RESUMO
Recently, there has been an upward trend in the occurrence of hand-foot-mouth disease, which is correlated with Coxsackie A6 and A10 infections. Although two separate diagnostic reagents are available for these two viral strains, the protocol and diagnosis efficiency still need to be improved. More importantly, as co-infection with these viruses is common, the development of a single test kit that can diagnose both viruses would be most beneficial for clinical practice. In our study, specific primers targeting viral nucleic acids were designed and modified. Viral nucleic acids were extracted from fecal or throat swab samples by ultrasonic rupture and silicon membrane purification. The consistency, specificity, and sensitivity of the tests were further optimized by adjusting the polymerase chain reaction (PCR) conditions. The efficiency of viral nucleic acid extraction was significantly enhanced by the ultrasonic rupture and silicon membrane elution approach. Specific amplifications of both viral nucleic acids were achieved using modified primers. The optimal conditions for PCR were also determined (60°C for 30 min and 95°C for 2 min, followed by 40 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 60°C, and elongation for 50 s at 72°C). Amplified products were confirmed as viral specific nucleotides by agarose gel electrophoresis and sequencing. The minimal nucleic acid concentration required for detection was 0.2 ng/L, which was adequate to yield satisfactory specificity and consistency. This novel diagnostic method has many advantages, including rapid protocols and accurate results, and can be promoted for large-scale clinical trials.
Assuntos
Enterovirus/patogenicidade , Doença de Mão, Pé e Boca/diagnóstico , Doença de Mão, Pé e Boca/virologia , Kit de Reagentes para Diagnóstico , Humanos , RNA ViralRESUMO
The development of diabetic peripheral neuropathy (DPN) is always followed by changes in vascular endothelial cells that are related to the reactivity of the homocysteine (Hcy) sulfhydryl group. In this meta-analysis, we investigated the association of Hcy with the pathogenesis and progression of DPN. We screened the Embase, Ovid, PubMed, Web of Science, Wangfang, and China National Knowledge Infrastructure databases. All analyses were performed by using the STATA software, version 12.0 (StataCorp, College Station, TX, USA) and the Comprehensive Meta-analysis 2.0 software (Biostatic Inc., Englewood, NJ, USA). The standardized mean difference (SMD) and 95% confidence interval (95%CI) were further calculated. The electronic literature search identified six articles that included 603 patients with DPN and 687 healthy controls. The pooled SMD of those six studies revealed that increased serum levels of Hcy may be correlated with DPN (SMD = 1.23, 95%CI: 1.09-1.36, P < 0.001). Subgroup analysis according to ethnicity indicated that high serum Hcy levels might be an important risk factor for DPN in both Asian and Caucasian populations (Asians: SMD = 0.62, 95%CI: 0.45-0.79, P < 0.001; Caucasians: SMD = 2.32, 95%CI: 2.10-2.55, P < 0.001; respectively). Elevated serum levels of Hcy indicate the risk of development of DPN in patients, suggesting that Hcy levels could be used as a marker for new therapeutic approaches to DPN.