SYBR Green I 结合碳纳米管对金黄色葡萄球菌的快速检测

【目的】建立一种快速、准确检测金黄色葡萄球菌tst 基因的方法,为预防和控制金黄色葡萄球菌感染提供检测手段。【方法】采用一段特异识别金黄色葡萄球菌的DNA探针来识别基因组DNA,通过识别荧光染料SYBR Green I的信号来实现检测。通过对此方法的可行性、单壁碳纳米管最适浓度、目标链最适浓度、特异性等进行验证,建立产TSST-1金黄色葡萄球菌tst 基因的检测体系。【结果】在仅加入终浓度为1.07 nmol/L目标DNA的条件下,荧光值的恢复率达91%,荧光值的增强值为4.764。最适碳纳米管浓度为30 μg/mL,最适目标DNA浓度与探针DNA浓度相等,为1.07 nmol/L。特异性检测结果表明,该检测方法可以区分具有高相似性的序列（2-或12-nt的区别）。【结论】SYBR Green I结合碳纳米管适用于环境、食品及临床检验中对携带tst 基因的金黄色葡萄球菌的快速检测。     

4种报春苣苔属植物叶插繁殖技术研究

以河池报春苣苔(Primulina hochiensis)、线叶报春苣苔(P.linearifolia)、永福报春苣苔(P.yungfuensis)、尖萼报春苣苔(P.pungentisepala)为研究对象,采用多因素完全随机区组试验设计,探讨3种扦插方式和3种扦插基质对4种报春苣苔属植物扦插生根的影响。结果表明,4种报春苣苔属植物在珍珠岩:草炭=1∶1(V/V)基质中的扦插效果最好,形成的子株数最多(19.67~29.67),不定根发生时间最短(28.33~41 d),不定芽发生时间最快(63.67~79.67 d);河池报春苣苔以全叶插方式繁殖效果最好,线叶报春苣苔、永福报春苣苔、尖萼报春苣苔以半叶插方式繁殖效果最好。     

甜菜碱对热应激肉鸡生长性能、十二指肠消化酶活性及盲肠微生物区系的影响

本试验通过在饲粮中添加不同剂量的甜菜碱研究其对热应激肉鸡生长性能、十二指肠消化酶活性及盲肠微生物区系的影响。选取270只14日龄爱拔益加(AA)肉鸡随机分成6组:常温组、热应激组,常温+0.1%甜菜碱组、热应激+0.1%甜菜碱组、热应激+0.2%甜菜碱、热应激+0.4%甜菜碱组。热应激肉鸡饲养在(32±1)℃的高温环境中。每组3个重复,每个重复15只。预试期7 d,正试期21 d。结果表明:1)饲粮添加甜菜碱可极显著增加28~42日龄热应激肉鸡的体重(P<0.01),极显著降低22~35日龄肉鸡料重比(P<0.01);2)除添加0.1%和0.2%甜菜碱饲粮对28日龄热应激肉鸡胰蛋白酶活性无显著影响(P>0.05)和添加0.1%甜菜碱对35日龄热应激肉鸡淀粉酶活性无显著影响(P>0.05)外,不同浓度的甜菜碱均能显著或极显著增加28、35、42日龄热应激肉鸡十二指肠胰蛋白酶、淀粉酶和胰脂肪酶活性(P<0.05或P<0.01);3)饲粮添加甜菜碱极显著提高了28、35、42日龄热应激肉鸡盲肠内乳酸杆菌的数量(P<0.01),极显著降低了产气荚膜梭菌的数量(P<0.01)。由此可见,热应激状态下,在基础饲粮中添加甜菜碱可改善AA肉鸡生长性能,提高十二指肠消化酶的活性,维持盲肠内微生物区系平衡,从而减轻热应激对肉鸡的危害。     

伪狂犬病病毒在NF-κB家族p65基因敲除细胞系的复制规律研究

试验旨在研究伪狂犬病病毒（PRV）在NF-κB家族p65基因敲除细胞系中的复制规律。利用慢病毒介导的CRISPR/Cas9基因定点修饰技术构建猪肺泡巨噬细胞（3D4/21）p65基因稳定敲除细胞系。通过构建p65-sgRNA重组质粒,转染至HEK293T/17细胞,收取慢病毒,感染3D4/21细胞后利用嘌呤霉素筛选获得多克隆细胞系,T7核酸酶检测敲除效率,再通过有限稀释法获得3D4/21-p65^-/-的稳定细胞系。CCK-8试剂盒检测3D4/21细胞中敲除p65基因后对细胞增殖的影响;流式细胞术检测PRV-GFP感染3D4/21及3D4/21-p65^-/-细胞后病毒增殖的差异;实时定量PCR检测PRV感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、TK基因mRNA表达水平及PRV感染细胞诱导的IL-1β和IL-6基因mRNA水平表达的变化;Western blotting检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后PRV gB、gE蛋白的表达;滴度测定检测PRV-QXX感染3D4/21及3D4/21-p65^-/-细胞后子代病毒滴度。结果表明,sgRNA2和sgRNA3的基因编辑效率较高,对其进行克隆化培养进而获得敲除p65基因的稳定表达细胞系;CCK-8试剂盒检测细胞活力表明,p65基因敲除对细胞活力无影响;流式细胞仪检测表明,同一时间点PRV-GFP在3D4/21-p65^-/-中的增殖显著高于对照细胞;实时荧光定量PCR表明在3D4/21细胞中敲除p65基因促进了PRV gB、TK基因的mRNA表达水平,而抑制了IL-1β、IL-6基因的mRNA表达;Western blotting结果表明,在3D4/21细胞中敲除p65基因促进了PRV gB、gE蛋白的表达;滴度测定结果表明,同一时间点PRV-QXX在3D4/21-p65^-/-细胞中子代病毒的复制显著高于对照细胞。以上结果均表明,p65基因敲除可促进PRV在3D4/21细胞中复制。     

盘江黄牛及其杂交牛育肥中干物质、粗蛋白、综合净能需要研究

将３６ 头盘江黄牛及其杂交牛分成１２ 组,采用不同的日粮,进行两期育肥试验,得到不同的增重水平和实际营养物质摄入量。用各组实际采食的干物质（ ＤＭ） 、粗蛋白（ＣＰ） 、综合净能（ＮＥｍｆ） 与我国试行的《肉牛饲养标准》进行比较。结果表明,除干物质与标准相差较多（ － ６ ．３ ％ ） 外,粗蛋白和综合净能与标准相当接近（ 仅相差－ １ ．５ ％ 和２ ．８ ％ ） ,说明盘江黄牛及其杂交牛在育肥中的营养需要基本与标准相符,在配制日粮时,可以参考我国的《肉牛饲养标准》。     

The use of polymerase chain reaction for determination of virulence factors of Escherichia coli strains isolated from pigs in Poland.

E. coli strains isolated from pigs with postweaning diarrhea or edema disease were tested by phenotypic and genotypic methods for the presence of virulence antigens and genes, respectively. The slide agglutination and ELISA analyses were used for determination of F4, F5, F6, F17, and F41 fimbriae whereas the prevalence of fimbrial fedA and toxin eltI, estI, estII, stx1, stx2 and stx2e genes were recorded by the means of PCR. Only F4 antigen (ac variant) was found in strains of the serogroup O149:K91 isolated from pigs with diarrhea. PCR analyses showed that the fedA gene encoding F18 fimbriae was present in 61.9% of strains isolated from pigs with diarrhea and in 84.2% of strains isolated from pigs with edema disease. The eltI genes encoding heat-labile toxin I (LTI) were present only in 9 out of 21 strains recovered from pigs with diarrhea. Shiga toxin 2 variant (stx2e) genes were found in six isolates from edema disease and also in one strain from diarrhea. The PCR test used in the study was a sensitive and valuable method for determination of virulence factors of E. coli strains.     

载畜率对荒漠草原植物群落影响的研究

对不同载畜率下短花针茅荒漠草原植物群落特征进行比较,结果表明:不同植物种群的盖度对载畜率的响应不同,无芒隐子草盖度在轻度放牧区最大,栉叶蒿盖度在7月份、8月份分别以中度放牧区和轻度放牧区最大;短花针茅、冷蒿、阿氏旋花、细叶葱、木地肤随载畜率增大高度下降明显,猪毛菜高度在四个载畜率下差异均不显著;随着载畜率的增大,植物群落生物量呈下降趋势,且重度放牧与不放牧、轻度放牧有显著差异;随着载畜率的增大,建群种、优势种地位改变不明显,但一年生的猪毛菜重要值增大,在重度放牧区达到最大并占据优势地位;Shannon-wiener指数、Simpson指数、Pielou均匀度指数均以轻度放牧区最大,Shannon-wiener指数、Simpson指数均为轻度放牧>中度放牧>重度放牧>不放牧,且不放牧与轻度放牧差异显著。     

Post-mortem examination and laboratory-based analysis for the diagnosis of bovine tuberculosis among dairy cattle in Ecuador

Veterinary inspection in slaughterhouses allows for the detection of macroscopic lesions reminiscent of bovine tuberculosis, but the presence of Mycobacterium bovis must be confirmed by laboratory methods. This study aimed at comparing the performances of the standard diagnostic tools used to identify M. bovis in tissue specimens sampled from suspicious animals. During a two years period, 1390 cattle were inspected at the Machachi abattoir in the Mejia canton - Ecuador. A total of 33 animals with granulomatous lesions were detected, representing 2.33% (16/687) and 2.42% (17/703) animals examined in 2007 and 2008, respectively. Ninety-four tissue specimens were sampled and screened for the presence of mycobacteria. Acid-fast bacilli were identified in one third of the suspicious cattle (11/33) and suggestive microscopic lesions in 27.3% (9/33) of the samples examined by direct microscopy and histopathology, respectively. Culturing on Stonebrink medium and 16S-rRNA-based polymerase chain reaction (PCR) yielded 36.4% (12/33) and 27.3% (9/33) of positives, respectively. Compared to culture, other diagnostic procedures displayed a lower sensitivity, with 56.5% for PCR, and 43.5% for direct microscopy and histopathology; however, the specificity was higher (94.4% for PCR and microscopy, and 97.2% for histopathology). We conclude that reliable post-mortem laboratory testing either requires the combination of a set of available diagnostic tools or necessitates the development of improved new-generation tools with better sensitivity and specificity characteristics.     

Compatibility of turkey herpesvirus-infectious bursal disease vector vaccine with Marek's disease rispens vaccine injected into day-old pullets

Mixtures of turkey herpesvirus (HVT) and Rispens poultry vaccines have been used worldwide for over 20 yr, mainly for vaccination of future layers and breeders. With increasing virulence of Marek's disease (MD) virus strains, vaccination strategies are evolving toward the use of vaccines combining HVT and Rispens. A single vaccination either in ovo or at 1 day of age with the HVT + infectious bursal disease (IBD) vector vaccine is efficient against IBD. However, with vaccination programs that include a hatchery administration of the HVT + IBD vaccine, additional protection against very virulent and very virulent-plus MD viruses is needed, especially for layers and breeders. This study looked at the combination of four commercially available Rispens vaccines with the HVT + IBD vector vaccine injected at 1 day of age. MD challenge tests that were superior to 90% in relative score in all the groups vaccinated with both vaccines showed that the mixture of HVT + IBD and Rispens vaccines had no effect on clinical protection against MD, and IBD challenge tests showed that the mixture of HVT + IBD and Rispens vaccines had no effect on clinical protection against IBD, which was equal to 100% protection in all the groups vaccinated with both vaccines.