Development of a Real-Time Recombinase-Aided Amplification Method to Rapidly Detect Methicillin-Resistant Staphylococcus aureus.

Methicillin-resistant staphylococcus aureus (MRSA) is a major pathogen responsible for human hospital and community-onset diseases and severe invasive livestock infections. Rapid detection of MRSA is essential to control the spread of MRSA. Conventional identification methods and antibacterial susceptibility tests of MRSA are time-consuming. The commonly used qPCR assay also has the disadvantages of being complicated and expensive, restricting its application in resource-limited clinical laboratories. Here, a real-time fluorescent recombinase-assisted amplification (RAA) assay targeting the most conserved regions within the mecA gene of MRSA was developed and evaluated to detect MRSA. The detection limit of this assay was determined to be 10 copies/reaction of positive plasmids. The established RAA assay showed high specificity for MRSA detection without cross-reactivities with other clinically relevant bacteria. The diagnostic performance of real-time RAA was evaluated using 67 clinical S. aureus isolates from dairy farms, which were detected in parallel using the TaqMan probe qPCR assay. The results showed that 56 and 54 samples tested positive for MRSA by RAA and qPCR, respectively. The overall agreement between both assays was 97.01% (65/67), with a kappa value of 0.9517 (p < 0.001). Further linear regression analysis demonstrated that the detection results between the two assays were significantly correlated (R2 = 0.9012, p < 0.0001), indicating that this RAA assay possesses similar detection performance to the qPCR assay. In conclusion, our newly established RAA assay is a time-saving and convenient diagnostic tool suitable for MRSA detection and screening.

Solomonseal polysaccharide and sulfated Codonopsis pilosula polysaccharide synergistically resist Newcastle disease virus.

Five combinations of three ratios (PS9-sPS1, PS7-sPS3 and PS6-sPS4) were prepared with polysaccharide (PS) and sulfated polysaccharide (sPS). The antiviral activities of these compounds were subsequently compared in vitro using the MTT assay, observation of the virus structure and immunofluorescence. The results demonstrated that SP9-sCP1, CP7-sCA3, EP7-sAP3, CA9-sEP1 and EP7-sCA3 presented higher activities, and SP9-sCP1 displayed the highest virus inhibition rate and clearly killed the virus and inhibited viral antigen expression. In an in vivo test, 28-day-old chickens were challenged with Newcastle disease virus (NDV) and were administered the five drug combinations. On day 14 after the challenge, the morbidity, mortality and cure rate in each group were calculated. The results indicated that SP9-sCP1 presented the lowest morbidity and mortality and the highest cure rate. These results indicate that Solomonseal polysaccharide and sulfated Codonopsis pilosula polysaccharide synergistically resist NDV. Moreover, SP9-sCP1 had the highest efficacy and may be used as a new antiviral drug.

Optimization of selenylation conditions for lycium barbarum polysaccharide based on antioxidant activity.

Lycium barbarum polysaccharide (LBP) was modified by HNO3-Na2SeO3 method according to L9(3(4)) orthogonal design to obtain nine selenizing LBPs (sLBPs), sLBP1-sLBP9. Their antioxidant activities in vitro were compared by free radical-scavenging test. sLBP6, sLBP8 and sLBP9 presented stronger activity. In vivo test, 14-day-old chickens were injected respectively with sLBP6, sLBP8 and sLBP9 taking LBP as control, and serum GSH-Px and SOD activities and MDA content were determined. The results showed that three sLBPs could significantly enhance GSH-Px and SOD activities and decrease MDA content. The actions of sLBPs were significantly stronger than that of unmodified LBP. These results indicated that selenylation modification could significantly enhance the antioxidant activities of LBP, sLBP6 possessed the best efficacy and could be exploited into an antioxidant. The optimal modification conditions were 400mg of sodium selenite for 500 mg of LBP, reaction temperature of 70 °C and reaction time of 6h.

Effects of selenylation modification on immune-enhancing activity of garlic polysaccharide.

The garlic polysaccharide was modified by HNO3-Na2SeO3 method according to orthogonal design L9(3(4)) to obtain nine selenizing garlic polysaccharides, sGPS1-sGPS9. Their effects on chicken peripheral lymphocytes proliferation in vitro were compared by MTT assay. The results showed that sGPSs could significantly promote lymphocytes proliferation, sGPS3, sGPS5 and sGPS6 presented stronger efficacy. In vivo experiment, 14-day-old chickens were injected respectively with sGPS3, sGPS5 and sGPS6 when they were vaccinated with ND vaccine taking unmodified GPS as control. The results showed that three sGPSs could significantly promote lymphocyte proliferation, enhance serum antibody titer, IFN-γ and IL-2 contents. These results indicated that selenylation modification could significantly enhance the immune-enhancing activity of GPS, sGPS6 possessed the best efficacy and could be as a candidate drug of immunoenhancer. Its optimal modification conditions were 400 mg of sodium selenite for 500 mg of GPS, reaction temperature of 70°C and reaction time of 6 h.

Effects of epimedium polysaccharide-propolis flavone oral liquid on mucosal immunity in chickens.

A previous study found that epimedium polysaccharide (EP)-propolis flavonoid (PF) injection (EPI) produced reliable immunoenhancement. In this study, we investigate the effects of EP-PF oral liquid (EFO) on mucosal immunity in the chicken small intestine while using EPI, EP and PF as controls. Groups of fourteen-day-old chickens were given EFO orally at one of the three doses when they were vaccinated with ND vaccine. On days 7, 21 and 35 after the first vaccination, six chickens were selected randomly from each group for measurements of the sIgA and IL-17 contents of the washing liquors of the duodenum and jejunum, counts of the lymphocytes in the duodenal endothelium and counts of the IgA(+) cells in the jejunal endothelium and cecum tonsil. The results indicated that EFO significantly promoted the secretion of sIgA and IL-17 and increased the numbers of lymphocyte and IgA(+) cells. Furthermore, EFO was more efficient than EPI at the high and medium doses. These findings indicate that EPO may enhance intestinal mucosal immunity and may be exploited as an oral immunopotentiator.

The preparation optimization and immune effect of epimedium polysaccharide-propolis flavone liposome.

The preparation conditions of epimedium polysaccharide-propolis flavone liposome (EPL) were optimized by response surface methodology taking entrapment rates of epimedium polysaccharide and propolis flavone as indexes. The immunoenhancement of EPL prepared with optimized condition was determined taking epimedium polysaccharide-propolis flavone suspension (EPS) and epimedium polysaccharide-propolis flavone watery solution (EPW) as control. The results showed that the optimized preparation condition was as follows: the ratio of drug to lipid was 14:1, the ratio of soybean phospholipid to cholesterol was 6:1, and the ultrasonic time was 19 min. EPL could significantly promote the proliferation of T and B lymphocytes singly or synergistically with PHA or LPS, mRNA expression of IL-2 and IL-6 and secretion of IgG and IgM as compared with EPS and EPW. These results indicated that liposome could significantly improve the immunoenhancement of epimedium polysaccharide-propolis flavone immunopotentiator (EPI) and would be as the suitable dosage form of EPI.

Immune-enhancing activity comparison of sulfated ophiopogonpolysaccharide and sulfated jujube polysaccharide.

The immune-enhancing activities of four sulfated polysaccharides, sOPS(t), sOPS(80), sJPS(t) and sJPS(50) picked out in our previous researches, were compared taking four corresponding unmodified polysaccharides as control. In vitro experiment, the effects of eight polysaccharides on chicken peripheral lymphocyte proliferation were determined by MTT assay. The result displayed that four sulfated polysaccharides could significantly stimulate lymphocyte proliferation, their actions were significantly or numerically stronger than those of corresponding unmodified polysaccharides, sOPS(80) presented better efficacy. In vivo experiment, 300 14-day-old chickens were averagely divided into ten groups. The chickens except blank control (BC) group were vaccinated with Newcastle disease vaccine, repeated vaccination at 28 days old. At the same time of the first vaccination, the chickens in eight polysaccharides groups were injected respectively with 0.5 ml (1mg) of eight polysaccharides, in vaccination control (VC) and BC group, with 0.5 mL of physiological saline, once a day for three successive days. On days 7, 14, 21 and 28 after the first vaccination, the peripheral lymphocyte proliferation and serum ND antibody titer were determined. The result showed that four sulfated polysaccharides could significantly promote lymphocyte proliferation and enhance serum antibody titer. Their actions were significantly or numerically stronger than those of corresponding unmodified polysaccharides, sOPS(80) possessed the best efficacy. These results indicated that sulfation modification could enhance the immune-enhancing activity of OPS and JPS, sOPS(80) possessed the best efficacy and would be expected as a component drug of new-type immunopotentiator.