Heteroalleles in Common Wheat: Multiple Differences between Allelic Variants of the Gli-B1 Locus

The Gli-B1-encoded γ-gliadins and non-coding γ-gliadin DNA sequences for 15 different alleles of common wheat have been compared using seven tests: electrophoretic mobility (EM) and molecular weight (MW) of the encoded major γ-gliadin, restriction fragment length polymorphism patterns (RFLPs) (three different markers), Gli-B1-γ-gliadin-pseudogene known SNP markers (Single nucleotide polymorphisms) and sequencing the pseudogene GAG56B. It was discovered that encoded γ-gliadins, with contrasting EM, had similar MWs. However, seven allelic variants (designated from I to VII) differed among them in the other six tests: I (alleles Gli-B1i, k, m, o), II (Gli-B1n, q, s), III (Gli-B1b), IV (Gli-B1e, f, g), V (Gli-B1h), VI (Gli-B1d) and VII (Gli-B1a). Allele Gli-B1c (variant VIII) was identical to the alleles from group IV in four of the tests. Some tests might show a fine difference between alleles belonging to the same variant. Our results attest in favor of the independent origin of at least seven variants at the Gli-B1 locus that might originate from deeply diverged genotypes of the donor(s) of the B genome in hexaploid wheat and therefore might be called “heteroallelic”. The donor’s particularities at the Gli-B1 locus might be conserved since that time and decisively contribute to the current high genetic diversity of common wheat.     

A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.     

水产品及其制品中大西洋鲑鱼实时荧光PCR检测方法建立

目的:三文鱼营养丰富,含有ω-3不饱和脂肪酸,深受大众喜爱,但存在产品标签标示错误、掺伪掺假、以次充好的现象。目前采用检测标准SN/T 3589.7—2013《出口食品中常见鱼类及其制品的鉴伪方法第七部》对鲑鱼成分检测,无法区分大西洋鲑鱼和其他种类的三文鱼。因此有必要建立一种用于区分上述几种三文鱼的快速检测方法。方法:研究建立了针对大西洋鲑鱼成分鉴定的实时荧光PCR法,并对该方法的特异性和检出限进行了验证。结果:该方法具有良好的特异性,检出限为1%(w/w)。结论:可应用于大西洋鲑鱼的日常检测,为监管部门提供技术支持。     

Application of the novel Droplet digital PCR technology for identification of meat species

The authenticity and traceability of meat products are issues of primary importance to ensure food safety. Unfortunately, food adulteration (e.g. the addition of inexpensive cuts to minced meat products) and mislabelling (e.g. the inclusion of meat from species other than those declared) happens frequently worldwide. The aim of this study was to apply a droplet digital PCR assay for the detection and quantification (copies μL⁻¹) of the beef, pork, horse, sheep, chicken and turkey in meat products. The analysis conducted on commercial meat showed the presence of traces of DNA from other animal species than those declared. We show that the method is highly sensitive, specific and accurate (accuracy = 100%). This method could be adopted by competent food safety authorities to verify compliance with the labelling of meat products and to ensure quality and safety throughout the meat supply chain, from primary production to consumption.     

PCR-DGGE技术检测速冻饺子馅料中微生物区系

以速冻饺子为研究对象,对速冻饺子冻前的微生物多样性进行研究分析,为速冻饺子馅料的存放期限及防腐技术提供参考,避免后期出现卫生问题。利用传统分离培养的方法,对饺子肉馅中的细菌进行分离,得到30株纯菌株。通过菌株形态学鉴定、基因组DNA提取、聚合酶链式反应(polymerase chain reaction,PCR)扩增及16S rDNA测序及序列比对分析来鉴定菌株的属种、构建种群结构进化树。结果表明:30株纯菌株中有14个属,22个种,优势菌分布在γ-变形菌纲中的假单胞菌属。     

SN/T 2980-2011在动物源性成分检测中的适用性分析

目的 探讨行业标准SN/T 2980-2011《动物产品中牛、山羊和绵羊源性成分三重实时荧光PCR检测方法》在动物源性成分检测中的适用性。方法 基于中华人民共和国出入境检验检疫行业标准SN/T 2980-2011和国家标准GB/T 25165-2010《明胶中牛、羊、猪源性成分的定性检测方法实时荧光PCR法》对鲜肉组织及加工肉制品进行动物源性成分检测实验, 建立依托上述标准的动物源性成分检测能力。结果 SN/T 2980-2011中牛源性成分检测特异性不佳, 山羊源性成分检测特异性良好, 绵羊探针仅适用于单通道实时荧光PCR检测; GB/T 25165-2010中的牛、羊源性成分检测均展示出良好的特异性。结论 建议SN/T 2980-2011行业标准的起草单位及起草人重新设计适用于三重实时荧光PCR的牛、绵羊引物及探针,既要保证引物和探针的特异性也要保证适用性。     

产志贺毒素大肠杆菌O26多重双启动寡核苷酸引物-PCR检测方法的建立

为建立一种三重DPO-PCR方法用于食品样品中的产志贺毒素大肠杆菌O26的检测。以志贺毒素stx1和stx2、O抗原基因wzx O26特异性和实际检测效果,设计引物,构建三重DPO-PCR反应体系,进行特异性、灵敏度、模拟样品验证和实际样品验证。结果表明,三对DPO引物对退火温度不敏感,在49~69℃之间均能发生扩增,且引物之间干扰较小,具有较高的特异性,除目的基因外非目标细菌均无扩增条带出现,纯菌灵敏度检测表明,三重DPO-PCR方法对O26的最低检测限为3.8×10^3 cfu/g。在模拟样品和实际样品中具有良好的检测效果。本研究基于DPO引物构建的三重DPO-PCR方法具有效率高,特异性强,不受退火温度限制等优点,可用于食品样品中产志贺毒素大肠杆菌O26的快速准确检测提供一种高效的辅助检测方法。     

大西洋三文鱼和虹鳟鱼双重PCR检测方法的建立及应用

为了实现大西洋三文鱼和虹鳟鱼的物种掺假快速检测，本研究建立了一种基于双重PCR（普通和荧光）的物种鉴定方法。通过序列分析，分别基于线粒体COⅠ和Cyt b基因设计大西洋三文鱼和虹鳟鱼特异性引物，验证引物的特异性并对双重PCR反应体系进行优化，建立大西洋三文鱼和虹鳟鱼的双重PCR快速鉴定方法。利用本研究设计的引物，仅大西洋三文鱼和虹鳟鱼可以分别扩增出108 bp和207 bp的特异性条带，而其他23种非目标物种均未有扩增条带。经验证，在双重PCR反应体系中，大西洋三文鱼和虹鳟鱼引物的最佳添加量分别为0.1和0.4 μmol/L，熔解温度分别约为81.5 ℃和85 ℃。利用该方法从29份市售“三文鱼”样品中检测到6份大西洋三文鱼和3份虹鳟鱼，且与DNA条形码比对结果一致。本研究建立的大西洋三文鱼和虹鳟鱼双重PCR（普通和荧光）鉴定方法具有良好的特异性和适用性，为大西洋三文鱼和虹鳟鱼物种鉴定提供了可靠的技术手段。     

食品中马源性成分定性检测能力验证结果与分析

摘要：目的提高实验室对食品中马源性成分定性鉴定的准确性和评估检测人员的专业技术水平，增强实验室竞争力。方法 通过核酸蛋白仪器分析法和单重实时荧光PCR法对样品真核生物18S rRNA基因扩增的循环阈值来分析DNA的提取质量。在利用标准SN/T 3730.5-2013《食品及饲料中常见畜类品种的鉴定方法第5部分：马成分检测实时荧光PCR法》检测过程中，对该标准扩增体系的引物探针浓度进行优化和检出限分析后，用优化的扩增体系对样品进行检测。再依据标准SB/T 10923-2012《肉与肉制品中动物源性成分的测定实时荧光PCR法》的要求，对样品进行检测。最后对2个标准的检测结果进行比对。结果 提取的DNA质量符合标准要求。标准SN/T 3730.5-2013的引物探针浓度优化后，能有效扩增阳性样品，检出限为0.1%，能有效检测马源性成分。2个标准检测20-M805和20-Q719待测样品，均未检出马源性成分。结论 本次能力验证获得满意评价。DNA提取质量的评估，反应体系中引物探针浓度的优化与选择，不同检测方法结果的相互验证和实验的质量控制都是影响能力验证结果的重要因素