环介导等温扩增技术快速检测肉中沙门氏菌

应用环介导等温扩增技术（loop-mediated isothermal amplification,LAMP）建立了一种快速、高效、灵敏的肉中沙门氏菌的检测方法。针对沙门氏菌的属特异性基因invA设计2对引物,对人工污染肉样以及实际肉样分别进行检测。结果表明：所建立的LAMP反应能够特异性的检测沙门氏菌,对沙门氏菌纯菌的检测灵敏度为普通PCR的100倍,可达到9.8×100CFU/mL。LAMP检测人工污染沙门氏菌肉样的检测限为9.8×101CFU/mL。因此,本实验利用LAMP建立的沙门氏菌检测方法具有快速、灵敏、特异、操作简便的特点,具有广泛发展前景。     

牡蛎单孢子虫环介导等温扩增可视化检测方法的建立

目的:建立基于环介导等温扩增(LAMP)技术的单孢子虫可视化检测方法。方法:根据尼氏单孢子虫的小亚基核糖体RNA保守序列,设计一套特异性LAMP引物,对反应条件如温度和试剂浓度进行优化,建立检测牡蛎单孢子虫的LAMP方法。结果:所建立的方法的敏感性可达1 fg,是常规PCR方法的100倍;全部反应可在1 h内完成;可通过肉眼观察颜色,直接判定结果;对其他牡蛎常见病原体的检测结果均为阴性。结论:建立的LAMP方法简便、快速、灵敏、特异,可用于牡蛎单孢子虫感染的快速检测。     

环介导等温扩增技术快速检测肉中单增李斯特菌

通过建立的环介导恒温扩增（Loop-Mediated Isothermal Amplification,LAMP）方法以达到肉中单增李斯特菌快速、灵敏的检出。以特异性的hlyA毒力基因作为靶基因,与6株非单增李斯特菌进行特异性试验,同时对不同培养浓度的单增李斯特菌进行了LAMP和PCR方法的灵敏度比较,进而应用LAMP法检测人工污染肉中的单增李斯特菌。结果表明：纯培养物中单增李斯特菌LAMP检出限为8.8×100CFU/mL,其灵敏度比普通PCR高100倍;在人工污染肉中单增李斯特菌的检出限为8.8×101CFU/mL,在1h内即可完成扩增反应。LAMP方法具备快速、特异、简单、灵敏度高等优势,在食品基质中单增李斯特菌的检测方面具有较好的应用前景。     

鸭瘟病毒LAMP可视化检测方法的建立

目的:建立一种快速、简便、特异性高的鸭瘟病毒(DPV)环介导等温扩增(LAMP)检测方法。方法:根据Gen Bank中DPVUI6基因的保守序列设计一套特异性引物,并对反应条件进行优化,建立DPV的LAMP可视化检测方法。结果:建立的LAMP方法对其他鸭常见病原体无扩增反应;可通过肉眼观察颜色直接判定结果;敏感性可达0.1fg,是常规PCR方法的100倍;扩增反应只须在常规水浴锅中进行,可在1 h内完成。结论:建立的DPV LAMP方法简便、快速、灵敏、特异,可用于DPV感染的快速检测。     

产肠毒素大肠杆菌快速检测方法的建立和评价

目的利用环介导等温扩增(LAMP)技术,建立产肠毒素大肠杆菌(ETEC)的快速、便捷、敏感、特异的检测方法,并对该方法的特异性和敏感性进行评价,为实验动物检测和细菌性腹泻的诊断提供技术支持。方法根据GenBank公布的产肠毒素大肠杆菌的LT毒素基因序列(S60731.1)设计外引物和内引物进行LAMP扩增,对LAMP特异性和敏感性与PCR方法做比较。结果建立的LAMP方法检测最低浓度为100 pg/μL,灵敏度是PCR的10倍以上并具有较高的特异性,利用该方法对27份猴腹泻样品进行LAMP和PCR方法检测,发现PCR检出率为33.3%,LAMP(60 min内)结果与PCR相同,而LAMP(90 min内)检出率为92.6%,约是PCR检出率的3倍。结论建立了一种用于检测肠毒性大肠杆菌(ETEC)的LAMP检测方法,该方法特异性强,灵敏度高,方便快捷,适合于ETEC临床快速检测。     

O1群霍乱弧菌实时荧光定量PCR快速检测方法的建立

目的:建立针对O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,并进行模拟粪便标本的检测评价。方法:根据O1群霍乱弧菌O抗原编码基因rfb的特异性序列设计引物和TaqMan探针,建立检测O1群霍乱弧菌的实时荧光定量TaqMan PCR快速检测方法,对所建立的方法分别进行实验室内的灵敏度及特异性评价;将O1群霍乱弧菌灭活菌株悬液倍比稀释后与健康成人新鲜粪便混匀,制备成模拟带菌者粪便标本,提取DNA,进行Taq-Man PCR检测,用以评价该方法。结果:建立了快速检测O1群霍乱弧菌的实时荧光定量TaqMan PCR方法,灵敏度为每反应体系104拷贝;该方法对其他14种肠道菌DNA没有扩增;该方法对模拟粪便标本的检测灵敏度为每反应体系102 CFU。结论:建立了一种快速、高效检测O1群霍乱弧菌的荧光定量PCR检测方法,该方法可用于O1群霍乱弧菌临床粪便标本的检测。     

DNA环介导恒温扩增技术快速检测霍乱弧菌

霍乱弧菌是一种重要的食源性致病菌,主要引起急性肠道传染病,其快速检测具有重要意义。根据霍乱弧菌的mdh管家基因序列,设计2对特异性检测引物,利用DNA环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP),经反应体系优化,成功建立了霍乱弧菌的LAMP快速检测方法。该方法最佳反应温度为65℃,60min完成检测,对培养菌的检测限为25CFU/mL,污染食品中霍乱弧菌的检测限为32CFU/g。对33株同种或近源细菌进行LAMP检测,仅霍乱弧菌得到阳性扩增。LAMP方法实践应用结果表明,对1057份虾、蟹、牡蛎、肉类、人腹泻物等样本进行检测,共检出85份阳性,与国际标准(ISO TS21872-1-2007)检测结果的符合率为100%。结果表明,本研究建立的霍乱弧菌LAMP检测方法特异性强、灵敏度高、操作简便,有利于霍乱弧菌疫情的监测。     

LAMP技术在食源性病原微生物检测中的应用

针对常见引起食物中毒细菌的特异保守基因设计环介导恒等温扩增(LAMP)引物,并以此套引物建立食源性病原微生物的检测方法。特异性及灵敏性试验显示,LAMP法最低检出限为10-5,Real-time LAMP最低检出限为10-7,而普通PCR的检出下限仅为10-3;且LAMP全部反应在1 h内完成;LAMP反应结果可通过肉眼直接观测判定结果。LAMP快速检测常见食物中毒菌的方法初步建立,同时结合Real-time LAMP,可提供更准确的检测结果并可实现仪器在线式实时监测。     

环介导等温扩增技术快速检测肉中金黄色葡萄球菌

应用环介导等温扩增（loop-mediated isothermal amplification,LAMP）技术建立了对肉中金黄色葡萄球菌检测的方法。实验中,使用了最新的Bst 20 WarmStart DNA聚合酶完成LAMP扩增反应,并针对金黄色葡萄球菌所特有的保守性耐热核酸酶基因（nuc）设计得到了一套LAMP扩增引物。对LAMP法和PCR法的检测灵敏度进行了比较,同时对人工污染肉中的金黄色葡萄球菌进行检测。结果表明：所建立的LAMP法能够特异性的检测金黄色葡萄球菌,并且检测金黄色葡萄球菌纯菌的灵敏度为201×100CFU/mL,是普通PCR检测灵敏度的100倍。在检测肉中金黄色葡萄球菌时,检测限为201×101CFU/mL。因此,本实验所建立的LAMP法检测肉中金黄色葡萄球菌的方法,具有灵敏、快速以及简便等的优点,是一种具有很好的发展前景的检测手段。     

基于颜色判定的环介导恒温扩增法快速检测副溶血性弧菌

利用DNA环介导恒温核酸扩增法（LAMP）针对副溶血性弧菌特异基因tlh基因设计4条引物,通过引物特异性识别tlh基因上的6个独立区域来快速检测副溶血性弧菌.LAMP反应的过程中会产生白色沉淀焦磷酸镁,故可以通过监测浊度来判定反应结果.实时浊度仪监测反应结果表明,LAMP反应在60～65℃恒温条件下50min内完成;如果在反应前添加羟基萘酚兰（HNB）,蓝色的阳性结果很明显区别于紫色阴性结果;LAMP方法的最低检出限为9.74pg/μL,PCR方法最低检出限为97.4pg/μL,LAMP方法检测灵敏度是PCR方法检测灵敏度的10倍,且具有良好的特异性.LAMP方法用于快速检测副溶血性弧菌具有检测过程简单、实验装置简便、反应结果肉眼可辨别、灵敏度高和特异性强的特点,所以LAMP方法检测副溶血性弧菌特别适合用于现场和基层检疫及医疗单位的快速诊断.     

On the origin of the Hirudinea and the demise of the Oligochaeta

The phylogenetic relationships of the Clitellata were investigated with a data set of published and new complete 18S rRNA gene sequences of 51 species representing 41 families. Sequences were aligned on the basis of a secondary structure model and analysed with maximum parsimony and maximum likelihood. In contrast to the latter method, parsimony did not recover the monophyly of Clitellata. However, a close scrutiny of the data suggested a spurious attraction between some polychaetes and clitellates. As a rule, molecular trees are closely aligned with morphology-based phylogenies. Acanthobdellida and Euhirudinea were reconciled in their traditional Hirudinea clade and were included in the Oligochaeta with the Branchiobdellida via the Lumbriculidae as a possible link between the two assemblages. While the 18S gene yielded a meaningful historical signal for determining relationships within clitellates, the exact position of Hirudinea and Branchiobdellida within oligochaetes remained unresolved. The lack of phylogenetic signal is interpreted as evidence for a rapid radiation of these taxa. The placement of Clitellata within the Polychaeta remained unresolved. The biological reality of polytomies within annelids is suggested and supports the hypothesis of an extremely ancient radiation of polychaetes and emergence of clitellates.     

On the ontogeny of the haptor and the evolution of the Monogenea

Data on the ontogeny of the posterior haptor of monogeneans were obtained from more than 150 publications and summarised. These data were plotted into diagrams showing evolutionary capacity levels based on the theory of a progressive evolution of marginal hooks, anchors and other attachment components of the posterior haptor in the Monogenea (Malmberg, 1986). 5 + 5 unhinged marginal hooks are assumed to be the most primitive monogenean haptoral condition. Thus the diagrams were founded on a 5 + 5 unhinged marginal hook evolutionary capacity level, and the evolutionary capacity levels of anchors and other haptoral attachement components were arranged according to haptoral ontogenetical sequences. In the final plotting diagram data on hosts, type of spermatozoa, oncomiracidial ciliation, sensilla pattern and protonephridial systems were also included. In this way a number of correlations were revealed. Thus, for example, the number of 5 + 5 marginal hooks correlates with the most primitive monogenean type of spermatozoon and with few sensillae, many ciliated cells and a simple protonephridial system in the oncomiracidium. On the basis of the reviewed data it is concluded that the ancient monogeneans with 5 + 5 unhinged marginal hooks were divided into two main lines, one retaining unhinged marginal hooks and the other evolving hinged marginal hooks. Both main lines have recent representatives at different marginal hook evolutionary capacity levels, i.e. monogeneans retaining a haptor with only marginal hooks. For the main line with hinged marginal hooks the name Articulon-choinea n. subclass is proposed. Members with 8 + 8 hinged marginal hooks only are here called Proanchorea n. superord. Monogeneans with unhinged marginal hooks only are here called Ananchorea n. superord. and three new families are erected for its recent members: Anonchohapteridae n. fam., Acolpentronidae n. fam. and Anacanthoridae n. fam. (with 7 + 7, 8 + 8 and 9 + 9 unhinged marginal hooks, respectively). Except for the families of Articulonchoinea (e.g. Acanthocotylidae, Gyrodactylidae, Tetraonchoididae) Bychowsky's (1957) division of the Monogenea into the Oligonchoinea and Polyonchoinea fits the proposed scheme, i.e. monogeneans with unhinged marginal hooks form one old group, the Oligonchoinea, which have 5 + 5 unhinged marginal hooks, and the other group form the Polyonchoinea, which (with the exception of the Hexabothriidae) has a greater number (7 + 7, 8 + 8 or 9 + 9) of unhinged marginal hooks. It is proposed that both these names, Oligonchoinea (sensu mihi) and Polyonchoinea (sensu mihi), will be retained on one side and Articulonchoinea placed on the other side, which reflects the early monogenean evolution. Except for the members of Ananchorea [Polyonchoinea], all members of the Oligonchoinea and Polyonchoinea have anchors, which imply that they are further evolved, i.e. have passed the 5 + 5 marginal hook evolutionary capacity level (Malmberg, 1986). There are two main types of anchors in the Monogenea: haptoral anchors, with anlages appearing in the haptor, and peduncular anchors, with anlages in the peduncle. There are two types of haptoral anchors: peripheral haptoral anchors, ontogenetically the oldest, and central haptoral anchors. Peduncular anchors, in turn, are ontogenetically younger than peripheral haptoral anchors. There may be two pairs of peduncular anchors: medial peduncular anchors, ontogentically the oldest, and lateral peduncular anchors. Only peduncular (not haptoral) anchors have anchor bars. Monogeneans with haptoral anchors are here called Mediohaptanchorea n. superord. and Laterohaptanchorea n. superord. or haptanchoreans. All oligonchoineans and the oldest polyonchoineans are haptanchoreans. Certain members of Calceostomatidae [Polyonchoinea] are the only monogeneans with both (peripheral) haptoral and peduncular anchors (one pair). These monogeneans are here called Mixanchorea n. superord. Polyonchoineans with peduncular anchors and unhinged marginal hooks are here called the Pedunculanchorea n. superord. The most primitive pedunculanchoreans have only one pair of peduncular anchors with an anchor bar, while the most advanced have both medial and lateral peduncular anchors; each pair having an anchor bar. Certain families of the Articulonchoinea, the Anchorea n. superord., also have peduncular anchors (parallel evolution): only one family, the Sundanonchidae n. fam., has both medial and lateral peduncular anchors, each anchor pair with an anchor bar. Evolutionary lines from different monogenean evolutionary capacity levels are discussed and a new system of classification for the Monogenea is proposed.In agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. EditorIn agreeing to publish this article, I recognise that its contents are controversial and contrary to generally accepted views on monogenean systematics and evolution. I have anticipated a reaction to the article by inviting senior workers in the field to comment upon it: their views will be reported in a future issue of this journal. Editor