Rapid detection of four major HFMD-associated enteroviruses by multiplex HiFi-LAMP assays.

Hand, foot, and mouth disease (HFMD) caused by various enteroviruses is a major public health concern globally. Human enterovirus 71(EVA71), coxsackievirus A16 (CVA16), coxsackievirus A6 (CVA6), and coxsackievirus A10 (CVA10) are four major enteroviruses responsible for HFMD. Rapid, accurate, and specific point-of-care (POC) detection of the four enteroviruses is crucial for the prevention and control of HFMD. Here, we developed two multiplex high-fidelity DNA polymerase loop-mediated isothermal amplification (mHiFi-LAMP) assays for simultaneous detection of EVA71, CVA16, CVA6, and CVA10. The assays have good specificity and exhibit high sensitivity, with limits of detection (LOD) of 11.2, 49.6, 11.4, and 20.5 copies per 25 µL reaction for EVA71, CVA16, CVA6, and CVA10, respectively. The mHiFi-LAMP assays showed an excellent clinical performance (sensitivity 100.0%, specificity 83.3%, n = 47) when compared with four singleplex RT-qPCR assays (sensitivity 93.1%, specificity 100%). In particular, the HiFi-LAMP assays exhibited better performance (sensitivity 100.0%, specificity 100%) for CVA16 and CVA6 than the RT-qPCR assays (sensitivity 75.0-92.3%, specificity 100%). Furthermore, the mHiFi-LAMP assays detected all clinical samples positive for the four enteroviruses within 30 min, obviously shorter than about 1-1.5 h by the RT-qPCR assays. The new mHiFi-LAMP assays can be used as a robust point-of-care testing (POCT) tool to facilitate surveillance of HFMD at rural and remote communities and resource-limited settings.

Capsaicin receptor TRPV1 maintains quiescence of hepatic stellate cells in the liver via recruitment of SARM1.

BACKGROUND & AIMS: Capsaicin receptor, also known as transient receptor potential vanilloid 1 (TRPV1), is involved in pain physiology and neurogenic inflammation. Herein, we discovered the presence of TRPV1 in hepatic stellate cells (HSCs) and aimed to delineate its function in this cell type and liver fibrosis. METHODS: TRPV1 expression was examined in liver biopsies from patients with liver fibrosis using quantitative real-time PCR and immunostaining. Its contribution to liver fibrosis was examined in Trpv1-/- mice, upon lentiviral delivery of the TRPV1 gene, and in human and mouse primary HSCs, using patch clamp, intracellular Ca2+ mobilization determination, FACS analyses and gain/loss of function experiments. Binding of sterile alpha and Toll/interleukin-1 receptor motif-containing protein 1 (SARM1) to TRPV1 was determined using mass spectrometry, co-immunoprecipitation, surface plasmon resonance, bioluminescence resonance energy transfer, and NanoBiT. RESULTS: TRPV1 mRNA levels are significantly downregulated in patients with liver fibrosis and mouse models, showing a negative correlation with F stage and α-smooth muscle actin expression, a marker of HSC activation. TRPV1 expression and function decrease during HSC activation in fibrotic livers in vivo or during culture. Genetic and pharmacological inhibition of TRPV1 in quiescent HSCs leads to NF-κB activation and pro-inflammatory cytokine production. TRPV1 requires binding of its N-terminal ankyrin repeat domain to the TIR-His583 (Toll/interleukin-1 receptor) domain of SARM1 to prevent HSCs from pro-inflammatory activation. Trpv1-/- mice display increased HSC activation and more severe liver fibrosis, whereas TRPV1 overexpression is antifibrotic in various disease models. CONCLUSION: The antifibrotic properties of TRPV1 are attributed to the prevention of HSC activation via the recruitment of SARM1, which could be an attractive therapeutic strategy against liver fibrosis. IMPACT AND IMPLICATIONS: We identified the neuronal channel protein TRPV1 as a gatekeeper of quiescence in hepatic stellate cells, a key driver of liver fibrogenesis and chronic liver disease. Physiologically expressed in healthy liver and consistently downregulated during liver fibrosis development, its therapeutic re-expression is expected to have few side effects, making it an attractive target diagnostic tool and drug candidate for industry and clinicians.

An HFman probe-based reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for HIV-1 detection.

Loop-mediated isothermal amplification (LAMP) is suitable for the development of a rapid and cost-effective nucleic acid technique for point of care (POC) applications. However, LAMP methods often generate non-specific amplification, therefore inevitably resulting in false positive results especially when sequence-independent dyes are used to indirectly reflect the results. In this study, we established and optimized a reverse transcription LAMP (RT-LAMP) assay with a high-fidelity DNA polymerase-mediated fluorescent probe (HFman probe) for human immunodeficiency virus-1 (HIV-1) detection. The assay showed high sensitivity and specificity. Using 101 plasma samples with different HIV-1 viral load, we demonstrated that our assay can detect the major HIV-1 subtypes circulating in China, including CRF01_AE, CRF07_BC, CRF08_BC, CRF55_01B, and unique recombinant forms (URFs). We also compared our assay with an approved commercial real-time quantitative polymerase chain reaction (RT-qPCR) kit and found the sensitivity, specificity and consistency was 88.8%, 100% and 89.1%, respectively. The HFman probe-based RT-LAMP assay is a high specific detection method that is rapid, variant-tolerant and simple to operate, and thus is of great significance for timely disclosure of HIV status and rapid POC diagnosis.

The interaction between free Ca2+ in host cells and invasion of E. tenella.

Eimeria tenella is the most pathogenic and common coccidia that causes chicken coccidiosis. The intracellular free Ca2+ of the host cell is closely related to the invasion, development, and proliferation of intracellular parasites. To determine the dynamic changes of intracellular free Ca2+ and its function in the process of E. tenella invading host cells, we established a chick embryo cecal epithelial cells model of E. tenella infection. Chick embryo cecal epithelial cells were treated with different Ca2+ signal inhibitor, respectively, and then infected with E. tenella. The results showed that extracellular Ca2+, Ca2+ channels on the cell membrane, IP3R ion channels on the endoplasmic reticulum membrane, and RyR ion channels regulated the free Ca2+ in cecal epithelial cells. Through fluorescence labeling and invasion rate detection, we found that the intracellular Ca2+ did not change significantly during the invasion of E. tenella, but its stability was critical to the invasion of parasites.

[Application of high content imaging technique in research on effective components of traditional Chinese medicine: a review].

High content imaging(HCI) technique that combines automatic high throughput with high-resolution cell imaging, is characterized by abundant data information, high imaging sensitivity, easy visualization and standardization, and is commonly used in the cellular(or subcellular) phenotypic analysis. Abundant phenotypic information can be obtained by using HCI in one experiment, including cellular morphology, cellular structure, and signal transduction pathways of related functions, on the basis of the maintenance of the integrity of cellular structures and functions. Multiple studies have shown that a series of dynamic spatio-temporal interactive change processes were induced by the disturbance of cells by specific factors, making cell phenotypes change accordingly, especially for the slight perturbation response of cells. Generally, the detection of one or several endpoint effect indicators is often difficult to accurately and comprehensively reflect the overall efficacy information of traditional Chinese medicine(TCM) because of its unique characteristics of multi-components and multi-targets. The application of HCI is thus helpful to discover the effective components and their action modes in the complex system of TCM. This paper reviewed the application progress in the HCI technique in the screening of active components and their regulation mechanism to provide references for further research.

A circular RNAs dataset landscape reveals potential signatures for the detection and prognosis of early-stage lung adenocarcinoma.

BACKGROUND: This study aimed to identify potential circular ribonucleic acid (circRNA) signatures involved in the pathogenesis of early-stage lung adenocarcinoma (LAC). METHODS: The circRNA sequencing dataset of early-stage LAC was downloaded from the Gene Expression Omnibus database. First, the differentially expressed circRNAs (DEcircRNAs) between tumour and non-tumour tissues were screened. Then, the corresponding miRNAs and their target genes were predicted. In addition, prognosis-related genes were identified using survival analysis and further used to build a network of competitive endogenous RNAs (ceRNAs; DEcircRNA-miRNA-mRNA). Finally, the functional analysis and drug-gene interaction analysis of mRNAs in the ceRNA network was performed. RESULTS: A total of 35 DEcircRNAs (30 up-regulated and 5 down-regulated circRNAs) were identified. Moreover, 135 DEcircRNA-miRNA and 674 miRNA-mRNA pairs were predicted. The survival analysis of these target mRNAs revealed that 60 genes were significantly associated with survival outcomes in early-stage LAC. Of these, high levels of PSMA 5 and low levels of NAMPT, CPT 2 and TNFSF11 exhibited favourable prognoses. In addition, the DEcircRNA-miRNA-mRNA network was constructed, containing 5 miRNA-circRNA (hsa_circ_0092283/hsa-miR-762/hsa-miR-4685-5p; hsa_circ_0070610/hsa-let-7a-2-3p/hsa-miR-3622a-3p; hsa_circ_0062682/hsa-miR-4268) and 60 miRNA-mRNA pairs. Functional analysis of the genes in the ceRNA network showed that they were primarily enriched in the Wnt signalling pathway. Moreover, PSMA 5, NAMPT, CPT 2 and TNFSF11 had strong correlations with different drugs. CONCLUSION: Three circRNAs (hsa_circ_0062682, hsa_circ_0092283 and hsa_circ_0070610) might be potential novel targets for the diagnosis of early-stage LAC.

A rapid variant-tolerant reverse transcription loop-mediated isothermal amplification assay for the point of care detection of HIV-1.

Human immunodeficiency virus (HIV) continues to be a major burden on public health globally with on-going increases in the number of new infections each year. Rapid and sensitive point-of-care tests allow timely interventions and are essential to control the spread of the disease. However the highly variable nature of the virus, resulting in the evolution of many subtypes and inter-subtype recombinants, poses important challenges for its diagnosis. Here we describe a variant-tolerant reverse-transcription RT-LAMP amplification of the virus's INT gene, providing a simple to use, rapid (<30 min) in vitro point-of-care diagnostic test with a limit of detection <18 copies/reaction. The assay was first validated in clinical studies of patient samples, using both established RT-LAMP and RT-qPCR assays for reference, with results showing that this new variant-tolerant HIV-1 RT-LAMP diagnostic test is highly sensitive without compromising its high specificity for HIV-1 subtypes. The diagnostic test was subsequently configured within an easy-to-read paper microfluidic lateral flow test and was validated clinically using patient samples, demonstrating its future potential for use in timely, effective, low cost HIV diagnostics in global regions where healthcare resources may be limited.

Incorporation of heparin/BMP2 complex on GOCS-modified magnesium alloy to synergistically improve corrosion resistance, anticoagulation, and osteogenesis.

The in vivo fast degradation and poor biocompatibility are two major challenges of the magnesium alloys in the field of artificial bone materials. In this study, graphene oxide (GO) was first functionalized by chitosan (GOCS) and then immobilized on the magnesium alloy surface, finally the complex of heparin and bone morphogenetic protein 2 was incorporated on the modified surface to synergistically improve the corrosion resistance, anticoagulation, and osteogenesis. Apart from an excellent hydrophilicity after the surface modification, a sustained heparin and BMP2 release over 14 days was achieved. The corrosion resistance of the modified magnesium alloy was significantly better than that of the control according to the results of electrochemical tests. Moreover, the corrosion rate was also significantly reduced in contrast to the control. The modified magnesium alloy not only had excellent anticoagulation, but also can significantly promote osteoblast adhesion and proliferation, upregulate the expression of alkaline phosphatase and osteocalcin, and enhance mineralization. Therefore, the method of the present study can be used to simultaneously improve the corrosion resistance and biocompatibility of the magnesium alloys targeted for the orthopedic applications.

Rational Design and Identification of Small-Molecule Allosteric Inhibitors of CD38.

CD38 is a multi-functional signaling enzyme that catalyzes the biosynthesis of two calcium-mobilizing second messengers: cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate. It also regulates intracellular nicotinamide adenine dinucleotide (NAD) contents, associated with multiple pathophysiological processes such as aging and cancer. As such, enzymatic inhibitors of CD38 offer great potential in drug development. Here, through virtual screening and enzymatic assays, we discovered compound LX-102, which targets CD38 on the side opposite its enzymatic pocket with a binding affinity of 7.7 µm. It inhibits the NADase activity of CD38 with an IC50 of 14.9 µm. Surface plasmon resonance (SPR) and hydrogen/deuterium exchange and mass spectrometry experiments verified that LX-102 competitively binds to the epitope of the therapeutic SAR 650984 antibody in an allosteric manner. Molecular dynamics simulation was performed to demonstrate the binding dynamics of CD38 with the allosteric ligand. In summary, we established that the cavity to which SAR 650984 binds was an allosteric site and was accessible for the rational design of small chemical modulators of CD38. The lead compound LX-102 that we identified in this study could also be a useful tool for probing CD38 functions and promoting drug discovery.

Effects of Salt Stress on Plant Growth, Antioxidant Capacity, Glandular Trichome Density, and Volatile Exudates of Schizonepeta tenuifolia Briq.

Salinity is a major abiotic factor affecting plant growth and secondary metabolism. However, no information is available about its effects on Schizonepeta tenuifolia Briq., a traditional Chinese herb. Here, we investigated the changes of plant growth, antioxidant capacity, glandular trichome density, and volatile exudates of S. tenuifolia exposed to salt stress (0, 25, 50, 75, 100 mM NaCl). Results showed that its dry biomass was reduced by salt treatments except 25 mM NaCl. Contents of antioxidants, including phenolics and flavonoids, increased at low (25 mM) or moderate (50 mM) levels, but declined at severe (75 and 100 mM) levels. On leaf surfaces, big peltate and small capitate glandular trichomes (GTs) were found. Salt treatments, especially at moderate and severe concentrations, enhanced the density of total GTs on both leaf sides. The most abundant compound in GT volatile exudates was pulegone. Under salinity, relative contents of this component and other monoterpenes decreased significantly; biosynthesis and accumulation of esters were enhanced, particularly sulfurous acid,2-ethylhexyl hexyl ester, which became the second major compound as salinity increased. In conclusion, salt stress significantly influenced the growth and secondary metabolism of S. tenuifolia, enabling us to study the changes of its pharmacological activities.

[Changes of ion absorption, distribution and essential oil components of flowering Schizonepeta tenuifolia under salt stress].

In this paper, a pot experiment using quartz sands was conducted to study the effects of different concentrations of NaCl (0, 25, 50, 75, 100 mmol·L⁻¹) on the ion absorption, distribution and essential oil components of flowering Schizonepeta tenuifolia. The results showed that as NaCl concentration increased, Na⁺ content of root, stem, leaf and flower increased significantly, and that of the aerial parts was in a higher level than in the root. Regarding the K⁺ content, it decreased in the root but increased in stem, leaf and flower. Some changes were detected in the Ca²âº content, but not significant on the whole. The value of K⁺/Na⁺ and Ca²âº/Na⁺ reduced as a result of increasing NaCl concentrations. The content of essential oil increased under medium salt treatment (50 mmol·L⁻¹ NaCl). However, the synthesis and accumulation of essential oil was inhibited by the serious salt treatment (100 mmol·L⁻¹ NaCl). Over 98% of the essential oil components were terpenes, in which pulegone and menthone were the most two abundant compounds. Varieties of essential oil components did not change significantly under salt stress but their relative proportions did. The transformation of pulegone to menthone was enhanced and the value of pulegone/menthone based on their relative contents decreased with NaCl concentration increasing. Consequently, menthone ranked the most abundant compound by replacing pulegone. Relative content of D-limonene increased under medium and serious salt stress, and that of ß-caryophyllene only increased under mild treatments. So our research could provide references for the standard cultivation on saline soil of S. tenuifolia.

[The synthesis of purine derivatives and its inhibitory activity on CD38 NADase].

CD38 is a multifunctional enzyme expressed in a variety of mammalian tissues, its catalytic activity was involved in a wide range of physiological processes. Based on the reported inhibitor of human CD38 NADase, 33 purine derivatives were designed and synthesized. The biological activity assay showed that compounds 20 and 38 exhibited almost the same extent of inhibitory activities on human CD38 NADase as the lead compound H2. The results also revealed that small substituents at C-6 of purine ring gave no obvious effect on inhibitory activity, but phenylpropionyl moiety at N-2 could affect the binding mode of the compound with CD38. This study provides a reliable basis for future rational design of inhibitors for CD38.

Design, synthesis and SAR studies of NAD analogues as potent inhibitors towards CD38 NADase.

Nicotinamide adenine dinucleotide (NAD), one of the most important coenzymes in the cells, is a substrate of the signaling enzyme CD38, by which NAD is converted to a second messenger, cyclic ADP-ribose, which releases calcium from intracellular calcium stores. Starting with 2'-deoxy-2'-fluoroarabinosyl-ß-nicotinamide adenine dinucleotide (ara-F NAD), a series of NAD analogues were synthesized and their activities to inhibit CD38 NAD glycohydrolase (NADase) were evaluated. The adenosine-modified analogues showed potent inhibitory activities, among which 2'-deoxy-2'-fluoroarabinosyl-ß-nicotinamide guanine dinucleotide (ara-F NGD) was the most effective one. The structure-activity relationship of NAD analogues was also discussed.

A novel extraction-free dual HiFi-LAMP assay for detection of methicillin-sensitive and methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a leading cause of bacteremia and blood stream infections. Methicillin-resistant S. aureus (MRSA) that first appeared in 1961 often caused hospital-acquired infections (HAIs) and community-acquired infections (CAIs) and was associated with high mortality rate. Accurate and rapid point-of-care testing (POCT) of MRSA is crucial for clinical management and treatment of MRSA infections, as well as the prevention and control of HAIs and CAIs. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of methicillin-susceptible S. aureus and MRSA. The dual HiFi-LAMP assay can detect 30 copies/reaction of nuc and mecA genes with detection limits of 147 and 158 copies per 25 µL reaction, respectively. A retrospective clinical evaluation with 107 clinical S. aureus isolates showed both sensitivity and specificity of 100%. A prospective clinical evaluation with 35 clinical samples revealed a specificity of 100% and a sensitivity of 92.3%. The dual HiFi-LAMP assay can detect almost all S. aureus samples (141/142; 99.3%) within 20 min, implying that the entire HiFi-LAMP assay (including sample process) can be completed within 40 min, extremely significantly shorter than 3-5 days by the traditional clinical microbial culture and antibiotic susceptibility testing. The novel extraction-free dual HiFi-LAMP assay can be used as a robust POCT tool to promote precise diagnosis and treatment of MRSA infections in hospitals and to facilitate surveillance of MRSA at hospital and community settings.IMPORTANCEMethicillin-resistant Staphylococcus aureus (MRSA) was associated with high mortality rate and listed as a "priority pathogen" by the World Health Organization. Accurate and rapid point-of-care testing (POCT) of MRSA is critically required for clinical management and treatment of MRSA infections. Some previous LAMP-based POCT assays for MRSA might be questionable due to their low specificity and the lack of appropriate evaluation directly using clinical samples. Furthermore, they are relatively tedious and time-consuming because they require DNA extraction and lack multiplex detection capacity. Here, we reported a novel extraction-free dual HiFi-LAMP assay for discriminative detection of MRSA and methicillin-susceptible S. aureus. The assay has high specificity and sensitivity and can be completed within 40 min. Clinical evaluation with real clinical samples and clinical isolates showed excellent performance with 100% specificity and 92.3%-100% sensitivity. The novel extraction-free assay may be a robust POCT tool to promote precise diagnosis of MRSA infections and facilitate surveillance of MRSA at hospital and community settings.

A novel fluorescent cell membrane-permeable caged cyclic ADP-ribose analogue.

Cyclic adenosine diphosphate ribose is an endogenous Ca(2+) mobilizer involved in diverse cellular processes. A cell membrane-permeable cyclic adenosine diphosphate ribose analogue, cyclic inosine diphosphoribose ether (cIDPRE), can induce Ca(2+) increase in intact human Jurkat T-lymphocytes. Here we synthesized a coumarin-caged analogue of cIDPRE (Co-i-cIDPRE), aiming to have a precisely temporal and spatial control of bioactive cIDPRE release inside the cell using UV uncaging. We showed that Co-i-cIDPRE accumulated inside Jurkat cells quickly and efficiently. Uncaging of Co-i-cIDPRE evoked Ca(2+) release from endoplasmic reticulum, with concomitant Ca(2+) influx in Jurkat cells. Ca(2+) release evoked by uncaged Co-i-cIDPRE was blocked by knockdown of ryanodine receptors (RyRs) 2 and 3 in Jurkat cells. The associated Ca(2+) influx, on the other hand, was abolished by double knockdown of Stim1 and TRPM2 in Jurkat cells. Furthermore, Ca(2+) release or influx evoked by uncaged Co-i-cIDPRE was recapitulated in HEK293 cells that overexpress RyRs or TRPM2, respectively, but not in wild-type cells lacking these channels. In summary, our results indicate that uncaging of Co-i-cIDPRE incites Ca(2+) release from endoplasmic reticulum via RyRs and triggers Ca(2+) influx via TRPM2.

Evolution of the Probe-Based Loop-Mediated Isothermal Amplification (LAMP) Assays in Pathogen Detection.

Loop-mediated isothermal amplification (LAMP), as the rank one alternative to a polymerase chain reaction (PCR), has been widely applied in point-of-care testing (POCT) due to its rapid, simple, and cost-effective characteristics. However, it is difficult to achieve real-time monitoring and multiplex detection with the traditional LAMP method. In addition, these approaches that use turbidimetry, sequence-independent intercalating dyes, or pH-sensitive indicators to indirectly reflect amplification can result in false-positive results if non-specific amplification occurs. To fulfill the needs of specific target detection and one-pot multiplex detection, a variety of probe-based LAMP assays have been developed. This review focuses on the principles of these assays, summarizes their applications in pathogen detection, and discusses their features and advantages over the traditional LAMP methods.

Subroutine Embedding and Finite Element Simulation of the Improved Constitutive Equation for Ti6Al4V during High-Speed Machining.

The Johnson-Cook (J-C) constitutive model is not suitable for Ti-6Al-4V alloy in the high-speed cutting finite element simulation, as it has no response dynamic recrystallization softening effect under heavy impact and high temperature. In this paper, an improved constitutive model considering the recrystallization effect was established, and the parameters were fitted with the data of flow stress-strain of the Split Hopkinson Pressure Bar (SHPB) test. The relevant theories of cutting finite element simulation were studied, such as nonlinear constitutive elastic-plastic deformation, strain state, and material yield. A subroutine that included the Recht shear failure instability criterion and the improved model was coded in Fortran and embedded in the finite element simulation software AdvantEdge FEM, along with the return mapping stress integration algorithm. The simulated stress of the improved model dropped dramatically from 460 MPa to 220 MPa when the temperature rises from 950 °C to 1000 °C, and its decline reached 46.7%, while the J-C model only decreased by 10%. Comparative studies indicate that the stress change of the improved constitutive simulation is closer to the SHPB test results than the J-C constitutive, and the new one is more suitable when it expresses the high temperature and heavy impact in the high-speed milling.

Structural Design and Optimization of the Milling Force Measurement Tool System Embedded with Thin-Film Strain Sensors.

A milling force measurement tool system is designed with an elastic beam structure, which is divided into a two-end ring hoop compression sensor mode and a two-end square hoop compression sensor mode to improve the strain sensitivity. A simplified mechanical model of the elastic beam is established, and the relationship between the strain and force of the elastic beam under the action of three cutting force components is investigated, which can act a guide for subsequent milling force measurement tool system calibration tests. Thin-film strain sensors occupy a central position in the milling force measurement tool system, which consists of a substrate, transition layer, insulating layer and resistance grid layer. The resistance grid layer has a particularly significant effect on the thin-film strain sensor's performance. In order to further improve the sensitivity of thin-film strain sensors, the shapes of the substrate, the transition layer, the insulating layer and the resistance grid layer are optimized and studied. A new thin-film strain sensor is designed with a resistance grid beam constructed from an insulating layer and a resistive grid layer double-end-supported on the transition layer. The flow of the wet-etching process of thin-film strain sensors is studied and samples are obtained. The surface microforms of the sensor samples are observed by extended depth-of-field microscopy, confocal microscopy and atomic force microscopy. It can be seen that the boundary of the resistance grid layer pattern is tidy and has high dimensional accuracy, thus enabling the basic achievement of the expected effect of the design. The electrical performance of the samples is tested on an experimental platform that we built, and the results show that the resistive sensitivity coefficient of the samples is increased by about 20%, to 51.2%, compared with that of the flat thin-film strain sensor, which fulfils the design's requirements.

Rapid detection of human influenza A viruses by HFman probe-based loop-mediated isothermal amplification assays.

Since China abandoned the zero-COVID policy at the end of 2022, a wave of severe Flu pandemic emerged in China. Rapid and accurate diagnosis of Influenza A virus (IAV) is critical for clinical management and therapeutic decision-making of patients with fever. Here, we reported a novel IAV HF-LAMP assay, which can be performed with purified RNA or directly using clinical samples. The assays with purified RNA and clinical samples have high sensitivity with limit of detection (LOD) of 9.6 copies/reaction, 9900 copies/mL, and short sample-to-answer times of 36 and 50 min, respectively. Both assays showed high specificity and significantly higher IAV detection rate than the rapid antigen detection (RAD) assays. Furthermore, we found the vast majority (91.2 %) of children with fever during the pandemic were infected by IAV, and current IAV infection has a very narrow detectable window. The novel IVA HF-LAMP assays will provide robust tools to facilitate early diagnosis of IAV infection in current and future seasonal influenza epidemics.