Application of multiplex PCR combined with invasive reaction and chromogenic reaction catalyzed by gold nanoparticles in detection of encephalitis and meningitis virus / 中国人兽共患病学报

We developed a method for detecting encephalitis and meningitis virus by using multiplex PCR combined with invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles.Primers were designed based on the conservative regions of encephalitis and meningitis virus (Eastern equine encephalitis virus,EEEV;Western equine encephalomyelitis virus,WEEV;West Nile virus,WNV;Nipah virus,NiPA;Japanese encephalitis virus,JEV).Multiplex PCR system,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were established to detect different encephalitis and meningitis virus in one reaction.Tick-borne encephalitis virus (TBEV),St Louis encephalitis virus (StLEV),Chikungunya virus (CHIKV) and Dengue virus(DV) were used to test its specificity.Quantitative RNA transcribed in vitro and PCR fragments were used to assess its sensitivity.Clinical specimens collected from JEV patients were detected by this method.A method for detecting encephalitis and meningitis virus by using multiplex PCR,invasive reaction and a chromogenic reaction catalyzed by gold nanoparticles were successfully established.This method can detect targeted pathogens specifically,and it has no cross reaction with TBEV,StLEV,CHIKV and DV.The detecting limitation for different targets was 103 copies/μL.Clinical samples were positive for JEV nucleic acids for above assay.The method presented here has characteristic of high specificity,sensitivity and throughput.The results can be observed by visual inspection.This method has broad application prospects in pathogen detection.

Ultra-sensitive quantification of the colorectal cancer-specific NDRG4 gene methylation levels in stool / 医学研究生学报

Objective The NDRG4 gene methylation in stool is a candidate biomarker for non?invasive diagnosis of colorectal cancer. However, the traditional methods for methylation detection could not be well applied to stool samples due to the low sensitivity and low specificity. The aim of this study was to develop a highly sensitive and specific method for quantifying the methylated NDRG4 gene in stools. Methods Forty one stool samples were collected from 12 colorectal cancer patients, 4 adenoma patients and 25 nor?mal persons. The invasive reaction was combined with real?time PCR and the relative quantification was performed by 2-ΔCT method to develop the highly sensitive and specific methylated DNA detection method, which was used for detecting NDRG4 methylation levels in 41 of stool samples. Results The sensitivity of the method was as low as 10 copies of methylated NDRG4 gene fragments. The specificity was high enough to distinguish 0.01% of methylated fragments from un?methylated fragments and 105 copies of unmethylated NDRG4 fragments gave noamplification signals. The detection results from 41 of stool samples showed that detection rate of the NDRG4 gene in stool from adenoma and colorectal cancer groups had a significant difference comparing to that from the normal group. Conclusion The 2-ΔCT method could accurately quantify the methylation levels of the NDRG4 gene in stool samples, and provide an efficient tool for non?invasive colorectal cancer detection.