多重LAMP—熔解曲线法检测食品中两种食源性致病菌

针对沙门氏菌的侵袭蛋白基因(inv A)和金黄色葡萄球菌的耐热核酸酶基因(nuc)设计LAMP引物,优化反应条件,并分别对反应产物进行熔解曲线分析,判断扩增结果,从而建立食品中沙门氏菌和金黄色葡萄球菌多重LAMP—熔解曲线检测方法。对9株目标菌和23株非目标菌的检测未出现假阳性和假阴性结果,并可通过熔解曲线的熔解温度分析确定所含目标菌;对产物的测序分析表明所得序列与目的基因序列吻合;对目标菌的检测灵敏度均可达到10 fg/μL;对232份样品的检测结果显示,其中2份生猪肉中检出金黄色葡萄球菌。该方法具有良好的检测特异性,并可为食源性致病菌的快速检测提供一种重要技术手段。     

Rapid Detection of Salmonella enterica Subspecies enterica serovar Typhimurium by Loop Mediated Isothermal Amplification (LAMP) Test From Field Chicken Meat Samples

Considering the importance of Salmonella enterica subsp. enterica serovar Typhimurium in the foodborne diseases, a Typhimurium specific loop mediated isothermal amplification (LAMP) test was standardized for its rapid detection in chicken meat. The Optimum results were obtained at 64^oC and 70 min temperature-time combination. The sensitivity of LAMP and PCR were compared with serial 10-fold dilution of the 100 ng of DNA. The LAMP test detected 2 pg DNA per reaction tube, whereas PCR detected 200 pg DNA per reaction. Therefore, the LAMP test was considered 100 times more sensitive than the PCR. The specificity of LAMP and PCR analyzed with six different isolates of non-Salmonella and 22 serovars of non-Typhimurium. None of these isolates were found positive by both LAMP and PCR. Twenty-eight pure isolates of Salmonella Typhimurium from diverse sources were also examined by Typhimurium specific LAMP and were all found positive. Two-hundred twenty-five field chicken meat samples were screened by cultural, PCR, and LAMP methods. The LAMP and PCR tests were performed by using DNA isolated from 8 h and 18 h enrichment samples, respectively. Typhimurium specific LAMP was shown to be in 100% correlated with cultural and PCR methods. However, LAMP test delivered the results within 26 h without sophisticated equipment while PCR and cultural methods took 48 h and 6 d, respectively. The LAMP test developed in this study has potential to detect and as well as differentiate S. Typhimurium from other Salmonella serovars.     

基于PHP的在线请假系统的设计与实现

通过对系统功能模块进行需求分析及设计．采用LAMP技术进行学生在线请假管理系统的开发。本系统可实现教师对学生请假信息的在线管理，促进高校学生工作管理的信息化．方便服务于广大师生。     

可视化环介导等温扩增技术检测鸡鸭源性成分

目的建立生肉及熟肉制品中鸡、鸭源性成分的特异性基因LAMP可视化快速检测方法。方法根据鸡鸭物种特异性cytb、COX3基因的6个特异性区域设计LAMP引物,在反应前加入羟基萘酚蓝(HNB),在63℃条件下反应45 min;反应结果可以根据颜色变化直接判断,阳性为天蓝色,阴性为紫罗兰色。并对30个肉制品样品进行可视化LAMP和PCR方法平行检测。结果 LAMP方法的最低检出限为1 pg,特异性良好,同时对肉制品中目标物检测的检出限可达到0.01%。结论该方法用于肉制品中鸡鸭源性成分的快速检测,具有操作简单、无需贵重仪器、反应结果肉眼可观察、灵敏度高和特异性强等特点,适合于基层监管部门进行肉制品现场快速检测。     

环介导等温核酸扩增技术快速检测食物中的大肠杆菌O157

建立了环介导等温核酸扩增技术(LAMP)检测食源大肠杆菌O157,并对该方法的灵敏度和特异性进行了评价。分别针对大肠杆菌O157三个特异基因rfbE,stx1和stx2的8个独立靶区域设计了外引物、内引物和环引物进行LAMP扩增检测,同时将检测结果与PCR方法做比较。研究结果表明,rfbE,stx1和stx2基因的LAMP方法检测限分别为100,100和10 fg DNA/管,灵敏度是PCR方法的10倍以上;将建立的环介导等温扩增法用于417株食物分离的大肠杆菌的检测,发现LAMP检测rfbE,stx1和stx2基因的灵敏度分别为100%,95.3%和96.3%,对3个靶基因的阴性预测率分别为100%,96.7%和97.1%,特异性和阳性预测率均为100%。结果表明,该方法用于大肠杆菌O157的检测具有特异性强、灵敏度高、操作简便的优越性,在食品安全检测方面具有良好的实际应用前景。     

基于荧光及可视化逆转录环介导等温扩增检测食源性甲型肝炎病毒的方法开发

目的 基于荧光及可视化RT-LAMP开发食源性甲型肝炎病毒(hepatitis a virus, HAV)的检测方法。方法 从NCBI中选取世界范围内具有代表性的HAV基因信息, 设计LAMP引物, 开发了一种荧光曲线RT-LAMP和可视化RT-LAMP检测方法。以HAV质粒为模板, 评估了方法的扩增效率、灵敏度和稳健性, 并与实时荧光RT-PCR进行检出限及实际样品验证比较。结果 荧光及可视化LAMP方法检测灵敏度为10.76 copies/μL, 与实时荧光RT-PCR一致; 在95%置信水平下经概率回归分析检出限(limit of detection, LOD), 荧光曲线RT-LAMP的LOD95%为17.46 copies/μL (7.00~511.29 copies/μL, 95%), 实时荧光RT-PCR的LOD95%为79.30 copies/μL (24.68~3208.71 copies/μL, 95%); 稳健性分析表明方法稳定; 与实时荧光RT-PCR比较, 检测30份实际样品验证比对符合率为100%。结论 该检测方法经济便捷, 可通过肉眼直接观察结果, 适合于食品安全现场监测, 具有很好的应用前景。     

Autophagic Upregulation Is Cytoprotective in Ischemia/Reperfusion-Injured Retina and Retinal Progenitor Cells

The cytoprotective versus cytotoxic role of macroautophagy in ocular ischemia/reperfusion injuries remains controversial and its effects under hyperglycemia are unclear. We investigated the involvement of autophagy in in vitro and in vivo normoglycemic and hyperglycemic models of retinal ischemia/reperfusion injury. Retinal ischemia (2 h) and reperfusion (2 or 22 h) was induced in wild-type and type I diabetic Ins2^Akita/+ mice using a middle cerebral artery occlusion model. R28 retinal precursor cells were subjected to CoCl₂-induced hypoxia with or without autophagic inhibitor NH₄Cl. Autophagic regulation during ischemia/reperfusion was assessed through immunohistochemical detection and Western blotting of microtubule-associated protein 1A/1B-light chain 3 (LC3) and lysosomal associated membrane protein 1 (LAMP1). Effect of autophagic inhibition on cell viability and morphology under hypoxic conditions was also evaluated. Upregulation of autophagic markers in the inner retinae was seen after two hours reperfusion, with tapering of the response following 22 h of reperfusion in vivo. LC3-II turnover assays confirmed an increase in autophagic flux in our hypoxic in vitro model. Pharmacological autophagic inhibition under hypoxic conditions decreased cell survival and induced structural changes not demonstrated with autophagic inhibition alone. Yet no statistically significant different autophagic responses in ischemia/reperfusion injuries were seen between the two glycemic states.     

利用环介导等温扩增技术对沙门氏菌进行检测

提出食品中沙门菌的环介导等温扩增技术(LAMP)检测方法.针对沙门菌特异性基因invA,设计了4条沙门菌LAMP检测特异性引物,建立食品中沙门菌LAMP检测方法.用16个血清型45株沙门菌,58株近源菌验证试验表明,所建立的LAMP方法准确且灵敏度高.加菌试验表明,食品样品中沙门菌检测低限为1.5 CFU/100g;新建的LAMP方法与FDA BAM方法比较试验表明,二种方法的检测结果完全符合.本方法特异性好、灵敏度高、与传统方法相比可节省大量时间且对实验仪器和操作人员的要求低,非常适于在实际工作推广.