Second round of an interlaboratory comparison of SARS-CoV2 molecular detection assays used by 45 veterinary diagnostic laboratories in the United States

The COVID-19 pandemic presents a continued public health challenge. Veterinary diagnostic laboratories in the United States use RT-rtPCR for animal testing, and many laboratories are certified for testing human samples; hence, ensuring that laboratories have sensitive and specific SARS-CoV2 testing methods is a critical component of the pandemic response. In 2020, the FDA Veterinary Laboratory Investigation and Response Network (Vet-LIRN) led an interlaboratory comparison (ILC1) to help laboratories evaluate their existing RT-rtPCR methods for detecting SARS-CoV2. All participating laboratories were able to detect the viral RNA spiked in buffer and PrimeStore molecular transport medium (MTM). With ILC2, Vet-LIRN extended ILC1 by evaluating analytical sensitivity and specificity of the methods used by participating laboratories to detect 3 SARS-CoV2 variants (B.1; B.1.1.7 [Alpha]; B.1.351 [Beta]) at various copy levels. We analyzed 57 sets of results from 45 laboratories qualitatively and quantitatively according to the principles of ISO 16140-2:2016. More than 95% of analysts detected the SARS-CoV2 RNA in MTM at ≥500 copies for all 3 variants. In addition, for nucleocapsid markers N1 and N2, 81% and 92% of the analysts detected ≤20 copies in the assays, respectively. The analytical specificity of the evaluated methods was >99%. Participating laboratories were able to assess their current method performance, identify possible limitations, and recognize method strengths as part of a continuous learning environment to support the critical need for the reliable diagnosis of COVID-19 in potentially infected animals and humans.     

蛋白质组学研究技术及其在果树学中的应用

蛋白质组研究是当今生命科学发展的一个新的增长点,它能阐明基因组所表达的真正执行生命活动的全 部蛋白质的表达规律和生物功能。简要介绍了蛋白质组学产生的科学背景、研究方法和研究内容。蛋白质组学研究 方法主要有双向聚丙烯酰胺凝胶电泳(2D-PAGE)、质谱(Mass-spectrometric)技术、蛋白质芯片(Protein chips)技术、酵母 双杂交系统(Yeast two-hybrid system)、双向高效柱层析和生物信息学等。其应用的范围包括植物群体遗传学、在个体 水平上植物对生物和非生物环境的适应机制、植物的发育和组织器官的分化过程,以及不同亚细胞结构在生理生态 过程中的作用等诸多方面。同时展望了植物蛋白质组学研究前景以及蛋白质组学技术在果树学中的应用前景。     

2-硝基-1-芳乙烯类化合物的设计合成及其对稻瘟病菌的抑制活性

为了筛选得到对稻瘟病菌Magnaporthe grisea具有较高抑菌活性的新型化合物,根据稻瘟病菌中1,3,8-三羟基萘酚还原酶（3HNR）的结构信息,设计合成了系列2-硝基-1-芳乙烯（2a~2e）和2-溴-2-硝基-1-芳乙烯（3a,3b）目标化合物,并测试了其对3HNR和稻瘟病菌的抑制活性,同时运用分子对接方法对目标化合物与3HNR可能的结合模式进行了分析。结果表明:大部分目标化合物对3HNR都有很好的抑制作用（IC503的抑制活性最好,IC50值为0.53 μmol/L。在50 μg/mL下,目标化合物对稻瘟病菌的生长具有不同程度的抑制作用,其中2e、3a和3b的抑制率高于96%;3a和3b的EC50值分别为16.4和11.6 μg/mL。分子对接方法分析结果表明,硝基苯乙烯骨架结构与稻瘟病菌的3HNR活性空腔的氨基酸残基有较好的相互作用,其中化合物3中的溴原子可与3HNR中Tyr223和Tyr178的羟基形成氢键,从而解释了化合物3对3HNR有较好抑制作用的原因。     

猪圆环病毒2型灭活疫苗中病毒抗原含量实时荧光定量PCR检测方法的建立

依据GenBank公布的猪圆环病毒2型Cap基因序列,在保守区域设计特异性引物和TaqMan探针,优化反应体系,建立评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,对方法的特异性、敏感性和重复性进行试验,并验证灭活剂用量和灭活时间对检测结果的影响。结果显示：该方法只对猪圆环病毒2型基因有特异性扩增,其他3种对照病毒基因的扩增结果均为阴性;检测灵敏度达到10^2.0 TCID₅₀/mL,比普通PCR方法高100倍;方法的重复性好,对同一样品进行10次检测,变异系数为2.28%;不同灭活剂用量和灭活时间对结果的影响较小,不会因各厂家使用的灭活剂用量和灭活时间不同,影响疫苗对比实验的公平性。该研究成功建立了一种评价猪圆环病毒2型灭活疫苗中病毒含量的实时荧光定量PCR检测方法,用于猪圆环病毒2型灭活疫苗样品中病毒抗原含量的定量,其结果可以反映不同猪圆环病毒2型灭活疫苗样品中抗原含量差异,为研究猪圆环病毒2型灭活疫苗病毒抗原含量评估方法提供了新的思路。     

Polymorphism of IGFBP2 Gene and Its Association with Meat Quality Traits in Pigs

This study was aimed to analyze the genetic variation of insulin-like growth factor binding protein 2 (IGFBP2) gene effect on meat quality traits in pigs. A pair of primer was designed,which based on DNA sequence of IGFBP2 gene (GenBank accession No.:BV727778). The genetic polymorphism of IGFBP2 gene was detected by PCR-RFLP in Sujiang pig, Jiangquhai pig and Landrace pig, and the association between IGFBP2 gene polymorphism and meat quality traits in three pig breeds was analyzed. The results showed that three genotypes (AA, AB and BB) were detected. The Chi-square test results showed that the genotype distribution of three pig breeds was in Hardy-Weinberg equilibrium (P > 0.05). The polymorphism information content (PIC) were moderately polymorphic in three pig breeds. According to the association analysis of different genotypes, significant difference was found in color value, intramuscular fat content and marbling, the color value (except for Jianquhai pig) and intramuscular fat content of AA genotype were significantly higher than AB and BB genotypes (P < 0.05), the marbling of AA and AB genotypes was significantly higher than BB genotype (P < 0.05). In conclusion,the polymorphism of IGFBP2 gene intron 3 had significant effects on meat quality traits, and was candidate gene for the potential impact on meat quality traits of pigs.     

西藏自治区首次发现螟黄赤眼蜂

为发掘西藏地区本土的赤眼蜂资源,本研究利用灭活米蛾卵卡在西藏林芝嘎玛地区的梨园采集到一个赤眼蜂种群。采用ITS2序列比对鉴定所采集的赤眼蜂种类,并测定该蜂的生物学指标。结果表明：西藏林芝嘎玛地区所诱集的赤眼蜂鉴定为螟黄赤眼蜂Trichogramma chilonis Ishii,羽化率为80%,雌性比为57.14%,耐饥饿时间为1.08 d,后足径节长度为157.6μm。本研究将为发掘西藏地区赤眼蜂资源提供参考,并为利用赤眼蜂在西藏自治区开展生物防治实践奠定基础。     

PCV2 Rep蛋白N-糖基化位点突变对病毒复制的影响

N-糖基化对病毒的感染与增殖均具有重要作用,与PCV2复制相关的Rep蛋白含有三个N-糖基化位点。为了分析PCV2Rep蛋白N-糖基化位点突变对病毒复制的影响,本试验构建了三个双拷贝突变体感染性克隆2M23、2M256、2M286,并成功拯救病毒。通过间接免疫荧光检测病毒的拯救效果,TCID50测定病毒的感染力,荧光定量PCR检测细胞病毒的载量。结果显示,PCV2Rep蛋白的23~25aa、256~258aa N-糖基化位点突变后降低病毒的复制能力,而286~288aa突变后增强病毒的复制能力,为进一步阐明PCV2的复制及致病机制提供参考。     

2014年-2016年广西地区猪繁殖与呼吸综合征病毒Nsp2和ORF5基因的遗传多样性分析

为了解广西地区猪繁殖与呼吸综合征病毒(PRRSV)流行毒株的遗传进化情况,对2014年-2016年来自广西各地的部分PRRSV阳性病料进行Nsp2和ORF5基因的扩增和测序分析.结果获得34个Nsp2基因序列和45个ORF5基因序列,均属于美洲型毒株.Nsp2基因间核苷酸序列的同源性为91.8％~100％,与PRRSV美洲型毒株VR-2332、CH-1a、JXA1及NADC30株核苷酸序列的同源性分别为81.3％~84.3％、88.9％~92.1％、94.3％~99.3％和73.5％~75.1％,而与PRRSV欧洲型毒株LV株核苷酸序列的同源性为51.5％~53.2％.ORF5基因间核苷酸序列的同源性为82.8％~100％,与PRRSV美洲型毒株VR-2332、CH-1a、JXA1及NADC30株核苷酸序列的同源性分别为83.7％~99.5％、85％~95％、83.8％~99.7％和83.2％~86.4％,而与PRRSV欧洲型毒株LV株核苷酸序列的同源性为62.4％~64.5％.基于Nsp2和ORF5基因推导的氨基酸序列绘制的遗传进化树中,广西地区的毒株主要分布在以JXA1为代表的Ⅳ亚群.表明当前广西PRRSV流行毒株以JXA1株为代表的高致病性美洲型毒株为主,各毒株Nsp2和ORF5基因序列存在一定的差异,尚未发现欧洲型毒株和美洲型NADC30类毒株.     

Effect of miR-181b on cell proliferation and expression of Bcl-2 and SP1 in HepG2 cells

AIM: To investigate the role of miR-181b in the expression of Bcl-2 and SP1 at mRNA and protein levels in the human hepatoma G2 cells (HepG2), and to explore the effect of miR-181b on the regulation of HepG2 cell proliferation. METHODS: The synthetic double-strand complementary DNA based on the sequence of miR-181b was inserted into the vector of miRNASelect^TM pEGP-miR. The microRNA high-expression plasmid was cloned, and the sequences were identified. The miR-181b plasmid was transfected into HepG2 cells with liposomes. The stable cell line was screened by puromycin. The mRNA and protein levels of Bcl-2 and SP1 were measured by RT-PCR and Western blotting, respectively. Methyl thiazolyl tetrazolium (MTT) method was used to analyze the proliferation of HepG2 cells. RESULTS: The Western blotting results showed that miR-181b inhibited the protein expression of Bcl-2 and SP1. The result of RT-PCR also indicated that the mRNA expression of Bcl-2 and SP1 was suppressed. Compared with the control, the growth rate of HepG2 with high expression of miR-181b was significantly decreased.CONCLUSION: miR-181b inhibits the proliferation of HepG2, which may be related to the down-regulation of Bcl-2 and SP1.     

Greenhouse gas emissions in response to straw incorporation,water management and their interaction in a paddy field in subtropical central China

A field experiment was conducted to study the effects of combination of straw incorporation and water management on fluxes of CH₄, N₂O and soil heterotrophic respiration (Rh) in a paddy field in subtropical central China by using a static opaque chamber/gas chromatography method. Four treatments were set up: two rice straw incorporation rates at 0 (S1) and 6 (S2) t ha^?1 combined with two water managements of intermittent irrigation (W1, with mid-season drainage) and continuous flooding (W2, without mid-season drainage). The cumulative seasonal CH₄ emissions for the treatments of S1W2, S2W1 and S2W2 increased significantly by 1.84, 5.47 and 6.63 times, respectively, while seasonal N₂O emissions decreased by 0.67, 0.29 and 1.21 times, respectively, as compared to S1W1 treatment. The significant increase in the cumulative Rh for the treatments S1W1, S2W1 and S2W2 were 0.54, 1.35 and 0.52 times, respectively, in comparison with S1W2. On a seasonal basis, both the CO₂-equivalents (CO₂e) and yield-scaled CO₂e (GHGI) of CH₄ and N₂O emissions increased with straw incorporation and continuous flooding, following the order: S2W2>S2W1>S1W2>S1W1. Thus, the practices of in season straw incorporation should be discouraged, while mid-season drainage is recommended in paddy rice production from a point view of reducing greenhouse gas emissions.