基于qPCR和LAMP技术的马铃薯晚疫病菌快速检测方法

马铃薯晚疫病菌（Phytophthora infestans）能侵染多种茄科植物,它引起的马铃薯晚疫病,是马铃薯生产中的第一大病害。为了开发能在田间快速检测马铃薯晚疫病病原的方法,利用P. infestans T30-4基因组测序数据的contig 1.18131,设计qPCR和LAMP引物,优化扩增条件后得到引物的特异性和灵敏度,最后通过检测田间收获薯块,比较形态学传统方法、qPCR及LAMP的差异。特异性检测结果发现,qPCR和LAMP仅在含有P. infestans DNA模板的体系有阳性扩增,在寄主和其他微生物DNA中均无扩增;在优化的条件下,qPCR和LAMP的检测下限可达1×10 ^-6ng/μL,在有寄主和其他微生物DNA存在的条件下,引物的灵敏度没有显著差异。利用两种快速方法对在大理、丽江及昆明3个地区田间收获薯块上检测发现,qPCR和LAMP方法得到的检出率差异极为不显著（P=0.420）,两种快速检测方法和形态学鉴定方法检出率差异极显著（P=0.009）。在大理、丽江及昆明3个地区的薯块中,两种分子检测方法检出率均比形态学方法高。其中,qPCR检测方法比形态学方法分别提高了12.00%、2.00%、8.70%;LAMP检测方法比形态学方法分别提高了11.30%、2.00%、8.70%。     

Temporal and spatial progress of the diseases caused by the crinivirus tomato chlorosis virus and the begomovirus tomato severe rugose virus in tomatoes in Brazil

Efficient management of whitefly-borne diseases remains a challenge due to the lack of a comprehensive understanding of their epidemiology, particularly of the diseases tomato golden mosaic and tomato yellowing. Here, by monitoring 16 plots in four commercial fields, the temporal and spatial distribution of these two diseases were studied in tomato fields in Brazil. In the experimental plots these diseases were caused by tomato severe rugose virus (ToSRV) and tomato chlorosis virus (ToCV), respectively. The incidence of each virus was similar in the plots within a field but varied greatly among fields. Plants with symptoms for both diseases were randomly distributed in three of four spatial analyses. The curves representing the progress of both diseases were similar and contained small fluctuations, indicating that the spread of both viruses was similar under field conditions. In transmission experiments of ToSRV and ToCV by Bemisia tabaci MEAM1 (former biotype B), these viruses had a similar transmission rate in single or mixed infections. It was then shown that primary and secondary spread of ToCV were not efficiently controlled by insecticide applications. Finally, in a typical monomolecular model of disease progress, simulation of the primary dissemination of ToSRV and ToCV showed that infected plants were predominantly randomly distributed. It is concluded that, although the manner of vector transmission differs between ToSRV (persistent) and ToCV (semipersistent), the main dispersal mechanisms are most probably similar for these two diseases: primary spread is the predominant mechanism, and epidemics of these diseases have been caused by several influxes of viruliferous whiteflies.     

基于土地利用变化的河北省坝上地区景观生态风险评价

[目的]对河北省坝上地区近40 a来的土地利用动态变化和生态风险进行分析评价并对未来趋势作出预测,为该地区生态建设和治理、可持续发展提供科学依据。[方法]基于坝上地区1980—2018年5期土地利用数据以及通过土地转移矩阵、空间相关性分析等方法揭示和预测该区1980—2026年的土地利用变化特征并评估该区生态风险水平。[结果](1)整个研究期间,坝上地区土地利用类型以耕地为主,所占比例近50%,其中,1980—2018年,耕地、林地扩张面积均超过300 km~2,草地减少近616.60 km~2,水域面积缩减36.04%,其中耕地、林地、草地之间的互相流转程度较为剧烈,空间变化上表现为各地类的重心在2000—2010年明显迁移。(2)1980—2026年,坝上地区6个时期内生态风险值全局空间自相关Moran’s I指数均在0.500左右,其空间分布表现出较高的趋同集聚性。(3)近40 a来,坝上生态风险水平升至为高风险级,其区域增加了123.22 km~2,较高风险区域分布在城镇地区,据CA-Markov模型预测,未来坝上地区中等及中等以上风险区域持续扩张,丰宁县和围场县将分别出现小规模高风险区和较高风险区。[结论](1)近40 a来坝上地区草地退化严重,水域面积显著减少,原因系安固里淖干涸所致。(2)该区生态风险水平与土地格局分布具有较强相关性,且在未来会继续升高。     

Comparison of the microbial communities of alpacas and sheep fed diets with three different ratios of corn stalk to concentrate

The objective of this study was to investigate the characteristics of ruminal microbial communities of alpacas (Lama pacos) and sheep (Ovis aries) fed three diets with varying ratios of roughage (corn stalk) to concentrate, 3:7 (LS), 5:5 (MS) and 7:3 (HS). Six alpacas (one-year-old and weighing 29.5 ± 7.1 kg) and six sheep (one-year-old and weighing 27.9 ± 2.7 kg) were used in this study, in a replicated 3 × 3 Latin square experiment. Total protozoa concentration was determined under the microscope; total fungi and methanogens were assessed using quantitative polymerase chain reaction and expressed as a percentage of total bacterial 16S rRNA gene copies; bacterial communities were investigated by targeted 16S rRNA gene (V3–V4 region) sequencing. The percentage of fungi was significantly higher in alpacas than in sheep under the LS diet, while the concentration of protozoa was significantly lower in alpacas under HS, MS and LS diets. The alpha diversity including Shannon, Chao l and ACE indices of bacterial communities was higher in alpacas than in sheep, under the LS diet. A total of 299 genera belonging to 22 phyla were observed in the forestomach of alpaca and sheep, with Bacteroidetes and Firmicutes dominating both animal species. Phyla Armatimonadetes and Fusobacteria, as well as 64 genera, were detected only in alpacas, whereas phyla Acidobacteria and Nitrospira, as well as 44 genera, were found only in sheep. The abundance of cellulolytic bacteria, including Butyrivibrio and Pseudobutyrivibrio, was higher in alpacas than in sheep under all three diets. These differences in the forestomach microbial communities partly explained why alpacas displayed a higher poor-quality roughage digestibility, and a lower methane production. Results also revealed that the adverse effects of high-concentrate diets (70%) were lesser in alpacas than in sheep.     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.     

苗后除草剂喷施时期对杂草防治及冬油菜产量和品质的影响

为了探究苗后除草剂不同施用时期对北方冬油菜田间杂草的防控效果及其对冬油菜生长、产量和品质的影响,以当地主栽甘蓝型冬油菜天油2266和白菜型冬油菜天油H614为试验材料,选用对冬油菜安全的苗后除草剂高效氟吡甲禾灵,设置4个喷施时期处理,于2018-2019年开展大田试验。结果表明,越冬后不同时期喷施苗后除草剂,甘蓝型冬油菜田间杂草总数较对照减少57.54%~92.56%,杂草总鲜重较对照降低75.40%~97.48%,第2时期（3月25日）处理产量较对照增加354.50kg/hm²;白菜型冬油菜田间杂草株防效和鲜重防效分别为55.91%~92.02%和37.17%~97.46%,产量较对照增加2.29%~16.77%。因此,在冬油菜大量返青但未封行前施用苗后除草剂,可显著防控田间杂草并增加冬油菜籽粒产量。     

安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征

猪伪狂犬病是由猪伪狂犬病毒(porcine pseudorabies virus,PRV)引起的一种急性、热性传染病。自2011年底以来,PRV发生了变异,传统的PRV弱毒疫苗已不能对PRV变异株提供完全保护,这给我国PR防控带来了巨大挑战。为了解安徽省PRV流行特征及其主要毒力基因遗传变异情况。利用PCR技术、细胞接种试验、电子显微镜观察、间接免疫荧光试验及兔体接种试验等方法,对安徽省临诊病例中疑似PRV感染的病猪进行病原检测及PRV分离鉴定,并通过设计6对特异性引物对PRV分离株主要毒力基因(gE、gI、TK、gB、gC、gD)进行克隆及测序分析。2016—2018年安徽省临诊病例中共分离鉴定15株PRV;PRV分离株主要毒力基因序列均与2011年后国内PRV变异株同源性较高;与2011年前国内PRV经典株序列比对,PRV分离株gE、gB、gC及gD基因存在多个位点的一致性替换、插入或缺失,且gE、gC基因多位点突变位于其重要的抗原表位区。本研究分离的15株PRV均为变异株,变异株已成为安徽省主要的流行毒株。15株PRV分离株的gI、TK基因序列较为保守,而gE、gC蛋白抗原表位区域氨基酸的突变可能导致其毒力及抗原性发生改变。部分PRV分离株与邻近地区PRV序列同源性均为100%,可能与频繁跨省调运生猪、跨区引种等原因有关。     

抗旱节水小麦分子遗传育种研究进展

干旱缺水等自然灾害会导致小麦产量年度间变幅较大,尤其在小麦主产区黄淮地区更为严重。小麦抗旱节水育种是应对干旱的重大措施。本综述对小麦抗旱节水常规育种、抗旱节水分子遗传育种相关性状QTL定位、抗旱相关功能基因克隆鉴定、转基因等方面的研究进展进行了综述。水旱亲本杂交与异地交叉选择是卓有成效的常规育种方法,通过分子标记鉴定了关于根重、根长、胚芽鞘、高水分利用效率等相关性状的大量QTL;42个抗旱相关基因被克隆并进行基因功能分析和验证,均从不同代谢通路上影响着小麦抗旱性;14个来自不同供体的抗旱相关基因被研究者导入小麦品种后,转基因小麦植株的抗旱能力均得到不同程度的提高,部分植株在产量和其它抗逆性方面也得到提高。以上研究进展为抗旱节水小麦分子设计育种提供了理论依据和发展方向。     

Versuche zur Zyklussynchronisation bei Kühen aus Beständen rnit Fertilitätsproblemen und bei Hybriden zwischen Wisent und Rind mit der PRID-Spirale*

Inhalt: An 81 Versuchs- und 92 Kontrolltieren der Rasse “Potnische Schwarzbunte” aus 3 Problembetrieben wurde ein Versuch zur Sterilitätsprophylaxe rnit der PRID-Spirale durchgeführt. Anhand klinischer Befunde und Milchprogesteronwerten wurde die Untergruppe aus Kühen mit Ovardystrophie (18 Versuchs- und 22 Kontrolltiere) gebildet und extra ausgewertet. Der Behandlungsbeginn lag zwischen 60 und 80 Tage post partum. Die Versuchstiere wurden mit der PRID-Spirale 12 Tage lang behandelt, die Kontrolltiere erhielten Injcktionen rnit Kochsalzlösung und solche rnit Ovardystrophie wurden zusätzlich mit einer Eierstocksmassage behandelt. Die Versuchstiere wurden 56 und 72 Stunden nach Entfernung der Spirale blind besamt. Die Brunstin-duktionsrate betrug insgesamt 90,1%, bei denen rnit Ovardystrophie 77,7%. Die An-wendung der PRID-Spirale führte zur Verbesserung der Fertilitätslage. Die Gesamt-trächtigkeitsrate lag mit 87,6% bei den Versuchskühenüber derjenigen der Kontrollkü-he mit 79,3%. Die Behandlung der Tiere rnit Ovardystrophie erbrachte eine Gesamt-trächtigkeitsrate von 88,8% bei den Versuchs- und 72,7% bei den Kontrolltieren. Die Zwischentragezeit betrug bei den Versuchstieren 101,2 Tage und bei den Kontrolltieren 113,3 Tage (p ≤ 0,05). Bei den azyklischen Tieren lag die Zwischentragezeit bei 104,6 Tage bzw. 134,7 Tage (p ≤ 0,05). In einem anderen Versuch wurde 20 Hybriden zwischen Wisent und Hausrind rwecks Zyklussynchronisation die PRID-Spirale verabreicht. Die Brunstinduktionsrate betrug 66,6%, das Erstbesamungsergebnis nach Doppelbesamung 30%, die Gesamtträchtig-keitsrate nach dem Decken der umrindernden Kreuzungstiere rnit einem Bullen 90%. Die Deckperiode konnte verkürzt werden. Contents: Investigations on the synchronization of estrus cycle in cattle from farms with fertility problems and in cross-breeds between bison and cattle with a PRID-de-vice In 81 experimental and 92 control animals of the “Polish Black and White” breed derived from 3 farms with fertility problems an investigation was carried out for sterility prophylaxis using the PRID-device. Based on clinical findings and milk progesterone values cattle with ovarian dystrophy (18 experimental and 22 control animals) from a special group were used. The begin of treatment was between 60 and 80 days post partum. All experimental animals had received a PRID-device for a duration of 12 days. Control animals were injected with physiological saline solution and animals with ovarian dystrophy were treated additionally with ovarian massage. Experimental animals were inseminated 56 and 72 hrs after removal of the device. All animals taken, estrus was induced in 90.1% of the animals, while only 77.7% of animals with ovarian dystrophy came to estrus. Application of the PRID-device led to an improvement of fertility. Pregnancy rates in experimental animals were 87.6%, while only 79.3% of the control animals were pregnant. Treatment of animals with ovarian dystrophy resulted in 88.8% pregnancies for treated and 72.7% for control animals. Experimental animals were non-pregnant for a duration of 101.2 days, while control animals remained non-pregnant for 113.3 days (p < 0.05). Acyclic cattle had a non-pregnant period of 104.6 days and 134.7 days (p < 0.05) respectively. In a second experiment 20 cross-breeds between bison and domestic cattle were treated with a PRID-device for synchronization of the estrus cycle. The rate of estrus induction was 66.6%, the fertility after double insemination 30%, the total pregnancy rate improved after mating with a bull of animals that had not conceived to 90%.     

Anaplasmosis in Uganda. I. Use of dried blood on filter papers and serum samples for serodiagnosis of anaplasmosis—A comparative study

Summary The suitability of blood collected on filter papers in comparison with corresponding conventional serum samples in the diagnosis of bovine anaplasmosis was studied using the complement fixation test, DOT-ELISA, Western immunoblot and rapid card agglutination test. Dried blood on Whatman filter paper no. 1 was eluted in PBS 0·05% Tween 20 giving an initial dilution of 1∶10. The reactivity of the eluted samples in both DOT-ELISA and Western immunoblotting were similar to those obtained with the corresponding straight serum sample dilutions. Filter paper samples gave lower reactivity in the remaining tests when compared with corresponding serum samples. There was no significant difference in the reactivity between the eluates from filter papers stored at temperatures ranging between 15·5 and 24°C and those kept refrigerated. Storage at 15·5 to 24°C did not significantly affect reactivity for up to six months. Eluates from filter papers stored for six months at 15·5 to 24°C continued to give similar reactivity as those from freshly prepared filter papers in both DOT-ELISA and Western blot, and in the rapid card agglutination test. It is concluded that collecting blood on filter papers is a suitable technique for large scale seroepidemiological studies on anaplasmosis and offers many advantages in developing countries where transport and cold chain facilities are a major constraint.

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Resumen Se estudió la efectividad de muestras de sangre colectadas en papel filtro, en comparación con las correspondientes muestras convencionales du suero, en el diagnóstico de anaplasmosis bovina, utilizando fijación de complemento, DOT-ELISA, Western, immunoblot y la prueba rápida de la tarjeta. La sangre seca en papel Whatman N^o 1 fue removida con PBS 0·05% entre 20, dando una dilución inicial de 1∶10. La reactividad de las muestras removidas de papel filtro, en la prueba de DOT-ELISA y Western immunoblotting, fueron similares a la obtenida con la correspondiente muestra de suero. Las muestras de papel filtro reaccionaron menos en las otras pruebas, cuando se compararon con las correspondients muestras du suero. No hubo diferencia significativa en la reactividad entre los lavados del papel filtro guardados a temperaturas entre 15·5 y 24°C y aquellos guardados en refrigeración. El almacenaje entre 15·5 y 24°C, no afectó la reactividad hastas seis meses. Los lavados de papel filtro guardados por seis meses entre 15·5 y 24°C, dieron la misma reactividad como los lavados frescos, en la prueba DOT-ELISA y Western blot, lo mismo que en la prueba de aglutinación rápida de tarjeta. Se concluye, qu la colección de sangre en papel filtro, es una buena técnica para estudios epidemiológicos de cierta magnitud, sobre anaplasmosis, ofreciendo ventajas considerables en paises en desarrollo en donde las cadenas de frío son deficientes.

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Résumé La fiabilité du sang récolté sur papier filtre comparée à celle des prélèvements conventionnels de sérum pour le diagnostic de l'anaplasmose a été étudiée à l'aide des tests suivants: fixation du complément, ELISA, immunoblot de Western, test rapide d'agglutination sur carte. Du sang séché sur papier filtre Whatman N^o 1 a fait l'object d'une élution dans une solution de PBS à 0,05 p. 100 (Tween 20) pour donner une dilution de base au dilution de base au 1∶10. Le réactivité des échantillons, autant avec le test ELISA que l'immunoblot Western, a été identique à celle obtenue par dilution directe de sérums homologue. Les échantillons sur papier filtre ont donné une réactivité plus faible pour les autres tests, comparée à celle des échantillons de sérum semblables. Aucune différence significative n'a été décelée quant à leur réactivité les éluats provenant de papiers filtres stockés à des températures comprises entre 15,5 et 24°C at ceux conservés au réfrigérateur. Le stockage entre 15,5 et 24°C n'a pas non plus affecté la réactivité de fa?on significative; les éluats conservés à partir des papiers filtres, à cette même température durant 6 mois, ont montré des réactions identiques que ceux provenant de papiers filtres fra?chement préparés, à la fois avec le test ELISA, celui de Western Blot et le test d'agglutination rapide sur carte. On peut conclure que la collecte du sang sur papier filtre est une technique adaptée à l'étude épidémiologique de l'anaplasmose à grande échelle. Elle offre de nombreux avantages dans les pays en développement où offre de nombreux avantages dans les pays en développement où les moyens de transports et la cha?ne du froid constituent des contraintes majeures.