致病性沙门菌SYBR GreenⅠ实时荧光定量PCR检测方法的建立

为建立一种灵敏、特异、快速地检测食品中致病性沙门菌的方法,针对沙门菌致病性菌株特有的invA基因设计引物,PCR扩增目的片段,构建重组质粒pMD19-T-inv285,将其作为标准品进行real-time PCR反应体系的优化和标准曲线的绘制,并进行灵敏度、特异性及稳定性检测;同时用该方法对90份肉品样品进行检测。结果显示,建立的致病性沙门菌SYBR GreenⅠ实时荧光定量PCR检测方法的标准曲线方程为y=-3.397 1 x+27.313,相关系数r2=0.999 6。该方法的灵敏度为5×100copies/μL,是普通PCR(5×102 copies/μL)的100倍。对10种常见食源性细菌的检测结果均为阴性;组内和组间重复试验的变异系数均低于1%。对90份肉品样品的检出率(6/90,6.67%)显著高于快速MALDI-TOF-MS法和传统的细菌分离鉴定方法(均为3/90,3.33%)。结果表明,本试验建立的致病性沙门菌SYBR GreenⅠ实时荧光定量PCR灵敏度高,且特异、稳定,适用于食品中致病性沙门菌的快速检测。     

沙门菌PCR快速检测试剂盒的研制与应用

为建立一种简单、快速、灵敏、准确的沙门菌检测方法,根据沙门菌fimY保守基因序列设计合成了1对引物,建立了快速检测沙门菌的PCR方法,并对反应条件进行优化,组装成快速检测试剂盒。结果显示,所有沙门菌均扩增出526 bp的特异性条带,而非沙门菌均未扩增出任何条带。检测灵敏度达93 CFU,还可检出含3 CFU的样品用BP预增菌的菌液或用MM增菌后的菌液,而且稳定性良好,冷冻至少能保存12个月。采用直接菌体加入法、热裂解法、反复冻融法、CTAB碱裂解法和柱式试剂盒提取法分别提取核酸模板,结果CTAB法效果最好,但操作烦琐,而直接菌体加入法和热裂解法可以达到和柱式试剂盒提取法相同的效果,且操作简便、快速、经济、效果好。通过对临床样品的检测,证实该试剂盒具有操作简便、快速、灵敏度高、特异性强、重复性好、稳定性高等优点,值得推广应用。     

进境动物源性饲料中沙门菌荧光定量PCR检测方法的建立及应用

为建立快速鉴别诊断进境动物源性饲料中沙门菌的实时荧光定量PCR方法,以inv A基因为靶基因,通过构建p UCm-inv A重组质粒建立的标准曲线确定了检测体系的灵敏度,并利用特异性试验和稳定性试验评价体系的检测性能。结果显示,该检测体系对基因组DNA的检测灵敏度为17 copies/L;由标准曲线的相关系数可知,初始拷贝数的对数值与Ct值之间具有良好的重现性(R2=0.988);进境动物源性饲料样品的检出率与国标法一致;30份模拟污染样品的检出率达100%。结果表明,建立的快速鉴别诊断方法具有检测限低、重复性好、定量准确、数小时内即可得出检测结果的优点,极大地提高了进境动物源性饲料中沙门菌检测的工作效率和口岸通关速度。     

肺炎克雷伯菌TaqMan荧光定量PCR检测方法的建立

为建立肺炎克雷伯菌TaqMan荧光定量PCR检测方法,根据肺炎克雷伯菌16SrRNA基因的保守序列设计特异性引物和TaqMan探针,通过反应条件优化和特异性、敏感性、重复性试验以及临床样品的检测,建立了肺炎克雷伯菌TaqMan荧光定量PCR。结果显示,该方法与猪肺炎支原体、猪鼻支原体、臭鼻克雷伯菌、鼻硬结克雷伯菌、多杀性巴氏杆菌、链球菌、葡萄球菌、大肠杆菌、沙门菌、肠球菌、乳杆菌、解淀粉芽胞杆菌、猪丹毒杆菌、奇异变形杆菌和化脓隐秘杆菌共15种细菌无交叉反应;标准品浓度在2.29×109~2.29×104 copies/μL范围内具有良好的线性关系,最低可检测到2.29×102 copies/μL的标准品阳性质粒;批内和批间变异系数均小于3%。临床样品的检测结果表明,该方法具有敏感性高、特异性好、稳定性强和检测快速的优点,可用于肺炎克雷伯菌感染的早期诊断、样品的快速检测以及肺炎克雷伯菌的定量分析。     

沙门菌和副溶血性弧菌双重荧光PCR快速检测方法的建立

根据沙门菌invA基因和副溶血性弧菌toxR基因的保守序列设计引物和探针,通过优化反应条件与参数,建立了可同时检测沙门菌和副溶血性弧菌的双重荧光PCR方法.结果显示,该方法敏感度高,特异性强,重复性好.对沙门菌和副溶血性弧菌的检测低限均低于每反应体系10CFU,经对29株非目标菌进行检测均呈阴性,而定量检测批内和批间的变异系数均小于2%;应用该方法检测人工染菌样品和实际样品,结果与SN标准方法的检测结果一致;应用该法可在8h内完成对样品中沙门菌和副溶血性弧菌的同步检验.     

丹毒丝菌属荧光定量PCR检测方法的建立

为建立高效快速的丹毒丝菌属荧光定量PCR(SYBR Green real-time PCR)检测方法,根据丹毒丝菌属16S r RNA基因保守序列设计特异性引物,通过优化确定最佳反应条件,并进行敏感性、特异性、重复性试验以及临床样品的检测,建立了丹毒丝菌属荧光定量PCR检测方法。结果显示,标准品浓度在1.06×106~1.06×101copies/L范围内具有良好的线性关系,最低可检测到1.06×10-2 copies/L的标准品阳性质粒;该方法与副猪嗜血杆菌、多杀性巴氏杆菌、金黄色葡萄球菌等22种细菌和病毒无交叉反应;批内和批间的变异系数(CV)均小于3%。对临床样品的检测结果表明,该方法具有敏感性高、特异性强、稳定性好、准确度高和检测快速等优点,可用于猪丹毒杆菌感染的早期诊断、样品的快速检测与定量分析。     

沙门菌毒力基因多重PCR检测方法的建立及应用

通过建立沙门菌不同毒力岛基因的多重PCR,对沙门菌毒力基因进行快速、灵敏的检测。根据GenBank中的序列设计合成16对引物,优化反应条件,建立了4组多重PCR,通过倍比稀释模板检测多重PCR的敏感性。利用建立的多重PCR对124株沙门菌进行毒力基因分布检测,验证多重PCR的实用性。电泳结果显示,多重PCR体系可有效扩增出每个目的基因。4组多重PCR体系的DNA敏感性分别为50、10、50、100pg;菌液敏感性分别为1×104、1×104、1×105、1×105 CFU。多重PCR与单基因PCR检测沙门菌毒力基因的结果一致。结果表明,本研究建立的多重PCR可有效检测沙门菌毒力岛基因,特异性强,灵敏度高,耗时短;还可用于沙门菌毒力基因的鉴定及分子流行病学调查。     

猪环曲病毒实时荧光定量RT-PCR检测方法的建立与应用

根据GenBank中猪环曲病毒(porcine torovirus,PToV)N基因的保守序列,设计合成1对针对PToV的特异性引物,建立了基于SYBR GreenⅡ检测PToV的实时荧光定量RT-PCR方法。结果显示,该方法能很好地将PToV与猪伪狂犬病病毒、猪嵴病毒、猪传染性胃肠炎病毒、猪流行性腹泻病毒、A群猪轮状病毒、猪瘟病毒、沙门菌和大肠杆菌区分开来,特异性强。检测PToV的N基因在8.65×101~8.65×108拷贝/μL范围内有很好的线性关系,其扩增相关系数为0.998 9,扩增产物的熔解曲线分析只有1个单特异峰。所建立的实时荧光定量RT-PCR方法组内变异系数为0.29%~1.30%,组间变异系数为0.45%~1.80%,重复性较好。利用该方法对采集自四川部分地区的43份临床样品进行检测,实时荧光定量RTPCR检出13份阳性样品,与常规PCR检测方法的阳性符合率为100%。结果表明建立的实时荧光定量RT-PCR方法特异性较强、灵敏度高,可用于该病的临床检测和流行病学调查。     

鸡烟曲霉菌SYBR GreenⅠ荧光定量PCR检测方法的建立

为建立鸡烟曲霉菌快速检测方法,根据GenBank已发表的benAfum基因序列设计1对特异性引物,建立了检测烟曲霉菌的SYBR GreenⅠ实时荧光定量PCR方法,并用该方法对烟曲霉菌分离株进行了检测。结果显示,该方法灵敏度高,对烟曲霉菌最低检出量为10 copies/L;重复性好,变异系数在0.7%~0.8%之间;特异性强,与黄曲霉、土曲霉、黑曲霉、塔宾曲霉及白色念珠菌等病原菌无交叉反应;该方法操作简单、耗时短,只需2 h即可完成整个试验过程。上述结果表明,该方法可应用于烟曲霉菌感染的快速检测。     

鸡白痢沙门菌等位基因特异性PCR检测方法的建立

根据鸡白痢沙门菌与鸡伤寒沙门菌的rfbS基因在第237和第598位碱基的不同,设计和合成了等位基因特异性PCR引物,建立了快速检测鸡白痢沙门菌的PCR方法,并应用该方法对鸡白痢沙门菌临床分离样品进行了PCR鉴定。结果显示,该PCR方法能够特异性地鉴定鸡白痢沙门菌,检测灵敏度达100 pgDNA。对35个经常规方法鉴定的鸡白痢沙门菌分离株应用等位基因特异性PCR方法进行鉴定,鉴定出33株鸡白痢沙门菌,符合率为94.3%。表明,建立的等位基因特异性PCR方法能够准确而快速地鉴定鸡白痢沙门菌。     

China＇s Rise vs. International Order Evolution

A heated debate over the rise of China has been going on unabated in the international community for nearly two decades. However, with the elapse of time, the initial divergence of views in the mainstream opinion has gradually diminished and the gap in China policy among nations has likewise narrowed down. Instead, reason and a realistic approach have eventually taken center stare.     

The U. S. Policy toward DPRK: Opportunities and Constraints

布什政府上台以来,始终未能阻止朝鲜拥核步伐.朝核试后,美对朝政策有了新的调整,六方会谈得以复会.但美朝双方受各自国家利益和政策目标的限制,在朝鲜半岛无核化问题上的尖锐分歧难以解决,朝核危机仍存在再度升级的可能.     

Marxist Philosophy vs. International Relations Research in China

This article argues for the confirmation of Marxist philosophy as the ideology that guides international relations （IR） studies in China. Justification comes through the following points. According to Marxism, correct theoretical guidance serves as the prerequisite for correct practice. Marxist philosophy embodies, by nature, righteousness, in opposition to political hegemony. Accordingly, practice under its guidance represents fruitful revolutionary realism. Finally, advanced studies of international relations in this country cannot be separated from philosophical thinking. Using these arguments as background, this article specifically advocates materialism, patriotism, and service to the people as the corner-stone of China＇s IR theory building.     

Excavating Concealed Tradeoffs: How Russians Watch the News

Citizens in democracies are expected to make better decisions if they understand policy tradeoffs. However, politicians rarely have incentives to communicate them; citizens are uncomfortable choosing among valued outcomes; and devising a common metric is difficult. It is not surprising that in the United States the environment provides relatively little cuing or priming of tradeoffs in television news. Russian citizens, on the other hand, face a media environment in which tradeoff cuing is intentionally suppressed by owners' agendas, yet viewers detect concealed tradeoffs even in the absence of tradeoff priming and viewpoint diversity. Analysis of discourse among ordinary Russians in 16 focus groups convened in four cities, differentiated by political reform and media market environments, showed that when watching news in which tradeoffs are thoroughly concealed, viewers challenge stories by offering a broad spectrum of uncued tradeoffs. Tradeoffs come from diverse policy domains and represent a range of cognitive strategies, some of which are considerably more abstract than others and link elements of their observations and assumptions (together with what they can extract from the stories) into complex reasoning outcomes.     

The U.S. Global Strategy as Seen from the Debate in the Primary Election

There may be more than one year to go before the 2008 U.S. presidential election, but the primary election of the two parties has already begun ahead of time. Both the Democratic and Republican candidates for the     

Young American China Watchers＇ Views on China

In this paper, the author has made a thorough study over the new generation of American China Watchers. Most of the pages are devoted to a detailed comparison between the old generation and the young generation of American China Watchers regarding to their background, education, and views over China. At the end of the paper, the author also gives some policy suggestions for both the Chinese and U.S. government based on the findings in this study.     

Constitution of the Chinese Associa tion for Interna tiona l Understa nding(Approved by the Plenary Session of the Eleventh Executive Council on April 2, 2014)

正Chapter I General Rules Article 1 The Chinese Association for International Understanding is a national non-governmental organization(NGO)in the People’s Republic of China.Article 2 The official name of the Association is"Zhong Guo Guo Ji Jiao Liu Xie Hui"in Chinese,or"Jiao Liu Xie Hui"for short.Its English translation is"Chinese Association for International Understanding",and abbreviated as"CAFIU".     

Guide to Authors

正Published by China Institute of International Studies,China International Studies is China’s first English-language journal on diplomacy and international politics for formal circulation.The magazine mainly publishes the latest research achievements of Chinese scholars and specialists on China’s diplomacy,international politics and international relations under such columns as China’s Diplomacy,International Relations,International Politics,International Security,Regional Issues and World Economy.     

My Journey to Artistic Peak

正I was born in a doctor’s family in a village in Heze,Shandong Province.Since the ancient times,Heze has been home to many painters and literature masters due to the influence of Confucianism.My grandfather and greatgrandfather were both good at calligraphy,not least to mention their medical skills.Even by now,senior people in our village still praise them for it.Both of them had deceased before I was born.     

Sidelights on Australian and New Zealand's Rotary Club Delegation's Visit to China

正At the invitation of the Chinese Association for International Understanding(CAFIU),a 6-member Australian and New Zealand’s Rotary Club delegation visited China from September 23 to September 27.As a staff member of CAFIU,I had the honor to take part in the reception of the delegation throughout this trip.