Brzo otkrivanje uzročnika virusnog proljeva goveda u mlijeku iz spremnika pomoću kombinacije metoda umnožene rekombinazne polimeraze i test-traka za „lateral flow“ analizu

Bovine viral diarrhea virus (BVDV) is one of the most prevalent and economically important pathogens of ruminants, and leads to significant financial losses to the livestock industry worldwide. Development of rapid and accurate diagnostic methods is of great importance for the control and eradication of BVDV infection. The aim of this study was to develop a novel isothermal recombinase polymerase amplification (RPA) method combined with a lateral flow dipstick (LFD), for rapid detection of BVDV. RPA primers and a probe targeting the specific conserved 5′-UTR of BVDV genome were designed. The RPA amplification could be finished at a constant temperature of 38 0000C for 15 min, and the amplification product was easily visualized on a simple LFD within 5 min. The detection limit of this assay was 20 copies per reaction, and there was no cross-reactivity with other bovine infectious viruses, such as infectious bovine rhinotracheitis virus (IBRV), bovine enterovirus (BEV), bovine coronavirus (BcoV), bovine parainfluenza virus type 3 (BPIV-3), bovine ephemeral fever virus (BEFV) and bovine respiratory syncytial virus (BRSV). The assay performance on bulk tank milk was also evaluated, and the sensitivity and accuracy of BVDV LFD RPA was compared with real-time RT-PCR. Of 284 pool or bulk tank milk samples, 51 were found to be positive by RPA assay, whereas 52 were positive by real-time RT-PCR. The coincidence rate between LFD RPA and real-time RT-PCR was 97.54% (277/284).Uzročnik virusnog proljeva goveda (BVDV) jedan je od najčešćih i ekonomski važnih patogena preživača koji uzrokuje znatne financijske gubitke u stočarskoj industriji širom svijeta. Razvoj brzih i točnih dijagnostičkih metoda iznimno je važan za kontrolu i iskorjenjivanje zaraze BVDV-om. Cilj ovog istraživanja bio je razviti novu metodu za brzo otkrivanje BVDV-a baziranu na kombinaciji metoda umnožene rekombinazne polimeraze i test-traka za „lateral flow“ analizu. Oblikovane su početnice i probe za umnažanje rekombinazne polimeraze usmjerene na specifični konzervirani 5’-UTR u genomu BVDV-a. Umnažanje se moglo završiti pri konstantnoj temperaturi od 38 °C tijekom 15 minuta i produkt umnažanja je lako vizualiziran na jednostavnoj test-traci za „lateral flow“ analizu unutar 5 minuta. Test je ograničen na 20 kopija po reakciji, pri čemu nije bilo križne reaktivnosti s drugim goveđim zaraznim virusima kao što su infektivni rinotraheitis virusa goveda (IBRV), goveđi enterovirus (BEV), goveđi koronavirus (BcoV), virus goveđe parainfluence tipa 3 (BPIV-3), virus gljivične ephemeralne groznice (BEFV) i goveđi respiratorni sincicijski virus (BRSV). Učinkovitost kombinacije navedenih metoda istražena je i s obzirom na usporedbu osjetljivosti odnosno točnosti koja se dobiva uporabom RT-PCR metode. Od 284 skupna uzorka mlijeka iz spremnika, kombinacijom metoda umnožene rekombinazne polimeraze i test-traka za „lateral flow“ analizu utvrđen je 51 pozitivan uzorak, a RT-PCR 52 pozitivna uzorka. Stopa podudarnosti između navedenih metoda bila je 97,54 % (277/284)

Shifts in dominant species modulate nitrogen effects on community temporal stability along a degradation gradient in Tibetan alpine grasslands

Grassland degradation is a widespread concern globally. Nutrient addition is widely recognized as an important management measure for degraded grassland restoration, but nutrient inputs may have important effects on temporal stability of communities. However, it is unclear how the degradation degrees regulate the impacts of adding nitrogen (N) on temporal stability of communities due to large variations in community composition and soil nutrient content. Herein, long-term experimental study was used to identify the N influences on temporal stability of community and driving factors of alpine grasslands at three degradational stages (none (ND), moderate (MD), and heavily degraded grassland (HD)). Elevated N could significantly enhance the aboveground biomass of ND, while there was no notable effect on MD and HD. However, N addition significantly lowered the temporal stability of all these three grasslands, although species richness was only pronouncedly reduced in MD. Our most conspicuous outcome is the major mechanism of temporal stability of community varied with degradation stages. N addition decreased temporal stability of the community predominantly by decrease in the richness of species and the stability of dominant species in ND and MD while decrease in dominant species stability, asynchrony, and richness of species in HD. This study highlights that impacts of N enrichment on temporal stability of community are potentially intermediated via community composition of alpine grasslands, which can offer guidance for scientific management and rehabilitation of degraded alpine grasslands

Rapid detection of infectious bovine Rhinotracheitis virus using recombinase polymerase amplification assays

Abstract Background Infectious bovine rhinotracheitis virus (IBRV) is a major pathogen in cattle and has led to significant economic losses to the dairy industry worldwide, and therefore a more optimal method for the rapid diagnosis of IBRV infection is highly needed. In this study, we described the development of a lateral flow dipstrip (LFD) of isothermal recombinase polymerase amplification (RPA) method for rapid detection of IBRV. Methods Distinct regions were selected as a candidate target for designing the LFD-RPA primers and probes. The analytical sensitivity of the RPA assay was determined using ten-fold serially diluted IBRV DNA. The specificity of the assay was assessed with other viral pathogens of cattle with similar clinic and other herpesviruses. The clinical performance was evaluated by testing 106 acute-phase high fever clinical specimens. Results RPA primers and probe were designed to target the specific conserved UL52 region fragment of IBRV. The detection could be completed at a constant temperature of 38 °C for 25 min, and the amplification products were easily visualized on a simple LFD. The detection limit of this assay was 5 copies per reaction of IBRV DNA and there was no cross-reactivity with other viruses causing bovine gastrointestinal and respiratory infections or other herpesviruses. The assay performance on acute-phase high fever clinical samples collected from cattle with no vaccine against IBRV, which were suspected to be infected with IBRV, was validated by detecting 24 fecal, 36 blood, 38 nasal swab and 8 tissue specimens, and compared with SYBR Green I based real-time PCR. The coincidence between IBRV LFD-RPA and real-time PCR was 100%. Conclusion IBRV LFD-RPA was fast and much easier to serve as an alternative to the common measures used for IBRV diagnosis, as there is reduction in the use of instruments for identification of the infected animals. In addition, this assay may be the potential candidate to be used as point-of-care diagnostics in the field

MiR-3470b promotes bovine ephemeral fever virus replication via directly targeting mitochondrial antiviral signaling protein (MAVS) in baby hamster Syrian kidney cells

Abstract Background Bovine ephemeral fever virus (BEFV), the causative agent of bovine ephemeral fever, is an economically important pathogen of cattle and water buffalo. MicroRNAs (miRNAs) are endogenous 21-23 nt small non-coding RNA molecules that binding to a multiple of target mRNAs and functioning in the regulation of viral replication including the miRNA-mediated antiviral defense. However, the reciprocal interaction between bovine ephemeral fever virus replication and host miRNAs still remain poorly understood. The aim of our study herein was to investigate the exact function of miR-3470b and its molecular mechanisms during BEFV infection. Results In this study, we found a set of microRNAs induced by BEFV infection using small RNA deep sequencing, and further identified BEFV infection could significantly up-regulate the miR-3470b expression in Baby Hamster Syrian Kidney cells (BHK-21) after 24 h and 48 h post-infection (pi) compared to normal BHK-21 cells without BEFV infection. Additionally, the target association between miR-3470b and mitochondrial antiviral signaling protein (MAVS) was predicted by target gene prediction tools and further validated using a dual-luciferase reporter assay, and the expression of MAVS mRNA and protein levels was negatively associated with miR-3470b levels. Furthermore, the miR-3470b mimic transfection significantly contributed to increase the BEFV N mRNA, G protein level and viral titer, respectively, whereas the miR-3470b inhibitor had the opposite effect on BEFV replication. Moreover, the overexpression of MAVS or silencing of miR-3470b by its inhibitors suppressed BEFV replication, and knockdown of MAVS by small interfering RNA also promoted the replication of BEFV. Conclusions Our findings is the first to reveal that miR-3470b as a novel host factor regulates BEFV replication via directly targeting the MAVS gene in BHK-21 cells and may provide a potential strategy for developing effective antiviral therapy

Development of a recombinase polymerase amplification combined with a lateral flow dipstick assay for rapid detection of the Mycoplasma bovis

Abstract Background Mycoplasma bovis (M. bovis) is a major etiological agent of bovine mycoplasmosis around the world. Point-of-care testing in the field is lacking owing to the requirement for a simple, robust field applicable test that does not require professional laboratory equipment. The recombinase polymerase amplification (RPA) technique has become a promising isothermal DNA amplify assay for use in rapid and low-resource diagnostics. Results Here, a method for specific detection of M. bovis DNA was established, which was RPA combined with lateral flow dipstick (LFD). First, the analytical specificity and sensitivity of the RPA primer and LF-probe sets were evaluated. The assay successfully detected M. bovis DNA in 30 min at 39 °C, with detection limit of 20 copies per reaction, which it was compared the real-time quantitative PCR (qPCR) assay. This method was specific because it did not detect a selection of other bacterial pathogens in cattle. Both qPCR and RPA-LFD assays were used to detect M. bovis 442 field samples from 42 different dairy farms in Shandong Province of China, also the established RPA-LFD assay obtained 99.00% sensitivity, 95.61% specificity, and 0.902 kappa coefficient compared with the qPCR. Conclusions To the author’s knowledge, this is the first report using an RPA-FLD assay to visualise and detect M. bovis. Comparative analysis with qPCR indicates the potential of this assay for rapid diagnosis of bovine mycoplasmosis in resource limited settings

Development and evaluation of serotype-specific recombinase polymerase amplification combined with lateral flow dipstick assays for the diagnosis of foot-and-mouth disease virus serotype A, O and Asia1

Abstract Background Foot-and-mouth disease (FMD) caused by foot-and-mouth disease virus (FMDV) is one of the most highly infectious diseases in livestock, and leads to huge economic losses. Early diagnosis and rapid differentiation of FMDV serotype is therefore integral to the prevention and control of FMD. In this study, a series of serotype-specific reverse transcription recombinase polymerase amplification assays combined with lateral flow dipstick (RPA-LFD) were establish to differentiate FMDV serotypes A, O or Asia 1, respectively. Results The serotype-specific primers and probes of RPA-LFD were designed to target conserved regions of the FMDV VP1 gene sequence, and three primer and probe sets of serotype-specific RPA-LFD were selected for amplification of FMDV serotypes A, O or Asia 1, respectively. Following incubation at 38 °C for 20 min, the RPA amplification products could be visualized by LFD. Analytical sensitivity of the RPA assay was then determined with ten-fold serial dilutions of RNA of VP1 gene and the recombinant vector respectively containing VP1 gene from FMDV serotypes A, O or Asia1, the detection limits of these assays were 3 copies of plasmid DNA or 50 copies of viral RNA per reaction. Moreover, the specificity of the assay was assessed, and there was no cross reactions with other viruses leading to bovine vesicular lesions. Furthermore, 126 clinical samples were respectively detected with RPA-LFD and real-time PCR (rPCR), there was 98.41% concordance between the two assays, and two samples were positive by RPA-LFD but negative in rPCR, these were confirmed as FMDV-positive through viral isolation in BHK-21 cells. It showed that RPA-LFD assay was more sensitive than the rPCR method in this study. Conclusion The development of serotype-specific RPA-LFD assay provides a rapid, sensitive, and specific method for differentiation of FMDV serotype A, O or Asia1, respectively. It is possible that the serotype-specific RPA-LFD assay may be used as a integral protocol for field detection of FMDV

Additional file 1: Figure S1. of Biopanning of polypeptides binding to bovine ephemeral fever virus G1 protein from phage display peptide library

Schematic drawing of BEFV G and the points linked to the primers of cloned G1 used. (A) The number of amino acids region about full length G gene and Region G1 (from 390 aa–529 aa) were indicated. (B) Presentation of G1 nucleotides sized and gene sequence amplification locus and positions of the forward (G1-F) and reversed (G1-R) primers used in this study. (TIFF 11694 kb