Search Results (26)

Duck viral enteritis (DVE) is an acute and highly contagious disease that affects waterfowl such as ducks, geese and swans. Duck enteritis virus (DEV) is the pathogen, causing huge economic losses to waterfowl farming in recent years. Establishing a rapid, simple, and visual detection should facilitate the early identification of DEV. After the amplification primers and reaction conditions were optimized, three multienzyme isothermal rapid amplification (MIRA) methods—basic MIRA, MIRA–quantitative PCR (MIRA–qPCR) and MIRA–lateral flow dipstick (MIRA–LFD)—were established to detect DEV. Specificity analyses showed that the three MIRA methods specifically detected DEV, with no cross-reaction with fowl adenovirus serotype 4, novel goose astrovirus, Muscovy duck reovirus, avian influenza virus subtype H9, or duck circovirus. The basic MIRA reaction was completed in 30 min at 35 °C, requiring only a pair of primers. Detection with MIRA–qPCR or MIRA–LFD was completed within 20 min, and the limits of detection were 1 × 10¹ copies/μL for both. MIRA–LFD required no specialized instruments, and the results could be viewed directly with the naked eye. Compared with the traditional PCR, MIRA assays are simple, rapid, and effective and therefore more suitable for the field detection of DEV. Full article

Aeromonas hydrophila is a prevalent opportunistic pathogen in aquaculture. To establish a rapid, convenient, and accurate detection method for A. hydrophila, this study developed and evaluated Multi-Enzyme Isothermal Rapid Amplification (MIRA) assays, which could complete amplification within 20 min at a constant temperature of 39 °C. The basic MIRA assay targeting the aerolysin (aerA) gene demonstrated high specificity, showing no cross-reactivity with six related bacterial species including Aeromonas veronii, Vibrio harveyi, Pseudomonas fluorescens, Bacillus subtilis, Bacillus cereus, and Lactiplantibacillus plantarum. The fluorescent MIRA assay achieved a detection limit of 1 fg/μL (3.1 × 10² copies/μL) when using the pUC57-aerA standard plasmid, while real-time quantitative PCR achieved a detection limit of 0.1 fg/μL (31 copies/μL). Thus, the MIRA assay exhibited 10-fold lower sensitivity than qPCR but shortened the reaction time from several hours (nearly two hours) to within one hour. Both the specificity and sensitivity of the MIRA reactions were evaluated with three independent experiments. These findings suggested that the developed MIRA assays provide a rapid, specific, and practical diagnostic tool for A. hydrophila detection in aquaculture environments, particularly suitable for resource-limited field applications. Full article

Eel populations are globally threatened by overfishing and illegal trade, making accurate species identification essential for resource conservation and regulatory enforcement. Conventional molecular identification methods are generally applied in the laboratory, with limited rapid on-site application. This study developed a field-deployable assay to identify the Japanese eel (Anguilla japonica), by incorporating multienzyme isothermal rapid amplification (MIRA) technology with a visually readable lateral flow assay (LFA). Species-specific primers targeting a 286 bp region within the mitochondrial genome of A. japonica were designed and labeled with fluorescein amidite and biotin, respectively. The performance of the MIRA-LFA was validated by assessing its specificity against four other major eel species and its analytical sensitivity, i.e., limit of detection (LoD), under optimized temperature and reaction-time conditions. The MIRA-LFA demonstrated 100% specificity, generating a positive signal only for A. japonica, with no cross-reactivity. A clear visual result was obtained within 10 min at the optimal reaction temperature of 39 °C. Under these optimal conditions, the assay showed a high sensitivity, with an LoD of 0.1 ng/μL of genomic DNA. The proposed assay is an effective tool for the rapid, specific, and sensitive identification of A. japonica. The ability to obtain fast, equipment-free visual results makes this assay an ideal point-of-care testing solution to combat seafood fraud and support the sustainable management of this economically important and vulnerable species. Full article

Mpox is a zoonotic disease caused by the Mpox virus (MPXV). The rapid and accurate diagnosis of MPXV is essential for the timely and effective prevention, control, and treatment of the disease. In this study, we combined Multienzyme Isothermal Rapid Amplification (MIRA) (at 42 °C) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 12b(CRISPR/Cas12b) (at 60 °C) to develop a single-tube two-step assay for rapid MPXV detection, leveraging the distinct physical states of tricosane at these temperatures. MIRA amplification primers and CRISPR/cas12b SgRNA were designed based on the MPXV F3L gene. After screening the primers and sgRNAs, the reaction conditions were optimized, and the performances of the assay were evaluated. The detection limit (LOD) of this single-tube two-step MIRA-CRISPR/Cas12b assay for MPXV is four copies of DNA molecules. No cross-reactivity with other pathogens (herpes simplex virus (HSV), Epstein–Barr virus (EBV), Coxsackievirus A16 (CVA16), Enterovirus A71 (EV-A71), and measles virus (MeV)) was found. The assay also showed good consistency with quantitative real-time PCR (qPCR) (Kappa = 0.9547, p < 0.05, n = 100) in the detection of clinical samples, with a sensitivity of 98.5% and a specificity of 97.0%. The single-tube two-step MIRA-CRISPR/Cas12b assay permits the rapid (within 45 min), sensitive, and specific detection of MPXV. The lack of need for opening the reaction tube eliminates the risk of product contamination. Full article

Vibrio parahaemolyticus is a foodborne pathogen commonly associated with the consumption of contaminated seafood, particularly oysters. While PCR and real-time PCR are widely used to detect its pathogenicity through tdh and trh gene detection, these methods may not be practical in resource-limited settings such as field environments. To address this limitation, a rapid, sensitive, and specific duplex detection method was developed using the multienzyme isothermal rapid amplification (MIRA) assay in combination with lateral flow dipstick (LFD) technology. The assay utilized specific primer sets and probes to simultaneously amplify tdh and trh fragments tagged with 3′-FAM and 5′-Digoxigenin or Biotin during MIRA amplification, enabling the detection via respective antibody capture on the LFD strip. This duplex MIRA-LFD assay demonstrated a detection limit of 100 fg of DNA, 300 CFU/reaction for bacterial culture, and 3000 CFU/reaction for seeded oyster samples at 40 °C within 20 min. Notably, the assay exhibited no cross-reactivity with nine other Vibrio species or 18 foodborne pathogens, confirming its high specificity. Due to its simplicity, rapid turnaround time, and high sensitivity, this duplex MIRA-LFD assay offers a valuable tool for the surveillance of V. parahaemolyticus pathogenicity, aiding in public health protection and supporting the local seafood industry. Full article

Bacillus cereus is a widespread foodborne pathogen that can cause food poisoning when present in food at certain levels. Ingesting contaminated food may lead to symptoms such as abdominal pain, diarrhea, and, in severe cases, life-threatening conditions. In this study, a simple and super-fast method for detecting B. cereus was developed, which combines cellulose filter paper-based DNA extraction, multienzyme isothermal rapid amplification (MIRA), and lateral flow dipstick (LFD) technology. Initially, PCR was adopted to evaluate the DNA extraction efficiency of the filter paper, followed by the optimization of the lysis formula and extraction conditions. With the above optimization, DNA that can be used for subsequent nucleic acid amplification can be obtained within 3 min. Then, the isothermal amplification of MIRA–LFD was established and optimized to evaluate the detection specificity and sensitivity. Finally, the developed method was applied to detect B. cereus in cooked rice samples. The results indicated that the entire amplification procedure of MIRA-LFD only takes 15 min at 39 °C. The whole super-fast detection system could be completed in less than 20 min, from DNA extraction to result interpretation, which achieved a detection limit of 12 fg/μL of DNA concentration, corresponding to approximately 115 CFU/mL in actual samples. Full article

Global climate change exerts great effects on plant distributions. However, the response of Prunus mira Koehne, one of the most important species for ecological protection in the southeast of the Qinghai–Tibet Plateau, to climate change remains unclear. To explore the ecological factors affecting the distribution of P. mira in the context of global climate change, the MaxENT model is used to predict suitable habitats for P. mira. Our study indicated that the distribution of Prunus mira Koehn is primarily influenced by temperature rather than precipitation, and warming can facilitate the growth of P. mira. When the temperature seasonality (bio4) ranges from 134 to 576 and the mean temperature of the coldest quarter (bio11) ranges from −2.6 °C to 2.7 °C, it is most conducive to the growth of P. mira. Among the four climate scenarios, the optimal habitat for P. mira is predominantly concentrated in river valley areas and is expected to expand into higher altitude regions, particularly in the north and southeast. SSP245 and SSP370 climate pathways are conducive to the growth and spatial expansion of P. mira. Our findings highlight the significant impact of temperature not precipitation on the distribution of P. mira, and this insight is crucial for the stability and conservation of this ecologically significant plant species. Full article

Background/Objectives: Teosinte branched1/Cycloidea/Proliferating cell nuclear antigen factors (TCPs) are plant-specific transcription factors involved in leaf development, flowering, branching, hormone signaling, and stress responses. Prunus a key temperate fruit tree with ornamental spring blooms, still lacks comprehensive TCP gene studies across many species. Methods: We identified 154 TCP genes in eight Prunus species: 19 in Prunus tenella var. tenella, 19 in P. amygdalus, 17 in P. armeniaca ‘Rojo Pasion’, 19 in P. mira, 20 in P. jamasakura var. jamasakura, 19 in P. fruticosa, 19 in P. mume var. tortuosa, and 22 in P. × yedoensis ‘Somei-yoshino’. These genes were classified into PCF, CIN, and CYC/TB1 groups. We examined segmental duplication, conserved motifs, and cis-acting elements. Expression patterns of 12 TCPs in P. tenella var. tenella were tested under low-temperature stress (25 °C, 5 °C, −5 °C, and −10 °C), and PtTCP9’s subcellular localization was determined. Results: TCP genes within the same groups showed similar motifs and cis-acting elements. Cold stress analysis identified multiple low-temperature-responsive elements in gene promoters. Four genes (PtTCP2, PtTCP6, PtTCP14, and PtTCP16) increased expression under cold stress, while six genes (PtTCP1, PtTCP5, PtTCP8, PtTCP9, PtTCP17, and PtTCP19) decreased. PtTCP9 was localized to the nucleus. Conclusions: This was the first genome-wide study of the TCP gene family in these eight Prunus species, providing valuable insights into the characteristics and functions of TCP genes within this important genus. Full article

The conditions in Mira variable atmospheres make them wonderful laboratories to study a variety of stellar physics such as molecule–grain formation, dust production, shock chemistry, stellar winds, mass loss, opacity-driven pulsation, and shocks. We were awarded an NSF grant to analyze over a decade of synoptic observations from the Palomar Testbed Interferometer (PTI) of 106 Miras to curate a Mira Reference Dataset. The Miras included in this dataset include M-types, S-types, and C-types, and span a wide range of pulsation periods. PTI measured K-band angular sizes that when combined with a distance allow us to directly determine fundamental stellar parameters such as effective temperature, radial size, and bolometric flux. Supplementing observations with interferometric measurements of the stars opens the Mira laboratory to a wealth of different experiments. We provide two case studies to serve as examples of the power of the Mira Reference Dataset. The first case study describes combining PTI measurements with Spitzer IRS spectra of M-type Miras, which allowed us to fully characterize ${\mathrm{CO}}_2$ gas in their atmospheres. The second case study examines how PTI narrow-band data can be used to study phase-dependent pulsation effects on the stellar atmosphere. We provide a list of all the Miras (with coordinates) included in the set for anyone who would like to add them to their observing programs. All the data we produce and collate for this Mira Reference Dataset will be hosted and curated on a website open to the public so that other researchers and citizen scientists can participate in expanding the utility and body of knowledge on this set of “wonderful” stars. Full article

Duck hepatitis B virus (DHBV) is widely prevalent in global ducks and has been identified in Chinese geese with a high prevalence; the available detection techniques are time-consuming and require sophisticated equipment. In this study, an assay combining multienzyme isothermal rapid amplification (MIRA) and lateral flow dipstick (LFD) was developed for the efficient and rapid detection of DHBV. The primary reaction condition of the MIRA assay for DHBV detection was 10 min at 38 °C without a temperature cycler. Combined with the LFD assay, the complete procedure of the newly developed MIRA assay for DHBV detection required only 15 min, which is about one-fourth of the reaction time for routine polymerase chain reaction assay. And electrophoresis and gel imaging equipment were not required for detection and to read the results. Furthermore, the detection limit of MIRA was 45.6 copies per reaction, which is approximately 10 times lower than that of a routine polymerase chain reaction assay. The primer set and probe had much simpler designs than loop-mediated isothermal amplification, and they were only specific to DHBV, with no cross-reactivity with duck hepatitis A virus subtype 1 and duck hepatitis A virus subtype 3, goose parvovirus, duck enteritis virus, duck circovirus, or Riemerella anatipestifer. In this study, we offer a simple, fast, and accurate assay method to identify DHBV in clinical serum samples of ducks and geese, which would be suitable for widespread application in field clinics. Full article

Vibrio parahaemolyticus causes severe gastroenteritis in humans after consuming contaminated raw or undercooked seafood. A species-specific marker, the thermolabile hemolysin (tlh) gene, and two pathogenic markers, thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes, have been used to identify V. parahaemolyticus and determine its pathogenicity using both PCR and qPCR assays. To enable testing in field conditions with limited resources, this study aimed to develop a simple and rapid method to detect the species-specific (tlh) and pathogenic (trh and tdh) genes of V. parahaemolyticus using multienzyme isothermal rapid amplification (MIRA) combined with a lateral-flow dipstick (LFD). The amplification of the tlh, trh, and tdh genes could be completed within 20 min at temperatures ranging from 30 to 45 °C (p < 0.05). The test yielded positive results for V. parahaemolyticus but produced negative results for nine Vibrio species and eighteen foodborne pathogenic bacterial species. MIRA-LFD could detect 10 fg of DNA and 2 colony-forming units (CFU) of V. parahaemolyticus per reaction, demonstrating a sensitivity level comparable to that of qPCR, which can detect 10 fg of DNA and 2 CFU per reaction. Both MIRA-LFD and qPCR detected seven tlh-positive results from thirty-six oyster samples, whereas one positive result was obtained using the PCR assay. No positive results for the trh and tdh genes were obtained from any oyster samples using MIRA-LFD, PCR, and qPCR. This study suggests that MIRA-LFD is a simple and rapid method to detect species-specific and pathogenic genes of V. parahaemolyticus with high sensitivity. Full article

We processed five years of measurements (2018–2022) of a vertically pointing radar MIRA 35c at the Milešovka meteorological observatory with the aim of analyzing the cloud structure of thunderstorms and comparing differences in measured data for cases when lightning discharges were observed very close to the radar position, and for cases when lightning discharges were observed at a greater distance from the radar position. The MIRA 35c radar is a Doppler polarimetric radar working at 35 GHz (Ka-band) with a vertical resolution of 28.9 m and a time resolution of approximately 2 s. For the analysis, we considered radar data whose radar reflectivity was at least 10 dBZ at 5 km or higher above the radar to ensure that there was a cloud above the radar. We divided the radar data into “near” data (a lightning discharge was registered up to 1 km from the radar position) and “far” data (a lightning discharge was registered from 7.5 to 10 km from the radar position). We compared the following quantities: (i) Power in co-channel (pow), (ii) power in cross-channel (pow-cx), (iii) phase in co-channel (pha), (iv) phase in cross-channel (pha-cx), (v) equivalent radar reflectivity (Ze), (vi) Linear Depolarization Ratio (LDR), (vii) co-polar correlation coefficient (RHO), (viii) Doppler radial velocity (V), (ix) Doppler spectrum width (RMS), and (x) Differential phase (Phi). Pow, pow-cx, pha, pha-cx, and V are basic data measured by the radar, while Ze, LDR, RHO, RMS, and Phi are derived quantities. Our results showed that the characteristics of the compared radar quantities are clearly distinct for “near” dataset from “far” dataset. Furthermore, we found out that there is a clear evolution close to the time of discharges of the observed radar quantities in the “near” dataset, which is not that obvious in the “far” dataset. Full article

Infectious diseases caused by Streptococcus iniae lead to massive death of fish, compose a serious threat to the global aquaculture industry, and constitute a risk to humans who deal with raw fish. In order to realize the early diagnosis of S. iniae, and control the outbreak and spread of disease, it is of great significance to establish fast, sensitive, and convenient detection methods for S. iniae. In the present study, two methods of real-time MIRA (multienzyme isothermal rapid amplification, MIRA) and MIRA-LFD (combining MIRA with lateral flow dipsticks (LFD)) for the simA gene of S. iniae were established, which could complete amplification at a constant temperature of 42 °C within 20 min. Real-time MIRA and MIRA-LFD assays showed high sensitivity (97 fg/μL or 7.6 × 10² CFU/mL), which were consistent with the sensitivity of real-time PCR and 10 times higher than that of PCR with strong specificity, repeatability simplicity, and rapidity for S. iniae originating from Trachinotus ovatus. In summary, real-time MIRA and MIRA-LFD provide effective ways for early diagnosis of S. iniae in aquaculture, especially for units in poor conditions. Full article

A point-of-care (POC) diagnostic is needed for both women and men to establish universal screening and surveillance for the number one, non-viral sexually transmitted infection (STI) caused by Trichomonas vaginalis. We developed a POC diagnostic for this STI using the MedMira Rapid Vertical Flow (RVF^®) Technology test cartridge with a membrane that includes a Vertical procedural/reagent control line (referred to as CVL) and spotted with 1 µg of a 72.4-kDa truncated version of α-actinin called ACT::SOE3. This protein is a specific diagnostic target for antibody in sera of individuals with trichomoniasis. Serum antibody to ACT::SOE3 is a positive reaction with the test spot. Specificity of ACT::SOE3 was revealed with monoclonal antibodies (MAbs) generated to ACT::SOE3. Addition of negative control serum with MAb 67B reactive to ACT::SOE3 shows detection of both ACT::SOE3 and the CVL. Only positive sera of individuals had antibody reactive with ACT::SOE3 and detected the presence of the spot and the CVL. Negative control sera were unreactive with ACT::SOE3 and only showed the presence of the CVL. Importantly, to show proof-of-principle for POC application, ACT::SOE3 was detected with the positive patient sera spiked with whole blood. Finally, packaged cartridges stored with desiccant packs at 37 °C for one year gave identical results with the positive and negative human sera. Our results show the validity of this new POC serodiagnostic for this STI. Full article

We report the syntheses and characterization of novel 3,7-bicycl[3.3.1]bispidines possessing an imidazolpropyl group attached to N-3, and at N-7 a Boc group, as well as a benzoylated-oximated group at C-9. These compounds were complexed with β-cyclodextrin [β-CD] and evaluated as seed protectors of selected wheat seedlings. Using strong acid, condensations of N-substituted piperidones with the appropriate imidazolpropyl groups at N-3 and N-7 led to bispidinones 6 and 7. These intermediates were reduced to the corresponding 3,7-diazabicyclo[3.3.1]nonane targets. The oxime at C-9 was benzoylated to yield 13. Heating these 3,7-diazabicyclo[3.3.1]nonanes in ethanol with β-CD generated the complexes required. We investigated the ability of such complexes as coatings on seedlings to protect and stimulate growth of three varieties of wheat, namely Kazakhstanskaya-10, Severyanka, and Miras. The complex of 3-[3-(1H-imidazol-1-yl)propyl]-7-(3-methoxypropyl)-3,7-diazabicyclo[3.3.1]nonane (2) promoted growth in the root systems of all three wheat varieties by more than 30% in Kazakhstanskaya-10, 30% in Severyanka and 8.5% in Miras. A complex of 3-Boc-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (9) increased both shoot and root length in only the Severyanka variety. The complex of 3-(3-butoxypropyl)-7-[3-(1H-imidazol-1-yl)propyl]-3,7-diazabicyclo[3.3.1]nonane (11) stimulated both shoot growth (0.8%, 12.3%, 13.5%) and root growth (12.3%, 9.4%, 21.7%) in all three varieties of wheat, respectively. The nature of substituents on the bispidine affect the activity. Solid complexes (1:1) were generated as powders which melted above 240 °C (dec) and were characterized via elemental analyses as 1:1 complexes. Full article