基于RAPD分子标记的烟草青枯病菌特异引物筛选及效果评价

为建立烟田土壤和烟株烟草青枯病菌的快速诊断方法,本研究采用RAPD方法筛选获得了6对可用于烟草青枯病菌PCR检测的特异性引物。经对6种细菌（烟草青枯病菌、多粘芽孢杆菌、枯草芽孢杆菌、节杆菌、荧光假单胞菌、野火病菌）和2个烟草品种（中烟100和云烟87）的检测验证,6对引物均具有良好的灵敏度、特异性和可靠性。对青枯菌菌液DNA的检测灵敏度可达0.42 pg/μL,对土壤青枯菌的最低检出率为6×102 cfu/g。应用特异引物A1T和S13T可对接种土壤和烟株样品的研磨悬浮液直接进行PCR快速检测,显著地缩短了检测时间,对烟叶产区烟草青枯病的预测预报及防控具有重要意义。      

试验土壤中烟草青枯病菌的RT-qPCR检测分析

为有效防治烟草青枯病,利用实时荧光定量PCR(Real-time quantity PCR,RT-qPCR)技术,对土壤中烟草青枯病菌进行了定量检测,并建立了土壤中烟草青枯病菌的RT-qPCR检测方法。结果表明,该方法对试验土壤青枯菌的最低检测浓度为5×10~2cfu/g土壤,RT-qPCR检测技术对土壤中烟草青枯病菌的灵敏度比常规PCR检测技术提高了10倍。     

皖南烟田三种土传病原物分子检测

为检测皖南烟田土传病害的分布,2011-2012年从皖南烟区烟田共采集土样51份,采用分子生物学方法对烟田土壤进行了烟草黑胫病、根黑腐病和青枯病检测。结果显示,烟田土壤中烟草黑胫病菌、根黑腐病菌和青枯病菌的阳性率分别为19.6%、41.2%和56.9%,3种烟草土传病原菌在皖南烟区多数烟田均有分布,其中青枯病菌检出率最高。本研究证明了应用分子生物学方法检测土传病原菌的可能性,生产上可为烟草种植提供理论依据。     

白肋烟栽培土壤中青枯病菌的鉴定与生化型分析

为建立一套成本低廉、简单快速的分离检测土壤青枯病菌的方法,采用半选择性培养基(PCCG)和PCR技术相结合,从白肋烟栽培土壤中分离鉴定出12个菌株,并分析了这12个菌株的ITS序列 .结果表明:分离出的12个菌株ITS序列与青枯病菌同源性最高,达到92.6％,该方法适合于土壤中青枯病菌的分离鉴定.Hayward的青枯病菌生化型鉴定结果表明,这12个菌株均为生化型Ⅲ.     

基于致病基因靶点的PCR法特异性检测土壤青枯菌

利用分子生物学手段,以R.solanacearum染色体上的16S rDNA ITS以及毒性质粒携带的致病性相关基因fliC为靶点,分别设计5对序列特异性引物,筛选得到了特异性扩增16SrDNA ITS保守序列的引物对RaITS-1/2以及特异性扩增病菌fliC基因片段的引物对RalfliC-F/R.比较这两对引物的扩增灵敏度、稳定性和特异性可以发现,它们均能够稳定、快速、灵敏地检测青枯菌DNA,检测灵敏度可以达到10 fg DNA/μL.在此基础上成功构建了直接检测土壤青枯菌DNA的PCR检测技术体系.     

烟草五种土传病原菌的多重PCR快速检测

  目的  建立一种可同时检测烟草黑胫病菌（Phytophthora parasitica）、青枯病菌（Ralstonia solanacearum）、立枯病菌（Rhizoctonia solani）、根腐病菌（Fusarium oxysporum）和根黑腐病菌（Thielaviopsis basicola）5种烟草重要土传病原菌的五重PCR快速检测方法。  方法  筛选5种病原菌的特异性引物组合,通过不同的引物浓度和退火温度对多重PCR体系进行优化,检测体系的灵敏度,并对土壤和植株样品进行测试,验证其实用性。  结果  根据青枯病菌fliC基因、立枯病菌RPB2基因、根腐病菌COI基因以及根黑腐病菌TEF1基因设计特异性引物,并结合已报道的黑胫病菌parA1基因的特异性引物,成功建立5种烟草土传病害的多重PCR检测方法。反应体系（25 μL）:Sf1/Sr1、Ff1/Fr1、Pf1/Pr1每条引物分别0.3 μL,Rf1/Rr1每条引物各1.8 μL,TBf1/TBr1每条引物各1 μL,2×PCR Mix 12.5 μL,退火温度为58℃,灵敏度可达到100 pg/μL。  结论  本研究建立的多重PCR体系能够同时快速检测烟草黑胫病菌、青枯病菌、立枯病菌、根腐病菌和根黑腐病菌以及田间带菌烟草病株和土壤,为田间烟草土传病害的早期诊断提供依据。      

烟草PVY Real-Time PCR定量检测体系的建立及应用

马铃薯Y病毒是近年来危害烟草生产的重要病毒之一,严重影响烟草的产量与品质。本研究利用DNAMAN软件对GenBank数据库中已登录的马铃薯Y病毒(Potato virus Y,PVY)全基因组序列进行序列比对,设计引物,以烟草肌动蛋白基因为内参,建立了烟草PVY的实时定量检测体系。获得的real-time PCR扩增基线平整,指数扩增明显,斜率大;稳定性和重现性好,变异系数小;循环阈值与PCR起始模板量对数之间存在良好的线性关系。与DAS-ELISA相比,该方法具有高效、灵敏、特异性强等优点,为从分子生物学水平上检测烟草中PVY提供了新的技术手段。     

3M Petrifilm~(TM)快速测试片与国标法检测饮料中霉菌和酵母的比较

本研究使用3M Petrifilm~(TM)快速霉菌酵母测试片以及国标法同时对某功能维生素饮料样品进行霉菌计数和酵母计数检测,比较两者结果是否具有显著性差异。试验筛选采用白色念珠菌(Candida albicans)、拟热带假丝酵母(Candida tropicalis)、汉逊德巴利酵母(Debaryomyces hansenii)、绿木霉(Trichoderma virens)、炭黑曲霉(Aspergillus carbonarius)5株标准菌株进行试验,分别进行了菌株的预选试验、快速测试片检测的重复性试验和特异性试验,以及快速测试片法和国标法的比较试验。研究结果显示,菌株预选试验所筛选的菌株均能在测试样品中生长良好;重复性试验结果表明第二次结果均在第一次结果可接受范围内;特异性试验中表明快速测试片特异性良好;对比试验中使用两种方法对样品进行霉菌和酵母计数检测,结果使用F检验、t检验进行统计分析,结果表明3M Petrifilm~(TM)测试片法与国标法两种方法总体均值无显著差异。     

两种副溶血性弧菌检测方法的比对

目的比较国家标准中的定性检测法与快速检测法中的普通PCR法在检测食源性副溶血性弧菌上的区别与优劣,为今后针对副溶血性弧菌的检测方法的改进依据及应对突发食品安全事件时快速检测提供参考。方法采用GB4789.7-2013《食品安全国家标准食品微生物学检验副溶血性弧菌检验》与SN/T1869-2007《食品中多种致病菌快速检测方法 PCR法》对经阳性菌株污染的市售墨鱼干样品进行副溶血性弧菌的检测,并对2种方法进行对比。结果 2种方法在针对食源性副溶血性弧菌的检验上的比对结果均为检出。在实验耗时方面,国家标准法确认检出阳性需耗时4 d,快速检测法仅需耗时26 h。结论国家标准中的定性检测法与快速检测方法均能检出副溶血性弧菌,国标法在应对日常检验上标准且能够广泛应用,快速检测方法在快速、准确地应对食品安全突发事件方面具有优势。     

食品中沙门氏菌快速检测技术研究进展

沙门氏菌的检验在食品微生物检验中具有十分重要的意义。本文主要针对基于免疫学的快速检测方法-酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)、基于分子生物学的聚合酶链反应(polymerase chain reaction,PCR)、环介导等温扩增(loop-mediated isothermal amplification,LAMP)和基于蛋白质的基质辅助激光解析电离飞行时间质谱法(matrix assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF MS)在食品中沙门氏菌快速检测的应用进行了分析与比较。基于3种不同原理的检测方法各有其优缺点,ELISA方法成本低,操作简单但检出限高;PCR方法成本较高,但检测快速、灵敏度高且检出限低;MALDI-TOF MS检出限低但需要完善的细菌库进行比对。因此建议将含内控的荧光定量PCR与LAMP法作为食品中检测沙门氏菌的主要方法。     

A detection method for recombinant DNA from genetically modified potato (NewLeaf Y potato)

A detection method using the polymerase chain reaction (PCR) was developed to detect genetically modified (GM) potato (NewLeaf Y potato; NL-Y), of which the mandatory assessment has not yet been completed in Japan. The potato sucrose synthase gene was used as an internal control. We designed a primer pair to specifically detect NL-Y without false-positive results in processed potato foods infected with the potato virus Y (PVY). The DNA introduced into NL-Y using the primer pair could be detected from potato powder samples containing 0.05% NL-Y. In addition, we designed primer pairs for recognizing the CryIIIA gene to detect the NewLeaf potato (NL), NewLeaf Plus potato (NL-P) and NL-Y and for recognizing p-FMV in order to detect NL-P and NL-Y. The proposed method was applied to the detection of NL-Y in 26 processed potato foods and NL-Y was not detected in any samples.     

A detection method of recombinant DNA from genetically modified potato (NewLeaf Plus potato) and detection of NewLeaf Plus potato in snack

A detection method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) potato (NewLeaf Plus potato; NL-P), which has not been authorized as safe in foods in Japan. The potato sucrose synthase gene was used as an internal control. The DNA from NL-P specifically provided an amplified band using PCR with a primer pair recognizing PLRV-rep gene. In addition, to prevent false-positive results in processed potato foods infected with PLRV, we designed a primer pair recognizing sequences derived from two organisms to detect specifically NL-P in processed potato. The PCR product obtained using the designed primer pair was specific for NL-P. The DNA introduced into NL-P could be detected from potato powder samples containing 0.05% NL-P. The proposed method was applied to the detection of NL-P in 25 processed potato foods. NL-P was detected in 3 snack products.     

基于机器视觉图像提取的马铃薯内部病虫害特征识别

构建二维马铃薯内部病虫害视觉图像采集模型,对采集的马铃薯内部病虫害视觉图像进行分块融合检测,根据马铃薯绿叶素纹理分布进行病虫害的特征检测,提取马铃薯内部病虫害视觉分形特征量,采用表面纹理配准和分块自适应检测方法进行病虫害的特征点标定,结合小波变换方法进行马铃薯内部病虫害视觉图像的特征分解,根据颜色梯度变化的差异性实现机器视觉下的马铃薯内部病虫害特征识别。仿真结果表明采用该方法进行马铃薯内部病虫害特征识别的准确率接近90%,提高了马铃薯内部病虫害的防治和识别能力。     

The HPLC analysis of thiamin and riboflavin in potatoes

A simple HPLC method for the analysis of thiamin (as thiochrome) and riboflavin in both raw and cooked potato is described. The potato extract, after a minimum clean up, is injected onto a C₁₈ reverse phase column and the vitamins are separated isocratically with water:methanol. The use of fluorescence detection, which is highly specific and sensitive, minimises the number of interfering peaks.Recoveries of both vitamins, when taken through the method or added to potato samples before extraction, are consistently better than 90%. The results for thiamin in the potato are slightly higher than those obtained by the AOAC (1980) chemical method, whereas the reverse is true for riboflavin. The method may have application to other food matrices and is more rapid than the AOAC (1980) chemical method.     

Determination of the phosphorus content in potato starch using an energy-dispersive X-ray fluorescence method

Potato starch is unique because of its high starch phosphorus content. The textural characteristics of potato starch change due to the presence of the starch phosphate. Thus, the measurement of phosphorus in potato starch is needed, but conventional methods require a considerable amount of time and labour. In this investigation, a simple and fast analytical procedure has been developed for the determination of the phosphorus content of potato starch with a non-destructive energy dispersive X-ray fluorescence (ED-XRF) technique. Potato starch samples were analyzed as pressed pellets using detection times of 200 s. Reference values, measured by a conventional method, namely, wet chemical analysis, were used to calibrate the ED-XRF. Calibration was done using 20 potato starch samples, and the results were validated using a second set of 15 samples. The results indicated the validity of ED-XRF as a rapid and non-destructive method for the quantitative determination of phosphorus content of potato starch. Based on the combined results of ED-XRF and Rapid Visco-Analyzer (RVA), ED-XRF is promising for predicting the peak viscosity, by RVA, of potato starch paste through the measurement of starch phosphorus content.     

甘薯酮检测方法研究进展

甘薯酮是甘薯黑斑病产生的主要有毒代谢物质,该病害分布范围广,世界各个地区的甘薯产业都受到不同程度的侵染,严重威胁人类健康,因此甘薯酮的检测尤为重要。目前的检测技术存在检测灵敏度低、误差大、操作复杂等问题。本文阐述了甘薯酮的合成途径、结构、生理毒性，总结了目前已有的检测方法：高效薄层色谱法、比色定量法、顶空式气相色谱质谱联用法，并展望了甘薯酮快速准确检测方法的发展。