创伤弧菌的优化培养及快速检测

研究了创伤弧菌优化培养的方案,用以提高该菌的检出率。利用Design-Expert软件中的Box-Behnken中心组合实验原理,设计一组3因素3水平实验,通过响应曲面分析,得到优化培养参数为:含盐量3.65%,pH6.75,培养温度37.00℃,在该条件下培养液的OD595nm为0.520。利用创伤弧菌优化培养条件对市售海产样品进行了培养,并通过聚合酶链式反应（Polymerase Chain Reaction,PCR）对样品进行快速检测,且该菌的检出率高达23.3%。结果表明:应用本实验的优化培养方案、PCR法能快速有效检测水产品中存在的创伤弧菌。      

不同原料组成对黏豆包酸面团的真菌菌群及流变学特性的影响

为探究原料组成对东北地区黏豆包酸面团中的真菌菌群构成及流变学特性的影响,收集东北地区手工制作的黏豆包酸面团16份并依据原料组成不同分为A(江米+大米)、B(江米+黏玉米)、C(黏玉米、黄米和大米)和D(黄米+黏玉米)四组,采用高通量测序技术结合聚合酶链式反应-变性梯度凝胶电泳技术(PCR-DGGE)研究黏豆包酸面团中真菌菌群构成,并测定酸面团的流变学特性、pH和总滴定酸度(TTA)。结果显示:在属水平上,A和C组检出的菌属除共有菌属[微囊藻属(Cystofilobasidium)、隐球酵母菌(Cryptococcus)、德巴利氏酵母属(Debaryomyces)和丝孢酵母属(Trichosporon)]外,A组中还发现曲霉属(Aspergillus)、青霉属(Penicillium)、附球菌属(Epicoccum)、威克汉姆酵母属(Wickerhamomyces)和根霉属(Rhizopus),而C组中还发现汉逊氏酵母属(Hanseniaspora)。B组有隐球酵母属和德巴利氏酵母属;D组中仅含有汉逊氏酵母属。汉逊氏德巴利酵母(Dabaryomyces hansenii)为优势酵母。此外,添加玉米或黄米能提高酸面团的黏性和弹性。生酸面团的弹性模量、复合黏度与拟威尔酵母(Cyberlindnera)有显著正相关性(P<0.05),拟威尔酵母的发酵作用能增强酸面团的弹性。生酸面团的损耗因子与汉逊氏酵母属呈显著正相关(P<0.05),与隐球酵母菌及Naumovozyma呈显著负相关(P<0.05),即真菌可影响生酸面团的流体性质和固体性质;丝孢酵母属等真菌能加强熟酸面团的流体性质。pH与掷孢酵母属、汉纳酵母属和亚球壳属等真菌之间呈显著正相关(P<0.05),TTA值与微囊藻属、隐球酵母菌和青霉属等8个菌属呈显著负相关性(P<0.05),说明pH低和总酸度高的酸性环境抑制掷孢酵母属和微囊藻属等真菌的生长。因此,原料组成对黏豆包酸面团中真菌菌群构成有影响(江米和大米混合制备酸面团中酵母菌多样性最丰富),对酸面团的流变学特性也有一定的影响,且流变学特性、pH和TTA与酸面团中真菌有相关性。     

水产品中创伤弧菌ddPCR定量方法的建立

选取创伤弧菌单拷贝基因met为靶基因,设计引物探针,建立对创伤弧菌准确定量的微滴式数字聚合酶链式反应(droplet digital polymerase chain reaction,ddPCR)方法,并进行特异性、灵敏度和重复性实验,同时与实时PCR(real-time PCR)方法进行比较。结果显示,所建立的ddPCR方法可以快速、高效地检测出创伤弧菌,细菌纯培养物中其定量限可达323拷贝数/mL,检测限可达61拷贝数/mL,人工污染牡蛎样品中能最低能检测到1.13×102拷贝数/g的目标菌。对人工污染样品中目标菌的检测,ddPCR的定量结果约为平板计数结果的1.4倍,比real-time PCR方法的检测更加稳定准确。本研究建立的ddPCR检测方法能快速准确、特异、灵敏地定量检测创伤弧菌。     

多重PCR技术在食源性致病菌检测中应用的研究进展

食源性致病菌的多重PCR检测技术是指能够同时扩增得到同种或多种致病菌的不同基因片段的技术。因该技术特异、灵敏且分析效率高,现已被广泛应用于食源性致病菌的检测工作中。本文介绍了多重PCR在食源性致病菌检测中应用的研究近况,并对影响因素及存在的问题进行了阐述。      

State-of-the-Art Molecular Dynamics Simulation Studies of RNA-Dependent RNA Polymerase of SARS-CoV-2

Molecular dynamics (MD) simulations are powerful theoretical methods that can reveal biomolecular properties, such as structure, fluctuations, and ligand binding, at the level of atomic detail. In this review article, recent MD simulation studies on these biomolecular properties of the RNA-dependent RNA polymerase (RdRp), which is a multidomain protein, of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are presented. Although the tertiary structures of RdRps in SARS-CoV-2 and SARS-CoV are almost identical, the RNA synthesis activity of RdRp of SARS-CoV is higher than SARS-CoV-2. Recent MD simulations observed a difference in the dynamic properties of the two RdRps, which may cause activity differences. RdRp is also a drug target for Coronavirus disease 2019 (COVID-19). Nucleotide analogs, such as remdesivir and favipiravir, are considered to be taken up by RdRp and inhibit RNA replication. Recent MD simulations revealed the recognition mechanism of RdRp for these drug molecules and adenosine triphosphate (ATP). The ligand-recognition ability of RdRp decreases in the order of remdesivir, favipiravir, and ATP. As a typical recognition process, it was found that several lysine residues of RdRp transfer these ligand molecules to the binding site such as a “bucket brigade.” This finding will contribute to understanding the mechanism of the efficient ligand recognition by RdRp. In addition, various simulation studies on the complexes of SARS-CoV-2 RdRp with several nucleotide analogs are reviewed, and the molecular mechanisms by which these compounds inhibit the function of RdRp are discussed. The simulation studies presented in this review will provide useful insights into how nucleotide analogs are recognized by RdRp and inhibit the RNA replication.     

沙门氏致病菌标准阳性模板的构建及实时荧光定量聚合酶链式反应检测

目的:构建重组质粒作为标准阳性模板,建立食品中沙门氏致病菌实时荧光定量聚合酶链式反应检测方法。方法:以致病性沙门氏菌inv A基因上特异性片段为目标,设计并合成引物和Taq Man探针,将目标片段连接到PGM-T载体上构建重组质粒,建立实时荧光定量检测体系,并考察方法的灵敏性、特异性、重复性和准确性。结果:构建出致病性沙门氏菌特异性基因片段的重组质粒,能够作为实时荧光定量聚合酶链式反应检测方法的标准阳性模板,标准曲线方程为Y=-3.151 lg X+42.86(R2=0.999),灵敏度80拷贝反应体系,能特异区分沙门氏菌与类型的细菌,同时,批内和批间的变异系数均小于5%,具有良好的重复性。结论:本方法能够实现对食品中致病性沙门氏菌进行定性定量检测。     

利用可视基因膜芯片技术与实时荧光定量聚合酶链式反应技术分析肉制品中动物源性成分

为调查了解肉制品中动物源性成分,以帮助判别掺假情况,应用可视基因膜芯片检测技术对市售的肉松、香肠、肉卷、预制调理肉、肉干及肉脯等23份样品动物源性成分进行筛查分析,同时,针对筛查结果采用实时荧光定量聚合酶链式反应（polymerase chain reaction,PCR）法进一步确证。结果表明：可视基因膜芯片检测法与实时荧光定量PCR法检测结果一致,提高了未知样品的筛查效率;在本次随机分析的样品中,动物源性成分检测结果与标签标示不一致的情况占比高达21.7%,肉制品掺假虚标情况不容忽视。     

Cover Picture: Chemical Restriction: Strand Cleavage by Ammonia Treatment at 8‐Oxoguanine Yields Biologically Active DNA (ChemBioChem 7/2003)

The cover picture shows a chemical restriction method in which DNA double strands are cleaved at the position of the artificial base 8‐oxoguanine (G^oxo). As as result of the mild cleavage conditions, the DNA fragments remain biologically active and can be used in gene cloning experiments. As an example, the lac Z′ gene was amplified by PCR with 8‐oxoguanine‐modified primers, restricted by treatment with ammonia, ligated into a plasmid vector, transformed in Escherichia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure, and allows the design of any 3′ overhang, independent of the sequence of the cloned DNA. Further information can be found in the article by Giese et. al. on pp. 610–614