天芥菜(Heliotropium europaeum L.)是一种严重危害牧场的杂草,也是谷物田的主要杂草.天芥菜危害小麦、玉米、大豆等农作物,在一些国家,被列为检疫性杂草.此外,天芥菜中含有5种吡咯里西啶生物碱(pyrrolizidine alkaloids,PAs),对羊、牛、马、猪和家禽有毒,引起肝损伤.有毒的生物碱可通过受其污染的小麦或鸡蛋等不同方式被人类摄入,进而影响人体健康.天芥菜可混杂在粮谷中进行远距离传播,需高度重视.本文介绍了天芥菜的分布、形态特征、生物学特性、风险评估,以期对检疫、监管、监测和防控有所帮助.

[目的]为完善我国转基因检测方法体系,建立转基因大豆MON87751品系特异性实时荧光聚合酶链式反应(real time polymerase chain reaction,PCR)检测方法.[方法]根据MON87751的3′端邻接区序列设计特异性引物和探针,建立MON87751品系特异性实时荧光PCR检测方法,并测定该方法的灵敏度、特异性及可重复性.[结果]灵敏度测试显示,其定量下限为40拷贝;重复性试验显示其相对标准偏差在可接受范围内.[结论]该研究建立的MON87751品系特异性实时荧光PCR检测方法特异性良好,灵敏度高,有良好的可重复性,适合对转基因大豆MON87751品系进行检测鉴定.

为实现转基因甜菜品系GTSB77的标识管理和精准定量,根据GTSB77的3'端边界序列和甜菜谷氨酰胺合成酶(GS)基因设计引物探针建立双重微滴数字PCR检测方法.结果显示:建立的转基因甜菜GTSB77数字PCR检测方法特异性强,在20 μL反应体系中定量下限(LOQ)均为1.84拷贝/μL,检测下限(LOD)均为0.61拷贝/μL,可应用于转基因甜菜GTSB77成分的精确定量检测.

Objective For implementation of labeling regulations, an event-specific real-time polymerase chain reaction (PCR) method for quantitative detection of sugarbeet GTSB77 was established. Methods The specific primer pairs and probe based on the 3’flanking sequence of GTSB77 were designed. The specificity, sensitivity and repeatability of the developed method were examined, respectively. Results The specificity test showed it specific for GTSB77 detection. The limit of quantification (LOQ) was 16 copies and the amplification efficiency was 102%. Reapeatability of the established method was assessed and the relative standard deviation (RSD) ranged from 0.21%-1.66%. Conclusion This event-specific real-time PCR method is suitable for identification of GTSB77.

以水果上3种重要的褐腐病菌,即美澳型核果褐腐菌(Monilinia fructicola)、核果褐腐菌(M.laxa)和仁果褐腐菌(M.fructigena)为检测对象,研究开发适用于口岸水果检疫的快速检测试纸.以硝酸纤维素膜为固相载体,以病菌翻译延长因子基因(Translation elongation factor 1-alpha,EF-1α)为检测靶标,生物素标记的PCR扩增产物为检测标记物,采用DNA-DNA杂交方式对3种褐腐病菌进行试纸法快速检测.分别以褐腐病菌DNA和本研究构建的EF-1α 重组质粒DNA为阳性标准品检验试纸的特异性和灵敏度.试验结果表明,本研究开发的检测试纸能够同时特异地检出以上3种褐腐病菌,整个检测流程(包括DNA提取和扩增过程)在2 h内即可完成,最低检测限达到10 fg/μL.其特异性强、灵敏度高、稳定性好,且检测结果可肉眼判定,无需依赖昂贵设备,便于向基层检疫实验室推广使用.

在对中国苹果产业发展概况及苹果进出口贸易现状进行梳理的基础上,介绍了国际贸易绿色壁垒对中国苹果出口的影响,由进境苹果检疫中的重要事件,阐述了植物检疫对中国苹果产业健康发展的重要性.

目的 为实现转基因北极苹果(ArcticTM apple)目的标识管理,建立特异性实时荧光聚合酶链式反应(polymerase chain reaction,PCR)检测方法.方法 针对转基因北极苹果特异性序列设计引物和TaqMan探针,建立转基因北极苹果实时荧光PCR检测方法,并对该方法的特异性、灵敏度和重复性进行检测.结果 建立的转基因北极苹果实时荧光PCR特异性强,定量检测限为20拷贝,扩增效率为96％,检测重复性良好.结论 建立的特异性实时荧光PCR法可应用于转基因北极苹果的鉴定.

[目的]建立转基因棉花MON88701品系特异性实时荧光聚合酶链反应(polymerase chain reaction,PCR)检测方法.[方法]利用实时荧光PCR技术,根据转基因棉花MON88701品系特异性序列设计引物和探针,建立转基因棉花MON88701实时荧光PCR检测方法,并测定本方法的灵敏度、特异性及可重复性.[结果]建立的检测方法特异于转基因棉花MON88701成分的检测,灵敏度测试表明其定量下限为34拷贝;重复性试验显示其相对标准偏差在可接受范围内.[结论]本研究建立的转基因棉花MON88701品系特异性实时荧光PCR检测方法具有良好的特异性和高灵敏度,适合对转基因棉花MON88701品系进行检测.

A rolling-circle amplification (RCA) method with padlock probes targeted on EF-1α regions was developed for rapid detection of apple bull’s-eye rot pathogens, including Neofabraea malicorticis, N. perennans, N. kienholzii, and N. vagabunda (synonym: N. alba). Four padlock probes (PLP-Nm, PLP-Np, PLP-Nk, and PLP-Nv) were designed and tested against 28 samples, including 22 BER pathogen cultures, 4 closely related species, and 2 unrelated species that may cause serious apple decays. The assay successfully identified all the bull’s-eye rot pathogenic fungi at the level of species, while no cross-reaction was observed in all target species and no false-positive reaction was observed with all strains used for reference. This study showed that the use of padlock probes and the combination of probe signal amplification by RCA provided an effective and sensitive method for the rapid identification of Neofabraea spp. The method could therefore be a useful tool for monitoring bull’s-eye rot pathogens in port quarantine and orchard epidemiological studies.

北美刺龙葵(Solanum carolinense L.)是一种具有庞大根茎系统、极强繁殖力和竞争力的有毒杂草.介绍了北美刺龙葵的生物学、生态学特性、竞争能力和危害性,并提出了有效的防除方式,以期对检疫监管、后续监测和防控有所帮助.

Objective For implementation of labeling regulations,an event-specific real-time polymerase chain reaction (PCR) method for the detection of genetically modified AquAdvantage salmon was established in this study.Methods Primers and TaqMan probe were designed based on the event-specific sequence of AquAdvantage salmon.The specificity,sensitivity and repeatability of the developed method were examined,respectively.Results The specificity test of this method showed it was specific to AquAdvantage salmon.The 600 000-60 copies range showed a good linear relationship with Ct values,and its linear regression equation was y =-3.2194x + 40.805 (R2 =0.997).The limit of quantification (LOQ) was 60 copies and the repeatability was good.Conclusion This event-specific real-time PCR method was suitable for the identification of genetically modified AquAdvantage salmon.

Weed seeds intercepted from imported American sorghum from 2014 to October 2016 in Huangpu and Nansha port were collected and seed germination experiment by using 9 quarantine weeds and 14 non-quarantine weeds with high intercepted frequency showed that all most of the intercepted weed seeds can germinate with high germination rate,such as Amaranthus palmeri and Pharbitis nil were more than 58％,Amaranthus tuberculatus were 19.17％,Sorghum halepense and Ambrosia artemisiifolia were 7.50％ and 5.00％.So we should pay attention to the weed quarantine and monitoring of imported American sorghum and prevent the weed spreading.

根据转基因低木质素苜蓿草品系KK179-25'端外源插入序列与苜蓿草基因组DNA之间的邻接区序列设计引物和探针,建立了KK179-2品系特异性实时荧光PCR检测方法,并对其特异性、灵敏度及可重复性进行了测定.结果表明:建立的定量实时荧光PCR检测方法特异性良好;标准曲线线性相关系数(R2)为0.99,扩增效率E为105%;检测限为20拷贝,定量限为200拷贝.重复性实验显示本方法的标准偏差(SD)及相对标准偏差(RSD)都在可接受范围内.综上,建立的KK179-2品系特异性定量PCR检测方法适于快速、准确、稳定地对转基因苜蓿草KK179-2品系进行检测.

[目的]近年来,美国高粱开始大量进入我国,其携带的杂草种子状况尚未有相关研究.通过对进境美国高粱携带的杂草种子现状进行分析,可为出入境检验检疫机构的检疫监管和后续监测提供依据.[方法]通过对2014—2016年进境美国高粱截获的杂草种子的研究,了解其携带的杂草种子状况.[结果]黄埔检验检疫局和南沙检验检疫局从进境的美国高粱中截获的杂草种子种类共涉及19个科106种.主要包括禾本科27种、菊科14种、大戟科3种、茄科2种、苋科15种、豆科10种、蓼科7种、锦葵科4种、旋花科7种、十字花科4种、藜科4种等,其中检疫性杂草共涉及5科25种,检出率高.[结论]美国高粱携带的杂草种子数量大,种类丰富,检疫性杂草含量大,应予以高度重视.

明孢盘菌属(Neofabraea)中很多种类是重要的植物病原菌.该属真菌分类历史较为复杂,系统发育关系也长期存在较大的争议.本文对明孢盘菌属的分类学研究历史做了系统回顾,包括属的建立、物种组成及其命名更替和系统发育,概述了该属重要的植物病原菌种类地理分布和经济为害性,讨论了该属真菌物种的传统分类方法及分子研究方法,指出了存在的问题及今后的研究方向.

苹果牛眼果腐病菌(Neofabraea malicorticis、N.perennans、N.alba和N.kienholzii)是我国检疫性植物病原真菌,为建立该病菌的实时荧光PCR检测法,根据病菌及其近缘种的翻译延伸因子(EF-1α)的保守序列设计了特异性探针,分别以苹果牛眼果腐病菌菌株和本研究构建的EF-1α重组质粒DNA为阳性标准品检验探针的特异性和灵敏度.结果显示探针MAL-P、PER-P、ALB-P和KIE-P分别对N.malicorticis、N.perennans、N.alba和N.kienholzii表现特异性阳性扩增,而与近缘种及其他常见的果腐病菌无交叉反应,单重探针和4种探针混合液的灵敏度分别达1 fg/μL和10 fg/μL DNA.总计从美国、智利、新西兰、法国进境截获的29批可疑病果的分离物中检测到PCR阳性荧光信号,包括20批N.perennans、8批N.alba和1批N.kienholzii.该方法在6h内即可完成整个检测流程,其特异性强、灵敏度高,适用于实际样品中苹果牛眼果腐病菌的快速检测.

本文针对转基因苜蓿草品系Jl01,设计引物及探针建立J101品系特异实时定量PCR检测体系.结果表明,本文建立的定量实时PCR检测方法特异性良好.实时荧光定量PCR检测方法的检测下限为10拷贝J101基因组DNA,定量检测下限为50拷贝的Jl01基因组DNA,本方法建立的标准曲线线性相关系数(R2)为0.993,扩增效率E为110％,重复性实验显示本方法的标准偏差(SD)及相对标准偏差(RSD)都在可接受范围内.本文建立的J101品系特异性定量PCR检测方法适于快速、准确、稳定地对转基因苜蓿草J101品系进行检测.

钻孔奸狡长蠹为非洲地区重要的林木害虫,也是近年来我国口岸进口木材中截获频次较多的昆虫种类.本文重点描述了钻孔奸狡长蠹雌虫和雄虫的形态特征并从背面、侧面、前面、后面和腹面图示了钻孔奸狡长蠹的性二型性,另外也提供了钻孔奸狡长蠹雌性和雄性生殖系统解剖结构和雌雄虫形态特征区别表,为进口木材中对钻孔奸狡长蠹的检疫鉴定提供了较详细的参考依据.

为了明确乳白蚁(Coptotermes spp.)形态分类中一些特征的分类价值,采用显微测量、显微观察及电镜扫描技术,对国内采集和口岸截获的8种乳白蚁兵蚁的形态进行了研究.结果显示,乳白蚁兵蚁囟孔侧毛数、头区刚毛数、头型、触角节数、上颚弯曲度、前胸背板刚毛数和前胸背板侧缘形状等7个特征可作为区分乳白蚁兵蚁的有效特征,并根据这些特征列出了8种乳白蚁的形态分类检索表.

The study was aimed to establish a specific and sensitive TaqMan Real-time PCR assay for detection of Arcanobacterium pyogenes(A.pyogenes).Based on the conservative sequence of pyolysin(PLO)gene of A.pyogenes published in GenBank,specific primers and TaqMan probes were designed.The TaqMan Real-time PCR assay was established,and the specificity and sensitivity were tested.The specificity test results showed that only A.pyogenes exhibited typical curves.The detection sensitivity of this assay was 77.6fg genomic DNA per 20μL reaction,and63CFU/mL for pure cultures.16 out of 23 clinical samples were positive detected by the TaqMan Real-time PCR assay,which were consistent with API Coryne identification.The TaqMan Real-time PCR assay developed in this study was specific and sensitive for detection of A.pyogenes,and it could be used for identification and epidemiological investigation of A.pyogenes.