry practices.

Numerous viruses, such as the bovine rotavirus (BRV), the bovine parvovirus (BPV), and the bovine viral diarrhea virus (BVDV), can cause bovine viral diarrhea syndrome. The global livestock industry has been subjected to significant consequences due to this condition. This results in considerable losses and hinders the production of crucial resources such as meat and milk, which are indispensable for sustaining the world's population. It is crucial to develop a quick and precise way of simultaneously detecting BVDV, BRV, and BPV, as they often occur together in mixed infections. A triplex loop-mediated isothermal amplification-lateral flow dipstick (LAMP-LFD) assay that can concurrently detect all three viruses was introduced in this study. The amplification process involved 30 minutes of incubation at 65 °C. The limits of detection (LODs) for BVDV, BRV, and BPV were 2.62 × 10¹ copies per μL, 2.43 × 10¹ copies per μL, and 2.50 × 10¹ copies per μL, respectively. The triplex LAMP-LFD assay was further evaluated in 156 anal swab samples, and the results were in agreement with the results of fluorescence quantitative PCR (qPCR) in more than 99% of the cattle. This assay is expected for the quick identification of triplex viruses in the field because it has high sensitivity and specificity and doesn't depend on laboratory equipment or conditions.

Trees in the genus Phoebe of the Lauraceae family are commonly known as “Nanmu” in traditional Chinese culture. As they have offered highly valued timbers for construction, furniture, and coffins since the pre-Qin Dynasty, it is crucial to identify and protect these Phoebe species. However, the accuracy of Phoebe species identification is frequently hampered due to the limitations of traditional morphological and wood anatomy methods as the marker characteristics are very similar between the species, alongside the requirement for specialized expertise. Here, we use DNA barcoding technology for the rapid and accurate identification of five endangered Phoebe species in China, including Phoebe bournei, P. chekiangensis, P. hui, P. sheareri and P. zhennan. Four highly divergent regions (petA-psbJ-psbL-psbF-psbE, Ψycf1-ndhF, rpl32-trnL^UAG and ycf1) were identified from a comparison of the 20 Phoebe plastomes downloaded from the database. Furthermore, phylogenetic analysis on 20 Phoebe species showed that rpl32-trnL^UAG + ycf1, as well as rpl32-trnL^UAG + ycf1 + Ψycf1-ndhF, effectively distinguished the fifteen Phoebe species. We further validated the usefulness of the core 2-locus barcode using wood and leaf samples from multiple sites for five target species. The study confirms the reliability of molecular diagnostics for five Phoebe species. It also establishes critical taxonomic protocols for conserving these endangered Nanmu species in southern China.