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1.
Biochem Biophys Res Commun ; 728: 150340, 2024 Jul 02.
Artículo en Inglés | MEDLINE | ID: mdl-38968770

RESUMEN

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy and poses a significant clinical challenge. Piperine, an alkaloid molecule extracted from Piper nigrum and Piper longum, has emerged as a promising anticancer agent. However, the molecular mechanisms of piperine' antitumor effects in CRC need to be further elucidated. METHODS: Human colorectal cancer cells were treated with piperine in vitro. CCK-8 and clone formation assays were adopted to detect cell viability. The accumulation of autophagosomes was assessed by Western blotting and immunofluorescence. Apoptosis and reactive oxygen species (ROS) levels were analyzed by flow. In vivo, a xenograft tumor mouse model was constructed using CT26 cells. RESULTS: Piperine inhibited CRC cell viability and suppressed tumor weight and volume in a mouse model. Additionally, piperine treatment induced the accumulation of autophagosomes in CRC cells. This effect was attributed to the inhibition of the AKT/mTOR pathway and the accumulation of ROS. activation of AKT or clearance of ROS attenuated piperine-mediated tumor suppression. CONCLUSION: This study shows that piperine induces autophagy-dependent cell death in CRC cells by increasing ROS production and inhibiting Akt/mTOR signaling.

2.
Chemistry ; 30(30): e202401010, 2024 May 28.
Artículo en Inglés | MEDLINE | ID: mdl-38517333

RESUMEN

Electrocatalytic nitrogen reduction reaction (NRR) has attracted much attention as a sustainable ammonia production technology, but it needs further exploration due to its slow kinetics and the existence of competitive side reactions. In this research, xAu/MIL-101(Fe) catalysts were obtained by loading gold nanoparticles (Au NPs) onto MIL-101(Fe) using a one-step reduction strategy. Herein, MIL-101(Fe), with high specific surface area and strong N2 adsorption capacity, is used as a support to disperse Au NPs to increase the electrochemical active surface area. Au NPs, with a high NRR activity, is introduced as the active site to promote charge transfer and intermediate formation rates. More importantly, the strong interaction between Au NPs and MIL-101(Fe) enhances the electron transfer between Au NPs and MIL-101(Fe), thereby enhancing the activation of N2 and achieving efficient NRR. Among the prepared catalysts, 15 %Au/MIL-101(Fe) has the highest NH3 yield of 46.37 µg h-1 mg-1 cat and a Faraday efficiency of 39.38 % at -0.4 V (vs. RHE). In-situ FTIR reveals that the NRR mechanism of 15 %Au/MIL-101(Fe) follows the binding alternating pathway and also indicates that the interaction between Au NPs and MIL-101(Fe) strengthens the activation of the N≡N bond in the rate-limiting process, thereby accelerating the NRR process.

3.
Bioorg Chem ; 142: 106958, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-37979322

RESUMEN

UPLC-TOF-MS/MDF directed phytochemical research of Chloranthus japonicus led to the isolation of 46 lindenane sesquiterpenoid dimers, which included 13 new analogs. Their structures with absolute configurations were elucidated by analysis of spectroscopic data. Fourteen compounds with ester chains significantly decreased PCSK9 protein level in medium of HepG2 cells, especially for compounds 14 and 29 (5 µM) with inhibition rates of 69.0% and 72.8%, respectively. Compound 14 in HepG2 cells was evaluated via DiI-LDL uptake assays and found to increase LDL uptake by upregulating LDLR mRNA and protein level. Meanwhile, 14 decreased the secretion of PCSK9 protein in medium and downregulated intracellular PCSK9 protein and mRNA level. The discovery of these natural small molecule compounds provides a novel structure basis for design PCSK9 regulators, making them a promising lead for development of new lipid-lowering agents.


Asunto(s)
Proproteína Convertasa 9 , Sesquiterpenos , Humanos , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Células Hep G2 , Sesquiterpenos/química , ARN Mensajero
4.
Nature ; 556(7700): 255-258, 2018 04.
Artículo en Inglés | MEDLINE | ID: mdl-29618817

RESUMEN

Cross-species transmission of viruses from wildlife animal reservoirs poses a marked threat to human and animal health 1 . Bats have been recognized as one of the most important reservoirs for emerging viruses and the transmission of a coronavirus that originated in bats to humans via intermediate hosts was responsible for the high-impact emerging zoonosis, severe acute respiratory syndrome (SARS) 2-10 . Here we provide virological, epidemiological, evolutionary and experimental evidence that a novel HKU2-related bat coronavirus, swine acute diarrhoea syndrome coronavirus (SADS-CoV), is the aetiological agent that was responsible for a large-scale outbreak of fatal disease in pigs in China that has caused the death of 24,693 piglets across four farms. Notably, the outbreak began in Guangdong province in the vicinity of the origin of the SARS pandemic. Furthermore, we identified SADS-related CoVs with 96-98% sequence identity in 9.8% (58 out of 591) of anal swabs collected from bats in Guangdong province during 2013-2016, predominantly in horseshoe bats (Rhinolophus spp.) that are known reservoirs of SARS-related CoVs. We found that there were striking similarities between the SADS and SARS outbreaks in geographical, temporal, ecological and aetiological settings. This study highlights the importance of identifying coronavirus diversity and distribution in bats to mitigate future outbreaks that could threaten livestock, public health and economic growth.


Asunto(s)
Alphacoronavirus/aislamiento & purificación , Alphacoronavirus/patogenicidad , Enfermedades de los Animales/epidemiología , Enfermedades de los Animales/virología , Quirópteros/virología , Infecciones por Coronavirus/veterinaria , Diarrea/veterinaria , Porcinos/virología , Alphacoronavirus/clasificación , Alphacoronavirus/genética , Enfermedades de los Animales/transmisión , Animales , Biodiversidad , China/epidemiología , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/transmisión , Diarrea/patología , Diarrea/virología , Reservorios de Enfermedades/veterinaria , Reservorios de Enfermedades/virología , Genoma Viral/genética , Humanos , Yeyuno/patología , Yeyuno/virología , Filogenia , Síndrome Respiratorio Agudo Grave/epidemiología , Síndrome Respiratorio Agudo Grave/veterinaria , Síndrome Respiratorio Agudo Grave/virología , Análisis Espacio-Temporal , Zoonosis/epidemiología , Zoonosis/transmisión , Zoonosis/virología
5.
J Nat Prod ; 85(5): 1294-1303, 2022 05 27.
Artículo en Inglés | MEDLINE | ID: mdl-35561431

RESUMEN

Seven new hexasaccharide resin glycosides, named calysepins I-VII (1-7), with 27-membered rings, were obtained from the aerial parts of Calystegia sepium. Their structures with absolute configuration were established on the basis of spectroscopic data interpretation analysis and the use of chemical methods. They were defined as hexasaccharides composed of one d-quinovose, four d-glucose, and one l-rhamnose unit, and their sugar moieties were partially acylated by (2S)-methylbutanoic acid in 1-7 and (2R,3R)-nilic acid in 1-5 and 7, which mainly differed at the positions of acylation. Additionally, calysepin IV (4) exhibited cytotoxicity against A549 cells with an IC50 value of 5.2 µM.


Asunto(s)
Antineoplásicos , Calystegia , Convolvulus , Calystegia/química , Glicósidos/química , Glicósidos/farmacología , Estructura Molecular , Resinas de Plantas/química
6.
Mol Cell Probes ; 50: 101494, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31863825

RESUMEN

Spring viremia of carp virus (SVCV) is a significant pathogenic agent that can cause large-scale outbreaks of spring viremia of carp (SVC) in many types of fish and bring huge economic losses to the aquaculture industry. A simple and convenient detection method is imperative for SVCV diagnosis. In this study, the real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and validated. Primers and probe targeting the conserved region of M gene were designed and applied to the real-time RT-RPA assay that performed at 39 °C for 20 min. The specificity analysis showed that no cross-reaction with other pathogenic viruses of fish was found, indicating appropriate specificity of the assay. In vitro transcribed RNA standards were used to estimate the sensitivity of the assay and the detection limit was 102copies/reaction. To further evaluate the assay, 65 clinical samples were tested using both real-time RT-RPA assay and real-time RT-PCR method. The same detection results were observed, suggesting the potential application of real-time RT-RPA assay in clinical sample detection. This is the first report on RPA assay for SVCV detection and this new developed assay would be useful in both laboratory and in the field for diagnosis of SVCV.


Asunto(s)
Carpas/virología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Recombinasas/metabolismo , Transcripción Reversa/genética , Rhabdoviridae/genética , Viremia/diagnóstico , Viremia/veterinaria , Animales , Cartilla de ADN/genética , Sensibilidad y Especificidad , Viremia/genética , Viremia/virología
7.
Arch Virol ; 164(6): 1639-1646, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30982935

RESUMEN

Rabbits are widely used as models in biological research, and the pathogen status of rabbits used in studies can directly affect the results of experiments. Serological surveillance is the common monitoring method used in laboratory animals. A rapid, sensitive, and cost-effective high-throughput Luminex xMAP assay could be an attractive alternative to labor-intensive enzyme-linked immunosorbent assay (ELISA) methods. In this study, recombinant proteins from rabbit hemorrhagic disease virus and rabbit rotavirus and whole viral lysates of Sendai virus were used as coating antigens in an xMAP assay for the simultaneous detection of antibodies against these pathogens. The xMAP assay showed high specificity, with no cross-reaction with other pathogens. The coefficient of variation for intra-assay and inter-assay comparisons was less than 3% and 4%, respectively, indicating good repeatability and stability of the assay. The xMAP assay exhibited similar limits of detection for rabbit hemorrhagic virus and Sendai virus and was less sensitive for the detection of rabbit rotavirus when compared with commercial ELISA kits. A total of 52 clinical samples were tested simultaneously using both the xMAP assay and ELISA kits. The results obtained using these two methods were 100% coincident. In summary, the novel xMAP assay offers an alternative choice for rapid and sensitive high-throughput detection of antibodies in rabbit serum and can be used as a daily monitoring tool for laboratory animals.


Asunto(s)
Anticuerpos Antivirales/sangre , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Rotavirus/inmunología , Virus Sendai/inmunología , Animales , Especificidad de Anticuerpos , Reacciones Cruzadas , Inmunoensayo/veterinaria , Conejos , Juego de Reactivos para Diagnóstico
8.
BMC Vet Res ; 15(1): 305, 2019 Aug 23.
Artículo en Inglés | MEDLINE | ID: mdl-31443656

RESUMEN

BACKGROUND: Porcine circovirus type 3 (PCV3) is an emerging circovirus species, that has been reported in major pig-raising countries including the United States, China, South Korea, Brazil, Spain, and Poland. RESULTS: A real-time loop-mediated isothermal amplification (LAMP) assay was developed for rapid detection of porcine circovirus 3 (PCV3). The method had a detection limit of 1 × 101 copies/µL with no cross-reactions with classical swine fever virus (CSFV) C strain, foot-and-mouth disease virus (FMDV), porcine circovirus 2 (PCV2) LG vaccine strain, porcine epidemic diarrhoea virus (PEDV), porcine respiratory and reproductive syndrome virus (PRRSV), or pseudorabies virus (PRV). The PCV3 positive detection rate of 203 clinical samples for the real-time LAMP assay was 89.66% (182/203). CONCLUSIONS: The real-time LAMP assay is highly sensitive, and specific for use in epidemiological investigations of PCV3.


Asunto(s)
Circovirus/genética , Circovirus/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Porcinos/virología , Animales , Técnicas de Amplificación de Ácido Nucleico/métodos , Sensibilidad y Especificidad
9.
Virol J ; 15(1): 146, 2018 09 21.
Artículo en Inglés | MEDLINE | ID: mdl-30241540

RESUMEN

BACKGROUND: A serological method to simultaneously detect antibodies against infectious laryngotracheitis virus (ILTV) and infectious bronchitis virus (IBV) is imperative for the differential diagnosis and evaluation of antibodies titers after vaccination. METHOD: The microspheres coated with purified recombinant glycoprotein D (gD) of ILTV or nucleocapsid (N) protein of IBV were incubated with serum samples. The simultaneous quantification of ILTV and IBV antibodies were achieved through the interrogation of microspheres by Luminex 200 detection system. . RESULTS: This xMAP detection demonstrated no nonspecific reactions with avian influenza virus (AIV), avian leukosis virus (ALV), newcastle disease virus (NDV), and Marek's disease virus (MDV). The results also demonstrated that the xMAP assay was four times more sensitive than the enzyme-linked immunosorbent assay (ELISA) for ILTV detection and two times more sensitive for IBV detection. A total of 90 chicken serum samples from a chicken farm were tested by xMAP and ELISA assays. The results showed that the coincidence rates were 84.44 and 100% for ILTV and IBV detection, respectively. CONCLUSION: This study exhibited an opportunity for the differential diagnosis through simultaneous detection of multiplex antibodies in serum and can be used for the multiplex antibodies evaluation after vaccination.


Asunto(s)
Anticuerpos Antivirales/sangre , Infecciones por Coronavirus/veterinaria , Infecciones por Herpesviridae/veterinaria , Herpesvirus Gallináceo 1/inmunología , Inmunoensayo/métodos , Virus de la Bronquitis Infecciosa/inmunología , Enfermedades de las Aves de Corral/diagnóstico , Animales , Pollos , Infecciones por Coronavirus/diagnóstico , Diagnóstico Diferencial , Infecciones por Herpesviridae/diagnóstico , Sensibilidad y Especificidad
10.
Mol Cell Probes ; 37: 1-5, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29054443

RESUMEN

Acute respiratory tract infections are of paramount importance in the poultry industry. We developed an xTAG bead assay for the simultaneous detection and discrimination of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV) and infectious laryngotracheitis virus (ILTV). The assay lacked nonspecific reactions with other common avian viruses and the limit of detection was 6.75 × 102- 3.52 × 103copies/µL. We examined 60 clinical specimens and found 18 positive for respiratory viruses. Our result demonstrated that xTAG-multiplex PCR method is a high-throughput, rapid, specific and sensitive assay for use in epidemiological studies and clinical detection of avian respiratory pathogens.


Asunto(s)
Herpesvirus Gallináceo 1/aislamiento & purificación , Virus de la Bronquitis Infecciosa/aislamiento & purificación , Virus de la Influenza A/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Virus de la Enfermedad de Newcastle/aislamiento & purificación , Enfermedades de las Aves de Corral/diagnóstico , Animales , Infecciones por Virus ADN/diagnóstico , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/virología , Herpesvirus Gallináceo 1/genética , Virus de la Bronquitis Infecciosa/genética , Virus de la Influenza A/genética , Límite de Detección , Virus de la Enfermedad de Newcastle/genética , Aves de Corral , Enfermedades de las Aves de Corral/virología , Infecciones por Virus ARN/diagnóstico , Infecciones por Virus ARN/veterinaria , Infecciones por Virus ARN/virología , Sensibilidad y Especificidad
11.
Mol Cell Probes ; 39: 25-32, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29609038

RESUMEN

Differentiation of classical strains and highly pathogenic strains of porcine reproductive and respiratory syndrome virus is crucial for effective vaccination programs and epidemiological studies. We used nested PCR and high resolution melting curve analysis with unlabeled probe to distinguish between the classical and the highly pathogenic strains of this virus. Two sets of primers and a 20 bp unlabeled probe were designed from the NSP3 gene. The unlabeled probe included two mutations specific for the classical and highly pathogenic strains of the virus. An additional primer set from the NSP2 gene of the highly pathogenic vaccine strain JXA1-R was used to detect its exclusive single nucleotide polymorphism. We tested 107 clinical samples, 21 clinical samples were positive for PRRSV (consistent with conventional PCR assay), among them four were positive for the classical strain with the remainder 17 for the highly pathogenic strain. Around 10 °C difference between probe melting temperatures showed the high discriminatory power of this method. Among highly pathogenic positive samples, three samples were determined as positive for JXA1-R vaccine-related strain with a 95% genotype confidence percentage. All these genotyping results using the high resolution melting curve assay were confirmed with DNA sequencing. This unlabeled probe method provides an alternative means to differentiate the classical strains from the highly pathogenic porcine reproductive and respiratory syndrome virus strains rapidly and accurately.


Asunto(s)
Desnaturalización de Ácido Nucleico/genética , Reacción en Cadena de la Polimerasa/métodos , Virus del Síndrome Respiratorio y Reproductivo Porcino/aislamiento & purificación , Animales , Cartilla de ADN/metabolismo , Sondas de ADN/metabolismo , Filogenia , Síndrome Respiratorio y de la Reproducción Porcina/diagnóstico , Síndrome Respiratorio y de la Reproducción Porcina/virología , Virus del Síndrome Respiratorio y Reproductivo Porcino/genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Porcinos
12.
Mol Cell Probes ; 41: 27-31, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30157451

RESUMEN

Theiler's murine encephalomyelitis virus (TMEV) is one of the most common viral pathogens that circulate widely in captive mouse colonies. A molecular biology detection method would be a useful tool to use in an integrated program to monitor and prevent TMEV infection and transmission. Thus, a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed to detect TMEV infection. The sensitivity of the RT-RPA assay approached 8 copies per reaction, which is equivalent to the sensitivity of RT-qPCR reactions. This assay did not detect RNA extracts from other murine pathogens included in this study or TMEV negative samples. Brain tissues and contaminated biological materials were used to assess the clinical performance of the RT-RPA. The detection results of RT-RPA and RT-qPCR were very similar, except that a contaminated biological material sample which was positive by RT-qPCR, with a CT value of 38, was negative by RT-RPA. In summary, the developed RT-RPA assay offers a rapid, sensitive and specific alternative method for monitoring of TMEV, especially in resource-limited conditions.


Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Recombinasas/metabolismo , Transcripción Reversa/genética , Theilovirus/aislamiento & purificación , Animales , Cartilla de ADN/metabolismo , Sondas de ADN/metabolismo , Ratones , Sensibilidad y Especificidad , Theilovirus/genética
13.
BMC Vet Res ; 14(1): 399, 2018 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-30547776

RESUMEN

BACKGROUND: Murine norovirus (MNV) is recognized as the most prevalent viral pathogen in captive mouse colonies. The rapid detection assay for MNV would be a useful tool for monitoring and preventing MNV infection. A recombinase polymerase amplification (RPA) assay was established in this study to provide a solution for rapid and sensitive detection of MNV. RESULTS: The detection limit of the RT-RPA assay for the detection of MNV was 1 × 102 copies of RNA molecules per reaction. The assay was specific since there was no cross-reaction with other common murine viruses. In addition, the broad reactivity of the RT-RPA assay was validated using the synthesized template carrying seven point mutations among several MNV strains. The MNV RT-RPA assay could detect as few as 1 × 102 copies of the mutant per reaction, suggesting the assay could be broadly reactive against a large diversity of MNV strains. Forty eight clinical samples including 16 gastric tissue specimens, 16 cecal tissue specimens and 16 fecal specimens were tested for the validation of the new developed RT-RPA assay. The detection results of RT-RPA and RT-qPCR for clinical samples were very similar, except that a gastric tissue sample which was positive by RT-qPCR, with a RNA titer of 27 copies, was negative by RT-RPA. CONCLUSIONS: A broadly reactive RT-RPA assay was successfully established for MNV detection.


Asunto(s)
Infecciones por Caliciviridae/veterinaria , Norovirus/genética , Enfermedades de los Roedores/diagnóstico , Animales , Animales de Zoológico , Infecciones por Caliciviridae/diagnóstico , Infecciones por Caliciviridae/virología , Ratones , Técnicas de Amplificación de Ácido Nucleico , Enfermedades de los Roedores/virología , Sensibilidad y Especificidad
14.
BMC Vet Res ; 14(1): 347, 2018 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-30442149

RESUMEN

BACKGROUND: Chicken anemia virus (CAV), avian reovirus (ARV), infectious bursal disease virus (IBDV), Marek's disease virus (MDV) and reticuloendotheliosis virus (REV) all cause immunosuppressive disease in birds through vertical or horizontal transmission. Mixed infections with these immunosuppressive pathogens lead to atypical clinical signs and obstruct accurate diagnoses and epidemiological investigations. Therefore, it is essential to develop a high-throughput assay for the simultaneous detection of these immunosuppressive viruses with high specificity and sensitivity. The aim of this study was to establish a novel method using a RT-PCR assay combined with fluorescence labeled polystyrene bead microarray (multiplex xTAG assay) to detect single or mixed viral infections. RESULTS: The results showed that the established xTAG assay had no nonspecific reactions with avian influenza virus (AIV), infectious bronchitis virus (IBV), newcastle disease virus (NDV), infectious laryngotracheitis virus (ILTV), Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS). The limit of detection was 1.0 × 103 copies/µL for IBDV and 1.0 × 102copies/µL for the other four viruses. Ninety field samples were tested and the results were confirmed using conventional RT-PCR methods. The detection results of these two methods were 100% consistent. The established multiplex xTAG assay allows a high throughput and simultaneous detection of five chicken immunosuppressive viruses. CONCLUSION: The multiplex xTAG assay has been showed to be an additional tool for molecular epidemiology studies of five chicken immunosuppressive viruses in the poultry industry.


Asunto(s)
Infecciones por Birnaviridae/veterinaria , Virus de la Anemia del Pollo , Infecciones por Circoviridae/veterinaria , Coinfección/veterinaria , Virus de la Enfermedad Infecciosa de la Bolsa , Mardivirus , Enfermedad de Marek/diagnóstico , Análisis por Micromatrices/veterinaria , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Orthoreovirus Aviar , Enfermedades de las Aves de Corral/diagnóstico , Infecciones por Reoviridae/veterinaria , Virus de la Reticuloendoteliosis Aviar , Infecciones por Retroviridae/veterinaria , Infecciones Tumorales por Virus/veterinaria , Animales , Infecciones por Birnaviridae/diagnóstico , Infecciones por Birnaviridae/virología , Pollos/virología , Infecciones por Circoviridae/diagnóstico , Infecciones por Circoviridae/virología , Coinfección/diagnóstico , Coinfección/virología , Enfermedad de Marek/virología , Análisis por Micromatrices/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Enfermedades de las Aves de Corral/virología , Infecciones por Reoviridae/diagnóstico , Infecciones por Reoviridae/virología , Reproducibilidad de los Resultados , Infecciones por Retroviridae/diagnóstico , Infecciones por Retroviridae/virología , Sensibilidad y Especificidad , Infecciones Tumorales por Virus/diagnóstico , Infecciones Tumorales por Virus/virología
15.
BMC Vet Res ; 14(1): 127, 2018 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-29625588

RESUMEN

BACKGROUND: Domestic rabbits especially New Zealand white rabbits play an important role in biological research. The disease surveillance and quality control are essential to guarantee the results of animal experiments performed on rabbits. Rabbit hemorrhagic disease virus, rabbit rotavirus and Sendai virus are the important pathogens that needed to be eliminated. Rapid and sensitive method focus on these three viruses should be established for routine monitoring. The Luminex x-TAG assay based on multiplex PCR and fluorescent microsphere is a fast developing technology applied in high throughput detection. Specific primers modified with oligonucleotide sequence/biotin were used to amplify target fragments. The conjugation between oligonucleotide sequence of the PCR products and the MagPlex-TAG microspheres was specific without any cross-reaction, and the hybridization products could be analyzed using the Luminex 200 analyzer instrument. Recombinant plasmids were constructed to estimate the detection limit of the viruses. Furthermore, 40 clinical samples were used to evaluate the efficiency of this multiplex PCR based Luminex x-TAG assay. RESULTS: According to the results, this new method showed high specificity and good stability. Assessed by the recombinant plasmids, the detection limit of three viruses was 100copies/µl. Among 40 clinical specimens, 18 specimens were found positive, which was completely concordant with the conventional PCR method. CONCLUSIONS: The new developed Luminex x-TAG assay is an accurate, high throughput method for rapid detection of three important viruses of rabbits.


Asunto(s)
Infecciones por Caliciviridae/veterinaria , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Conejos/virología , Infecciones por Respirovirus/veterinaria , Infecciones por Rotavirus/veterinaria , Animales , Animales de Laboratorio/virología , Infecciones por Caliciviridae/diagnóstico , Reacciones Cruzadas , Fluorescencia , Virus de la Enfermedad Hemorrágica del Conejo , Microesferas , Reacción en Cadena de la Polimerasa Multiplex/métodos , Infecciones por Respirovirus/diagnóstico , Rotavirus , Infecciones por Rotavirus/diagnóstico , Virus Sendai , Sensibilidad y Especificidad
16.
J Nanosci Nanotechnol ; 18(3): 1811-1817, 2018 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-29448664

RESUMEN

In the present work, the nanocomposite of the silver nanoparticles decorated partially unzipped carbon nanotubes (PUCNTs) (PUCNTs/Ag) was fabricated by in situ method, and its application as a sensitive non-enzymatic hydrogen peroxide (H2O2) sensor was then explored correspondingly. The measurements of transmission electron microscopy (TEM), fourier transform infrared (FTIR) spectroscopy and ultraviolet visible (UV-vis) absorption spectrum proved that the PUCNTs/Ag composite has been successfully prepared and the Ag nanoparticles dispersed on the surface of the PUCNTs and entered the inner of unzipped MWCNTs. Cyclic voltammetric (CV) measurement indicated that the PUCNTs/Ag nanocomposite showed the well-defined redox characteristics in 0.1 M phosphate buffer solution (PBS, pH = 7.5), and under the optimized experimental conditions, the current response of the as-obtained sensor towards electrochemical oxidation of H2O2 was linear with the concentration of H2O2 in the range of 0.0419 mM to 87 mM (R = 0.997) in the solution of 0.1 M PBS (pH = 7.5) at the applied potential of 0 V. The detection limit was 1 µM with the sensitivity calculated as 1.115 × 103 µA · M-1 · cm-2 and the fast response achieved within 3 s. The constructed non-enzymatic sensor is one of the promising candidates due to it's good sensitivity, selectivity, and reproducibility.

17.
Helicobacter ; 22(2)2017 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-27687595

RESUMEN

BACKGROUND: Enterohepatic Helicobacter species (EHS) are widespread in rodent species around the world. Several studies have demonstrated that infection with EHS can interfere with the outcomes of animal experiments in cancer research and significantly influence the study results. Therefore, it is essential to establish a rapid detection and identification of EHS for biomedical research using laboratory rodents. Our study aimed to develop a rapid and sensitive method to detect and distinguish five enterohepatic Helicobacter species. MATERIALS AND METHODS: Nested PCR followed by high-resolution melting curve analysis (HRM) was developed for identification of H. bilis, H. rodentium, H. muridarum, H. typhlonius, as well as H. hepaticus. To validate the accuracy of nested PCR-HRM analysis, quantitative real-time PCR methods for five different enterohepatic Helicobacter species were developed. A total of 50 cecal samples were tested using both nested PCR-HRM analysis and qPCR method. RESULTS: The nested PCR-HRM method could distinguish five enterohepatic Helicobacter species by different melting temperatures. The melting curve were characterized by peaks of 78.7 ± 0.12°C for H. rodentium, 80.51 ± 0.09°C for H. bilis, 81.6 ± 0.1°C for H. typhlonius, 82.11 ± 0.18°C for H. muridarum, and 82.95 ± 0.09°C for H. hepaticus. CONCLUSIONS: The nested PCR-HRM assay is a simple, rapid, and cost-effective assay. This assay could be a useful tool for molecular epidemiology study of enterohepatic Helicobacter infection and an attractive alternative for genotyping of enterohepatic Helicobacter species.


Asunto(s)
ADN Bacteriano/química , ADN Bacteriano/genética , Helicobacter/clasificación , Helicobacter/genética , Técnicas Microbiológicas/métodos , Reacción en Cadena de la Polimerasa/métodos , Temperatura de Transición , Animales , Ciego/microbiología , Costos y Análisis de Costo , Técnicas de Genotipaje/métodos , Helicobacter/aislamiento & purificación , Ratones , Factores de Tiempo
18.
Virus Genes ; 52(6): 877-882, 2016 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-27470155

RESUMEN

A new family of IFNs called type III IFN or IFN-λ has been described, and shown to induce antiviral activity against several viruses in the cell culture. In this study, the molecular cloning, expression, and antiporcine epidemic diarrhea virus (PEDV) activity of porcine IFN-λ3 (poIFN-λ3) were reported. The full-length poIFN-λ3 cDNA sequence encoded 196 amino acids with a 23 amino acid signal peptide. Sequence alignments showed that poIFN-λ3 had an amino acid sequence similarity to Ovis aries (78.1 %), Bos taurus (76.0 %), Tupaia belangeri (71.3 %), Equus caballus (69.9 %), and Homo sapiens (69.9 %). The phylogenetic analysis based on the genomic sequences indicated that poIFN-λ3 is located in the same branch as B. taurus and O. aries IFN-λ3. The poIFN-λ3 without a signal anchor sequence was efficiently expressed in Escherichia coli, and the purified recombinant poIFN-λ3 exhibited significant antiviral effects against PEDV in a dose- and time-dependent manner. This inhibitory effect of poIFN-λ3 on PEDV was observed under three different treatment conditions. The highest inhibition of PEDV was observed in Vero E6 cell cultures pretreated with poIFN-λ3 (prior to PEDV infection). In addition, poIFN-λ3 was able to induce the expression of IFN-stimulated genes, including ISG15, OAS1, and Mx1 in Vero E6 cells. These data demonstrate that poIFN-λ3 has antiviral activity against PEDV and may serve as a useful biotherapeutic candidate to inhibit PEDV or other viruses in swine.


Asunto(s)
Antivirales/farmacología , Interferón gamma/farmacología , Virus de la Diarrea Epidémica Porcina/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Clonación Molecular , Interferón gamma/química , Interferón gamma/genética , Interferón gamma/aislamiento & purificación , Filogenia , Virus de la Diarrea Epidémica Porcina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Análisis de Secuencia de ADN , Porcinos , Células Vero , Replicación Viral/efectos de los fármacos
19.
Virus Genes ; 51(2): 252-9, 2015 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-26329934

RESUMEN

Porcine epidemic diarrhea virus (PEDV) is a member of the coronaviridae family, which can cause acute and highly contagious enteric disease of swine characterized by severe entero-pathogenic diarrhea in piglets. Currently, the vaccines of PEDV are only partially effective and there is no specific drug available for treatment of PEDV infection. To exploit the possibility of using RNA interference (RNAi) as a strategy against PEDV infection, five shRNA-expressing plasmids targeting the N, M, and S genes of PEDV were constructed and transfected into Vero cells. The cytopathic effect and MTS assays demonstrated that two shRNAs (pSilencer4.1-M1 and pSilencer4.1-N) were capable of protecting cells against PEDV invasion with very high specificity and efficiency. The two shRNA expression plasmids were also able to inhibit the PEDV replication significantly, as shown by detection of virus titers (TCID50/mL). A real-time quantitative RT-PCR further confirmed that the amounts of viral RNAs in cell cultures pre-transfected with these two plasmids were reduced by 95.0 %. Our results suggest that RNAi might be a promising new strategy against PEDV infection.


Asunto(s)
Virus de la Diarrea Epidémica Porcina/crecimiento & desarrollo , Virus de la Diarrea Epidémica Porcina/genética , Interferencia de ARN , Animales , Supervivencia Celular , Chlorocebus aethiops , Efecto Citopatogénico Viral , Plásmidos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Viral/análisis , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Células Vero , Carga Viral
20.
Virus Genes ; 51(3): 361-6, 2015 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-26573282

RESUMEN

Since 2013, the second outbreak of peste des petits ruminants (PPR) caused by Peste des petits ruminants virus (PPRV) has spread over more than 20 provinces, municipalities, and autonomous regions in China, resulting in major economic losses for livestock industry. In 2014, we encountered a clinical PPR case on a goat farm in Guangdong province, southern China. The complete genome of this PPRV strain, named CH/GDDG/2014, was sequenced to determine its similarities and differences with other strains. The CH/GDDG/2014 genome comprised 15,954 nucleotides (six nucleotides more than classical PPRVs identified before 2013, but complying with the rule of six) with six open reading frames encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin, and large polymerase protein, respectively. The whole-genome-based alignment analysis indicated that CH/GDDG/2014 had the most proximate consensus (99.8 %) to China/XJYL/2013 and the least consensus (87.2 %) to KN5/2011. The phylogenetic analysis showed that CH/GDDG/2014 was clustered in one branch (lineage IV) with other emerging strains during the second outbreak. This study is the first report describing the whole-genome sequence of PPRV in Guangdong province, southern China and also suggests the PPR outbreak may be closely related to illegal cross-regional importation of goats.


Asunto(s)
Enfermedades de las Cabras/virología , Peste de los Pequeños Rumiantes/genética , Peste de los Pequeños Rumiantes/virología , Virus de la Peste de los Pequeños Rumiantes/genética , Filogenia , Animales , Secuencia de Bases , China/epidemiología , Análisis por Conglomerados , Brotes de Enfermedades , Genes Virales , Enfermedades de las Cabras/epidemiología , Cabras , Proteínas de la Nucleocápside/genética , Peste de los Pequeños Rumiantes/mortalidad , Virus de la Peste de los Pequeños Rumiantes/aislamiento & purificación , Alineación de Secuencia , Análisis de Secuencia de ADN
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