Aspleniumguodanum (Aspleniaceae), a distinct new fern species from northern Guangdong, China, based on morphological data and molecular phylogeny.

A new spleenwort species, Aspleniumguodanum, was found and described from Danxia landform region in Guangdong, China. The new species has close resemblance to A.subcrenatum Ching ex S.H.Wu in morphology, but can be distinguished by having plants small, stipes and rachises not covered with fibrous scales, relatively fewer pairs of pinnae, pinnae short, pinna margin weakly biserrate, pinna apex acute and lower pinnae obviously reduced. Phylogenetic analyses, based on six plastid markers (atpB, rbcL, rps4 & rps4-trnS and trnL & trnL-F) of the new species and its relatives, support a close relationship between A.guodanum and A.subcrenatum. Only one population with no more than 50 individuals were found and, therefore, it is recommended to be classified as Critically Endangered (CR) following IUCN Red List Criteria.

Transmission characteristics in Tuberculosis by WGS: nationwide cross-sectional surveillance in China.

China, with the third largest share of global tuberculosis cases, faces a substantial challenge in its healthcare system as a result of the high burden of multidrug-resistant and rifampicin-resistant tuberculosis (MDR/RR-TB). This study employs a genomic epidemiological approach to assess recent tuberculosis transmissions between individuals, identifying potential risk factors and discerning the role of transmitted resistant isolates in the emergence of drug-resistant tuberculosis in China. We conducted a population-based retrospective study on 5052 Mycobacterium tuberculosis (MTB) isolates from 70 surveillance sites using whole genome sequencing (WGS). Minimum spanning tree analysis identified resistance mutations, while epidemiological data analysis pinpointed transmission risk factors. Of the 5052 isolates, 23% (1160) formed 452 genomic clusters, with 85.6% (387) of the transmissions occurring within the same counties. Individuals with younger age, larger family size, new cases, smear positive, and MDR/RR were at higher odds for recent transmission, while higher education (university and above) and occupation as a non-physical workers emerged as protective factors. At least 61.4% (251/409) of MDR/RR-TB were likely a result of recent transmission of MDR/RR isolates, with previous treatment (crude OR = 2.77), smear-positive (cOR = 2.07) and larger family population (cOR = 1.13) established as risk factors. Our findings highlight that local transmission remains the predominant form of TB transmission in China. Correspondingly, drug-resistant tuberculosis is primarily driven by the transmission of resistant tuberculosis isolates. Targeted interventions for high-risk populations to interrupt transmission within the country will likely provide an opportunity to reduce the prevalence of both tuberculosis and drug-resistant tuberculosis.

Endemic medicinal plant distribution correlated with stable climate, precipitation, and cultural diversity.

Medicinal plants provide crucial ecosystem services, especially in developing countries such as China, which harbors diverse endemic medicinal plant species with substantial cultural and economic value. Accordingly, understanding the patterns and drivers of medicinal plant distribution is critical. However, few studies have investigated the patterns and drivers of endemic medicinal plants distribution in China. Here, we linked endemic medicinal plants distribution with possible explanatory variables, i.e., paleoclimate change, contemporary climate, altitudinal range and ethnic minority human population size at the prefecture city level in China. Our results show that endemic medicinal plants are concentrated in southern China, especially in southwestern China. Notably, both endemic medicinal plant species richness and the ratio of endemic medicinal plant species richness are negatively associated with glacial-interglacial anomaly in temperature, and positively associated with contemporary precipitation and altitudinal range. In addition, we found that endemic medicinal plant species richness is positively associated with ethnic minority population sizes as well as its ratio to the overall population size. These findings suggest that the distribution of endemic medicinal plants is determined by multiple drivers. Furthermore, our findings stress that dramatic future climate changes and massive anthropogenic activities in southern China pose great challenges to the conservation of China's endemic medicinal plants.

Geographical patterns and determinants in plant reproductive phenology duration.

Biodiversity is and always has been an important issue in ecological research. Biodiversity can reflect niche partitioning among species at several spatial and temporal scales and is generally highest in the tropics. One theory to explain it is that low-latitude tropical ecosystems are dominated by species that are generally only distributed over a narrow area. This principle is known as Rapoport's rule. One previously unconsidered extension of Rapoport's rule may be reproductive phenology, where variation in flowering and fruiting length may reflect a temporal range. Herein, we collected reproductive phenology data for more than 20,000 species covering almost all angiosperm species in China. We used a random forest model to quantify the relative role of seven environmental factors on the duration of reproductive phenology. Our results showed that the duration of reproductive phenology decreased with latitude, although there was no obvious change across longitudes. Latitude explained more of the variation in the duration of flowering and fruiting phases in woody plants than in herbaceous plants. Mean annual temperature and the length of the growing season strongly influenced the phenology of herbaceous plants, and average winter temperature and temperature seasonality were important drivers of woody plant phenology. Our result suggests the flowering period of woody plants is sensitive to temperature seasonality, while it does not influence herbaceous plants. By extending Rapoport's rule to consider the distribution of species in time as well as space, we have provided a novel insight into the mechanisms of maintaining high levels of diversity in low-latitude forests.

Engineering Outer Membrane Vesicles to Increase Extracellular Electron Transfer of Shewanella oneidensis.

Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogenShewanella oneidensis MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene pbpC for peptidoglycan integrity (Module 1) and the N-acetyl-d-mannosamine dehydrogenase-encoding gene wbpP involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m-2, 6.33- and 6.96-fold higher than that of the wild-typeS. oneidensis MR-1 (52.3 ± 0.6 mW m-2), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane c-type cytochromes (c-Cyts) including MtrC and OmcA and periplasmic c-Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of S. oneidensis, which paves the way for further study of OMV-mediated EET.

Soil nutrients and plant diversity affect ectomycorrhizal fungal community structure and functional traits across three subalpine coniferous forests.

The symbiotic relationship between ectomycorrhizal fungi (EMF) and the roots of host plants is significantly important in regulating the health and stability of ecosystems, especially of those such as the climate warming affected subalpine forest ecosystems. Therefore, from the coniferous forest systems located in the Southern Qinghai-Tibetan Plateau, root tips from three forest tree species: Pinus wallichiana, Abies spectabilis and Picea spinulosa, were collected to look for the local causes of EMF community composition and diversity patterns. The EMF colonization rate, diversity and taxonomic community structure were determined by morphotyping and sanger sequencing of the fungal ITS gene from the root tip samples. Soil exploration types were identified based on the morphologies of the ectomycorrhizas, coupled with soil properties analysis and plant diversity survey. Contrasting patterns of EMF community and functional diversity were found across the studied three forests types dominated by different coniferous tree species. In terms of associations between soil and EMF properties, the total phosphorus (TP) and nitrate (NO3 -) contents in soil negatively correlated with the colonization rate and the Shannon diversity index of EMF in contrast to the positive relationship between TP and EMF richness. The soil total nitrogen (TN), ammonium (NH4 +) and plant diversity together caused 57.6% of the total variations in the EMF taxonomic community structure at the three investigated forest systems. Whereas based on the soil exploration types alone, NH4 + and TN explained 74.2% of variance in the EMF community structures. Overall, the findings of this study leverage our understanding of EMF dynamics and local influencing factors in coniferous forests dominated by different tree species within the subalpine climatic zone.

Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing.

Whole genome sequencing provides rapid insight into key information about the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), such as virus typing and key mutation site, and this information is important for precise prevention, control and tracing of coronavirus disease 2019 (COVID-19) outbreak in conjunction with the epidemiological information of the case. Nanopore sequencing is widely used around the world for its short sample-to-result time, simple experimental operation and long sequencing reads. However, because nanopore sequencing is a relatively new sequencing technology, many researchers still have doubts about its accuracy. The combination of the newly launched nanopore sequencing Q20+ kit (LSK112) and flow cell R10.4 is a qualitative improvement over the accuracy of the previous kits. In this study, we firstly used LSK112 kit with flow cell R10.4 to sequence the SARS-CoV-2 whole genome, and summarized the sequencing results of the combination of LSK112 kit and flow cell R10.4 for the 1200bp amplicons of SARS-CoV-2. We found that the proportion of sequences with an accuracy of more than 99% reached 30.1%, and the average sequence accuracy reached 98.34%, while the results of the original combination of LSK109 kit and flow cell R9.4.1 were 0.61% and 96.52%, respectively. The mutation site analysis showed that it was completely consistent with the final consensus sequence of next generation sequencing (NGS). The results showed that the combination of LSK112 kit and flow cell R10.4 allowed rapid whole-genome sequencing of SARS-CoV-2 without the need for verification of NGS.

Inorganic Nitrogen-Containing Aerosol Deposition Caused "Excessive Photosynthesis" of Herbs, Resulting in Increased Nitrogen Demand.

The amount of atmospheric nitrogen-containing aerosols has increased dramatically due to the globally rising levels of nitrogen from fertilization and atmospheric deposition. Although the balance of carbon and nitrogen in plants is a crucial component of physiological and biochemical indexes and plays a key role in adaptive regulation, our understanding of how nitrogen-containing aerosols affect this remains limited; in particular, regarding the associated mechanisms. Using a fumigation particle generator, we generated ammonium nitrate solution (in four concentrations of 0, 15, 30, 60 kg N hm-2 year-1) into droplets, in 90% of which the diameters were less than 2.5 µm, in the range of 0.35-4 µm, and fumigated Iris germanica L. and Portulaca grandiflora Hook. for 30 days in April and August. We found that the weight percentage of nitrogen in the upper epidermis, mesophyll tissue, and bulk of leaves decreased significantly with the N addition rate, which caused a decrease of carbon:nitrogen ratio, due to the enhanced net photosynthetic rate. Compared with Portulaca grandiflora Hook., Iris germanica L. responded more significantly to the disturbance of N addition, resulting in a decrease in the weight percentage of nitrogen in the roots, due to a lower nitrogen use efficiency. In addition, the superoxide dismutase activity of the two plants was inhibited with a higher concentration of nitrogen sol; a reduction of superoxide dismutase activity in plants means that the resistance of plants to various environmental stresses is reduced, and this decrease in superoxide dismutase activity may be related to ROS signaling. The results suggest that inorganic nitrogen-containing aerosols caused excessive stress to plants, especially for Iris germanica L.

Extensive sharing of chloroplast haplotypes among East Asian Cerris oaks: The imprints of shared ancestral polymorphism and introgression.

Shared ancestral polymorphism and introgression are two main causes of chloroplast DNA (cpDNA) haplotype sharing among closely related angiosperms. In this study, we explored the roles of these two processes in shaping the phylogeographic patterns of East Asian Cerris oaks by examining the geographic distributions of randomly and locally distributed shared haplotypes, which coincide with the expectations of shared ancestry and introgression, respectively. We sequenced 1340 bp of non-coding cpDNA from Quercus acutissima (n = 418) and Q. chenii (n = 183) and compiled previously published sequence data of Q. variabilis (n = 439). The phylogenetic relationships among haplotypes were examined using a median-joining network. The geographic patterns of interspecifically shared haplotypes were assessed to test whether nearby populations have a higher degree of interspecific cpDNA sharing than distant ones. We identified a total of 27 haplotypes that were grouped into three non-species-specific lineages with overlapping distributions. Ancestral haplotypes were extensively shared and randomly distributed across populations of the three species. Some young haplotypes were locally shared in mountainous areas that may have been shared refugia. The local exchange of cpDNA resulted in an excess of similar haplotypes between nearby populations. Our study demonstrated that the haplotype sharing pattern among East Asian Cerris oaks reflected the imprints of both shared ancestral polymorphism and introgression. This pattern was also associated with the relatively stable climates and complex landscapes in East Asia, which not only allowed the long-term persistence of ancestral lineages but also connected the survived populations across refugia.

Nanopore sequencing technology and its application in plant virus diagnostics.

Plant viruses threaten crop yield and quality; thus, efficient and accurate pathogen diagnostics are critical for crop disease management and control. Recent advances in sequencing technology have revolutionized plant virus research. Metagenomics sequencing technology, represented by next-generation sequencing (NGS), has greatly enhanced the development of virus diagnostics research because of its high sensitivity, high throughput and non-sequence dependence. However, NGS-based virus identification protocols are limited by their high cost, labor intensiveness, and bulky equipment. In recent years, Oxford Nanopore Technologies and advances in third-generation sequencing technology have enabled direct, real-time sequencing of long DNA or RNA reads. Oxford Nanopore Technologies exhibit versatility in plant virus detection through their portable sequencers and flexible data analyses, thus are wildly used in plant virus surveillance, identification of new viruses, viral genome assembly, and evolution research. In this review, we discuss the applications of nanopore sequencing in plant virus diagnostics, as well as their limitations.

Mutations and Phylogenetic Analyses of SARS-CoV-2 Among Imported COVID-19 From Abroad in Nanjing, China.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a pandemic and is threatening human health globally. The rapid genome sequencing and bioinformatic analysis of SARS-CoV-2 have become a helpful tool in the battle against the COVID-19. Here, we report the genetic characteristics, variations and phylogenetic analysis of SARS-CoV-2 sequenced from 42 clinical specimens. The complete genomes sequencing of SARS-CoV-2 were performed using Oxford Nanopore sequencing. All genomes accumulated mutations compared to the Wuhan-Hu-1 (GenBank Accession No: MN908947.3). Our data of the 42 whole genomes revealed 16 different lineages. The B.1.1 lineage was the most frequent, and 5, 2, 2, 3, and 1 sequences were classified as lineages of B.1.1.7, B.1.351, P.1, B.1.617.2, and C.37, respectively. A total of 328 nucleotide mutation sites were found in 42 genomes, among which A23403G mutation (D614G amino acid change in the spike protein) was the most common substitution. The phylogenetic trees of 42 SARS-CoV-2 sequences and GISAID-available SARS-CoV-2 sequences were constructed and its taxonomic status was supported. These results will provide scientific basis for tracing the source and prevention and control of SARS-CoV-2 imported from abroad in Nanjing, China.

Identification of the first case of Penicillium marneffei infection in Shaanxi province by nanopore sequencing.

INTRODUCTION: Penicillium marneffei is one of the most common opportunistic infectious fungi associated with AIDS (Acquired Immunodeficiency Syndrome). It is prevalent in Southeast Asia and southern China (such as Guangdong), but rare in inland provinces of China. Penicillium marneffei infections are often misdiagnosed. CASE REPORT: Here, we report a patient with Penicillium marneffei infection from Shaanxi Province, China, who was previously repeatedly diagnosed as Kala-azar in two hospitals. The patient received medical treatment due to fever and multiple papules, worked as a tour guide, and had travel history in Southeast Asian countries. We performed nanopore genome sequencing on blood samples of the patient and obtained 84,000 reads in 3.5 hours. The average length of the sequences was 7088.7 bases and the longest sequence was 87471 bases. Through comparison with the pathogenic bacteria database, 13 homologous Penicillium marneffei sequences were identified. Furthermore, by using specific quantitative real time-polymerase chain reaction of Penicillium marneffei, fungal ITS (Internal Transcribed Spacer) sequencing, and fungal biphasic culture, we further confirmed the pathogen as Penicillium marneffei. Meanwhile, the patient was confirmed to be HIV (Human Immunodeficiency Virus) positive. Thus, the patient was diagnosed with AIDS combined with Penicillium marneffei infection, which, to the best of our knowledge, is the first report of Penicillium marneffei infection in Shaanxi Province, China. CONCLUSIONS: Metagenomic analysis based on nanopore sequencing provides an important reference for the diagnosis of Penicillium marneffei infection in this case.

Comparative analysis of complete Ilex (Aquifoliaceae) chloroplast genomes: insights into evolutionary dynamics and phylogenetic relationships.

BACKGROUND: Ilex (Aquifoliaceae) are of great horticultural importance throughout the world for their foliage and decorative berries, yet a dearth of genetic information has hampered our understanding of phylogenetic relationships and evolutionary history. Here, we compare chloroplast genomes from across Ilex and estimate phylogenetic relationships. RESULTS: We sequenced the chloroplast genomes of seven Ilex species and compared them with 34 previously published Ilex plastomes. The length of the seven newly sequenced Ilex chloroplast genomes ranged from 157,182 bp to 158,009 bp, and contained a total of 118 genes, including 83 protein-coding, 31 rRNA, and four tRNA genes. GC content ranged from 37.6 to 37.69%. Comparative analysis showed shared genomic structures and gene rearrangements. Expansion and contraction of the inverted repeat regions at the LSC/IRa and IRa/SSC junctions were observed in 22 and 26 taxa, respectively; in contrast, the IRb boundary was largely invariant. A total of 2146 simple sequence repeats and 2843 large repeats were detected in the 41 Ilex plastomes. Additionally, six genes (psaC, rbcL, trnQ, trnR, trnT, and ycf1) and two intergenic spacer regions (ndhC-trnV and petN-psbM) were identified as hypervariable, and thus potentially useful for future phylogenetic studies and DNA barcoding. We recovered consistent phylogenetic relationships regardless of inference methodology or choice of loci. We recovered five distinct, major clades, which were inconsistent with traditional taxonomic systems. CONCLUSION: Our findings challenge traditional circumscriptions of the genus Ilex and provide new insights into the evolutionary history of this important clade. Furthermore, we detail hypervariable and repetitive regions that will be useful for future phylogenetic and population genetic studies.

Corrigendum: Systematic benchmarking of nanopore Q20+ kit in SARS-CoV-2 whole genome sequencing.

[This corrects the article DOI: 10.3389/fmicb.2022.973367.].

Spatial phylogenetics of the Chinese angiosperm flora provides insights into endemism and conservation.

The flora of China is well known for its high diversity and endemism. Identifying centers of endemism and designating conservation priorities are essential goals for biodiversity studies. However, there is no comprehensive study from a rigorous phylogenetic perspective to understand patterns of diversity and endemism and to guide biodiversity conservation in China. We conducted a spatial phylogenetic analysis of the Chinese angiosperm flora at the generic level to identify centers of neo- and paleo-endemism. Our results indicate that: (i) the majority of grid cells in China with significantly high phylogenetic endemism (PE) were located in the mountainous regions; (ii) four of the nine centers of endemism recognized, located in northern and western China, were recognized for the first time; (iii) arid and semiarid regions in Northwest China were commonly linked to significant PE, consistent with other spatial phylogenetic studies worldwide; and (iv) six high-priority conservation gaps were detected by overlaying the boundaries of China's nature reserves on all significant PE cells. Overall, we conclude that the mountains of southern and northern China contain both paleo-endemics (ancient relictual lineages) and neo-endemics (recently diverged lineages). The areas we highlight as conservation priorities are important for broad-scale planning, especially in the context of evolutionary history preservation.

Influence of Pliocene and Pleistocene climates on hybridization patterns between two closely related oak species in China.

BACKGROUND AND AIMS: Contemporary patterns of genetic admixture reflect imprints of both ancient and recent gene flow, which can provide us with valuable information on hybridization history in response to palaeoclimate change. Here, we examine the relationships between present admixture patterns and past climatic niche suitability of two East Asian Cerris oaks (Quercus acutissima and Q. chenii) to test the hypothesis that the mid-Pliocene warm climate promoted while the Pleistocene cool climate limited hybridization among local closely related taxa. METHODS: We analyse genetic variation at seven nuclear microsatellites (1111 individuals) and three chloroplast intergenic spacers (576 individuals) to determine the present admixture pattern and ancient hybridization history. We apply an information-theoretic model selection approach to explore the associations of genetic admixture degree with past climatic niche suitability at multiple spatial scales. KEY RESULTS: More than 70 % of the hybrids determined by Bayesian clustering analysis and more than 90 % of the individuals with locally shared chloroplast haplotypes are concentrated within a mid-Pliocene contact zone between ~30°N and 35°N. Climatic niche suitabilities for Q. chenii during the mid-Pliocene Warm Period [mPWP, ~3.264-3.025 million years ago (mya)] and during the Last Glacial Maximum (LGM, ~0.022 mya) best explain the admixture patterns across all Q. acutissima populations and across those within the ancient contact zone, respectively. CONCLUSIONS: Our results highlight that palaeoclimate change shapes present admixture patterns by influencing the extent of historical range overlap. Specifically, the mid-Pliocene warm climate promoted ancient contact, allowing widespread hybridization throughout central China. In contrast, the Pleistocene cool climate caused the local extinction of Q. chenii, reducing the probability of interspecific gene flow in most areas except those sites having a high level of ecological stability.

Improved targeting of the 16S rDNA nanopore sequencing method enables rapid pathogen identification in bacterial pneumonia in children.

Objectives: To develop a rapid and low-cost method for 16S rDNA nanopore sequencing. Methods: This was a prospective study on a 16S rDNA nanopore sequencing method. We developed this nanopore barcoding 16S sequencing method by adding barcodes to the 16S primer to reduce the reagent cost and simplify the experimental procedure. Twenty-one common pulmonary bacteria (7 reference strains, 14 clinical isolates) and 94 samples of bronchoalveolar lavage fluid from children with severe pneumonia were tested. Results indicating low-abundance pathogenic bacteria were verified with the polymerase chain reaction (PCR). Further, the results were compared with those of culture or PCR. Results: The turnaround time was shortened to 6~8 hours and the reagent cost of DNA preparation was reduced by employing a single reaction adding barcodes to the 16S primer in advance. The accuracy rate for the 21 common pulmonary pathogens with an abundance ≥ 99% was 100%. Applying the culture or PCR results as the gold standard, 71 (75.5%) of the 94 patients were positive, including 25 positive cultures (26.6%) and 52 positive quantitative PCRs (55.3%). The median abundance in the positive culture and qPCR samples were 29.9% and 6.7%, respectively. With an abundance threshold increase of 1%, 5%, 10%, 15% and 20%, the test sensitivity decreased gradually to 98.6%, 84.9%, 72.6%, 67.1% and 64.4%, respectively, and the test specificity increased gradually to 33.3%, 71.4%, 81.0%, 90.5% and 100.0%, respectively. Conclusions: The nanopore barcoding 16S sequencing method can rapidly identify the pathogens causing bacterial pneumonia in children.

Long-Term Nitrogen Deposition Alters Ectomycorrhizal Community Composition and Function in a Poplar Plantation.

The continuous upsurge in soil nitrogen (N) enrichment has had strong impacts on the structure and function of ecosystems. Elucidating how plant ectomycorrhizal fungi (EMF) mutualists respond to this additional N will facilitate the rapid development and implementation of more broadly applicable management and remediation strategies. For this study, we investigated the responses of EMF communities to increased N, and how other abiotic environmental factors impacted them. Consequently, we conducted an eight-year N addition experiment in a poplar plantation in coastal eastern China that included five N addition levels: 0 (N0), 50 (N1), 100 (N2), 150 (N3), and 300 (N4) kg N ha-1 yr-1. We observed that excessive N inputs reduced the colonization rate and species richness of EMF, and altered its community structure and functional traits. The total carbon content of the humus layer and available phosphorus in the mineral soil were important drivers of EMF abundance, while the content of ammonium in the humus layer and mineral soil determined the variations in the EMF community structure and mycelium foraging type. Our findings indicated that long-term N addition induced soil nutrient imbalances that resulted in a severe decline in EMF abundance and loss of functional diversity in poplar plantations.

Reverse transcription recombinase-aided amplification assay for rapid detection of the influenza A(H1N1)pdm09 H275Y mutation that confers oseltamivir resistance.

The emergence of the influenza A(H1N1)pdm09 virus with the NA-H275Y mutation, which confers oseltamivir resistance, must be monitored, especially in patients undergoing neuraminidase inhibitor treatment. In this study, we developed a reverse transcription recombinase-aided amplification assay that has high sensitivity (detection limit: 1.0 × 101 copies/µL) and specificity for detecting the oseltamivir-resistant H275Y mutation; the assay is performed within 30 min at a constant temperature of 39° Celsius using an isothermal device. This method is suitable for the clinical application of targeted testing, thereby providing technical support for precision medicine in individual drug applications for patients with severe infection or immunosuppression.