Visual assay of Escherichia coli O157:H7 based on an isothermal strand displacement and hybrid chain reaction amplification strategy.

Here, we describe a simple, sensitive, and enzyme-free method for visual point-of-care detection of 16S rRNA of Escherichia coli O157:H7 based on an isothermal strand displacement-hybrid chain reaction (ISD-HCR) and lateral flow strip (LFS). In this study, the secondary structure of 16S rRNA of E. coli O157:H7 was unwound by two helper oligonucleotides to expose the single-strand-specific nucleic acid sequence. The free specific sequence promoted the toehold-mediated strand displacement reaction to output a large number of FITC-labeled single-stranded DNA probes (capture probe [CP]). The 3'-end sequence of the reporter probe propagated a chain reaction of hybridization events between the two hairpin probes modified with biotin to form long nicked DNA polymers with multiple biotins (RP-HCR complexes); the free CP and RP-HCR complexes then form CP/RP-HCR complexes. The biotin-labeled double-stranded DNA CP/RP-HCR polymers then introduced numerous streptavidin (SA)-labeled gold nanoparticles (AuNPs) on the LFS. The accumulation of AuNPs produced a characteristic red band, which enabled visual detection of changes in the signal of 16S rRNA of E. coli O157:H7. The current approach could detect E. coli O157:H7 at concentrations as low as 102 CFU mL-1 without instrumentation. This approach thus provides a simple, sensitive, and low-cost tool for point-of-care detection of pathogenic bacteria, especially in resource-limited countries.

Development of a Quantitative Real-Time PCR Assay for Detection of Cryptosporidium spp. Infection and Threatening Caused by Cryptosporidium parvum Subtype IIdA19G1 in Diarrhea Calves from Northeastern China.

Parasitic diarrheal disease is a major cause of morbidity and mortality in the developing world. Calves are highly susceptible to Cryptosporidium spp. infection that resulted in diarrhea, growth retardation, and weight loss, and was one of the most common enteropathogens. It is especially difficult for molecular detection of calves with inapparent or subclinical infections of cryptosporidiosis. In view of this, this study established a real-time quantitative PCR (RT-qPCR) detection method to clarify its epidemic characteristics, based on Cryptosporidium 18S rRNA gene with the 150 bp product length to investigate the infection of Cryptosporidium spp. in northeastern China The standard curve equation is Ct = -2.91 × lg (Cryptosporidium spp. copies) +10.18, with better sensitivity, stability, and reproducibility. A total of 148 out of 425 fecal samples (34.82%) were detected Cryptosporidium positive with RT-qPCR, including (36.11%) in Heilongjiang province (29.60%), (29.6%) in Jilin province, and (37.50%) in Liaoning province. The infection prevalence of Cryptosporidium parvum, Cryptosporidium ryanae, Cryptosporidium andersoni, and Cryptosporidium bovis from calves in order from high to low was 14.35% (95% confidence interval [CI], 11.2-18.1), 6.12 (95% CI, 4.0-8.8), 2.35 (95% CI, 1.1-4.3), and 0.47 (95% CI, 0.1-1.7), respectively, suggesting C. parvum was the predominant species in calves in northeastern China. Using 60-kDa glycoprotein gp60 gene, all of the 61 C. parvum-positive specimens were further precisely confirmed to IIdA19G1 subtype. This suggested that IIdA19G1 subtype of C. parvum could threaten to cause diarrhea calves from notheastern China (p < 0.01). The prevalence of 34.82% (148/425) using RT-qPCR had a significant difference compared with the prevalence of nested-PCR (23.29%) and microscopic examination (3.76%). The findings improved the epidemiological knowledge of calves infected with cryptosporidiosis in China, highlighting the importance of ongoing Cryptosporidium surveillance.

Irisin Protects Against Motor Dysfunction of Rats with Spinal Cord Injury via Adenosine 5'-Monophosphate (AMP)-Activated Protein Kinase-Nuclear Factor Kappa-B Pathway.

The aim of the present research was to investigate the effects of irisin, a skeletal muscle-derived myokine, on spinal cord injury (SCI) in rats and explore the possible mechanisms. SCI model was constructed in male SD rats. The effects of irisin on SCI rats were assessed via behavior tests including Basso, Beattie, and Bresnahan (BBB) scoring method and inclined plane test, followed by histomorphology tests including HE staining, Nissl staining, and transmission electron microscope examination. Biochemical analyses including PCR, Western blots and ELISA were employed to further evaluate the changes at molecular level of SCI rats. In addition, lipopolysaccharide (LPS)-induced cell damage model was established in PC12 cells to verify the mechanism of irisin's effect on nerve cells in vitro. Results showed that the BBB score and the angle of incline significantly decreased after SCI surgery, however, chronic irisin treatment improved SCI-induced motor dysfunction. HE and Nissl staining assays showed that SCI surgery induced histological injury of spinal cord, which could be reversed by irisin treatment. Morphological abnormality of nerve cells caused by SCI also could be alleviated by irisin. Further biochemical analyses showed that irisin inhibited SCI-induced overexpression of Interleukin-1ß (IL-1ß), Interleukin- 6 (IL-6), tumor necrosis factor alpha (TNF-α), inducible nitricoxidesynthase (iNOS) and Cyclooxygenase-2 (COX-2)], as well as nuclear factor kappa-B (NF-κB)p65 in rats, and the positive function of irisin could be reversed by Compound C treatment. In our in vitro study, LPS-induced declines of cell viability and neurite length of PC12 cell were inhibited by irisin treatment, and irisin inhibited LPS-induced overexpression of NF-κBp65, IL-1ß, IL-6, TNF-α, iNOS and COX-2. These changes could be reversed by activated protein kinase (AMPK) siRNA pre-treatment. Taken together, irisin could protect the rats from SCI, and its protection is associated with the regulation of adenosine 5'-monophosphate-activated protein kinase (AMPK)- NF-κB signaling pathway.