Research Note: Real-time fluorescence-based recombinase-aided amplification for rapid detection of Mycoplasma synoviae.

Mycoplasma synoviae (MS) is an essential pathogenic mycoplasma in poultry worldwide, posing a serious threat to the poultry industry's health. Timely detection is imperative for early diagnosis, prevention, and control of MS infection. Current laboratory methods for MS detection are generally complicated, time-consuming, and require sophisticated equipment. Therefore, a simple and rapid method is urgently needed. This study developed a novel real-time fluorescence-based recombinase-aided amplification (RF-RAA) technique for detecting MS nucleic acids, enabling target gene amplification within 20 min at 39°C. The RF-RAA outcomes are interpretable in 2 modalities: real-time fluorescence monitoring employing a temperature-controlled fluorescence detector or direct visual inspection facilitated by a portable blue light transilluminator. This method exhibits robust specificity, demonstrating no cross-reactivity with various common poultry pathogens, and achieves high sensitivity, detecting as low as 10 copies/µL for the standard plasmid. Seventy-one clinical samples of chicken throat swabs were detected by RF-RAA and real-time fluorescence quantitative polymerase chain reaction (qPCR) methods. The diagnostic coincidence rates of qPCR with RF-RAA (fluorescence monitoring) and RF-RAA (visual observation) were determined to be 100% and 97.2% (69/71), respectively. In conclusion, the RF-RAA method developed in this study provides a rapid and visually observable approach for MS detection, offering a novel technique to diagnosing MS infection, especially in resource-limited settings.

Rapid and Visual Detection of Type 2 Porcine Reproductive and Respiratory Syndrome Virus by Real-Time Fluorescence-Based Reverse Transcription Recombinase-Aided Amplification.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important diseases that has brought significant economic losses to the swine industry worldwide. Rapid and accurate PRRS virus (PRRSV) detection is one of the key factors for PRRS prevention and control. This study developed a real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) method for type 2 PRRSV (PRRSV-2) detection. The RF-RT-RAA assay could be performed at 42 °C for 20 min with the optimal primers and a probe. RF-RT-RAA results could be monitored using real-time fluorescence read-out or visually observed with the naked eye using a portable blue light transilluminator. The method had a strong specificity; no cross-reaction was identified with the detected common swine viruses. Moreover, the technique yielded high sensitivity with the lowest detection limit of 101 copies/µL and exhibited good repeatability and reproductively with the coefficients of variation (CV) less than 10%. Eighty-seven clinical samples were tested using RF-RT-RAA and a commercial PRRSV-2 RT-qPCR detection kit. The coincidence rate was 100% between RF-RT-RAA (real-time fluorescence read-out) and RT-qPCR, and 97.7% between RF-RT-RAA (visually observed) and RT-qPCR. The RF-RT-RAA assay provides a new method for rapid and visual detection of PRRSV-2.

Rapid and visual detection of Mycoplasma synoviae by recombinase-aided amplification assay combined with a lateral flow dipstick.

Mycoplasma synoviae (MS) is an important avian pathogen that has brought substantial economic losses to the global poultry industry. Fast and accurate diagnosis is one of the critical factors for the control of MS infection. This study established a simple, rapid and visual detection method for MS using a recombinase-aided amplification (RAA) combined with a lateral flow dipstick (LFD). The reaction temperature and time of the RAA-LFD assay were optimized after selecting the primers and probe, and the specificity and sensitivity rates were analyzed. The results showed that RAA could amplify the target gene in 20 min at a constant temperature of 38°C, and the amplification products could be visualized by LFD within 5 min. There was no cross-reaction with Mycoplasma gallisepticum (MG), Pasteurella multocida (P. multocida), Escherichia coli (E. coli), Newcastle disease virus (NDV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), and avian reovirus (ARV). Furthermore, the RAA-LFD assay exhibited high sensitivity with a detection limit of 10 copies/µL. A total of 128 clinical samples with suspected infection of MS were tested by RAA-LFD, PCR, and real-time fluorescence quantitative PCR (RFQ-PCR). The coincidence rate of the detection results was 95.3% between RAA-LFD and PCR, and 98.4% between RAA-LFD and RFQ-PCR. These results suggested that the RAA-LFD method established in the present study was easy to use and was associated with strong specificity and high sensitivity. This method was very suitable for the rapid detection of MS in clinical practice.

Potential Role of Macrophage Migration Inhibitory Factor in the Pathogenesis of Marek's Disease.

INTRODUCTION: Marek's disease virus (MDV) can cause malignant T-cell lymphomas and immunosuppression in chickens. Macrophage migration inhibitory factor (MIF) not only plays a critical role in inhibiting T-cell responses, but also contributes to multiple aspects of tumour progression. The aim of this study was to reveal the potential role of MIF in the pathogenesis of MDV infection. MATERIAL AND METHODS: MIF gene expression levels were measured by using real-time PCR. Expression was assayed at different times in chicken embryo fibroblast (CEF) cells and tissue samples of SPF chickens infected with different MDV strains and fold change was calculated by the 2-△△CT method. RESULTS: The expression of MIF was significantly downregulated (p < 0.05 and FC > 2) in CEF cells infected with the very virulent MDV RB1B strain at 48 h post infection (hpi) and in the skin and spleen at 14 days post infection (dpi). The reduction of MIF expression was also found in CEF cells infected by reticuloendotheliosis virus (REV), avian leukosis virus subgroup J (ALV-J), and MDV vaccine strain CVI988 or in HD11 cells stimulated with TLR2, 3, 4, and 7 ligands. Interestingly, MIF expression decreased continuously from 7 to 28 dpi in the thymus after RB1B virus infection while it increased after CVI988 virus infection. Upregulated expression of MIF was found in CEF infected with RB1B at 96 hpi and in the spleen and skin at 21 and 28 dpi. CONCLUSION: The present study revealed the different expression pattern of MIF in response to MDV infection and indicated that MIF level may be associated with MDV pathogenesis.

A tropomyosin-like Meretrix meretrix Linnaeus polypeptide inhibits the proliferation and metastasis of glioma cells via microtubule polymerization and FAK/Akt/MMPs signaling.

Glioblastoma (GBM) represents the most common, aggressive and deadliest primary tumors with poor prognosis as available therapeutic approaches fail to control its aberrant proliferation and high invasiveness. Thus, the therapeutic agents targeting these two characteristics will be more effective. In present study, a novel polypeptide (MM15), which was originally purified from Meretrix meretrix Linnaeus and has been proven to possess potent antitumor activity by our laboratory, was recombinant expressed and identified as a tropomyosin homologous protein. The recombinant polypeptide (re-MM15) could induce the U87 cell cycle arrest in G2/M phase and cell apoptosis by inducing tubulin polymerization. Additionally, re-MM15 displayed the significant inhibition to the migration and invasion of U87 cells through downregulating FAK/Akt/MMPs signaling. Furthermore, the in vivo analysis suggested that re-MM15 significantly blocked tumor growth in U87 xenograft model. Collectively, our results indicated that re-MM15, with anti-GBM properties in vitro and in vivo, has promising potential as a new anticancer candidate for GBM.

Molecular identification and expression analysis of natural resistance-associated macrophage protein (Nramp) gene from yellow catfish Pelteobagrus fulvidraco (Siluriformes: Bagridae).

Natural resistance associated macrophage protein genes (Nramp) is one of the important candidate genes responsible for regulating immune response against pathogen infection. The aim of the present was to quantify expression of Nramp gene in response to pathogen infection. Here, a Nramp was identified and molecularly characterized from Pelteobagrus fulvidraco (PfNramp). The obtained 3134 bp cDNA fragment of PfNramp comprised a 5'-untranslated region (UTR) of 81 bp, a 3'-UTR of 1403 bp and an open reading frame (ORF) of 1650 bp, encoding a polypeptide of 549 amino acids that contained a typical structural features of Nramp domain (Pfam01566). BLAST analysis exhibited that PfNramp shared sequence similarity to other organisms, in particular to Ictalurus furcatus (92%), Danio rerio (82%), and Homo sapiens (77%). Phylogenetic analysis revealed that PfNramp is close to Teleostei. Real-time quantitative reverse transcription-PCR (qRT-PCR) analysis showed that PfNramp was expressed in all examined tissues, with the highest abundance in liver. The mRNA expression of PfNramp was remarkably increased at different time points after lipopolysaccharide (LPS), and polyriboinosinic polyribocytidylic acid (poly I:C) challenge. These results suggest that PfNramp is an inducible protein in the innate immune reactions of P. fulvidraco and probably in other fish species.

Proteomics of DF-1 cells infected with avian leukosis virus subgroup J.

Avian leukosis virus subgroup J (ALV-J) is an avian oncogenic retrovirus that has led to severe economic losses in the poultry industry in China in recent years. The pathogenesis of virus infection and virus-host interactions are still not well elucidated. In this paper, we investigated the expression changes for cellular proteins in DF-1 cells infected with ALV-J. Comparative analyses revealed that the majority of the altered proteins in DF-1 cells appeared at 6-12h after ALV-J infection. Mass spectrometry identified 74 altered cellular proteins, including 30 up-regulated proteins and 44 down-regulated proteins. Some of these proteins are involved in cell cytoskeleton, metabolic processes, response to stimulus and immune responses. Other proteins, such as DJ-1, UCHL1, VDAC1 and HMGB1, have some relationship to apoptosis or oncogenesis. The changes in the transcriptional profile of DJ-1, UCHL1, VDAC1 and HMGB1 in infected as compared to uninfected DF-1 cells were confirmed by real-time RT-PCR. Our work gives some information about differential protein expression in cells infected with ALV-J, which will help us to understand viral pathogenicity.