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.     

Microbial community dynamics of a blue-veined raw milk cheese from the United Kingdom

A commercial blue-veined cheese made from unpasteurized milk was examined by conventional culturing and PCR denaturing gradient gel electrophoresis analysis of the bacterial community 16S rRNA genes using 3 primer sets, V3, V4V5, and V6V8. Genomic DNA for amplification was extracted directly from raw milk, starter culture, cheese at different stages of production, fully ripened cheese, and from the cultured cells grown on various media. The outer rind was sampled separately from the inner white core and blue veins. A diverse microbiota containing Lactococcus lactis ssp. lactis, Lactobacillus plantarum, Lactobacillus curvatus, Staphylococcus gallinarum, Staphylococcus devriesei, Microbacterium sp., Sphingobacterium sp., Mycetocola sp., Brevundimonas sp., Enterococcus faecalis, Proteus sp., and Kocuria sp. was detected in the raw milk using culturing methods, but only Lactococcus lactis ssp. lactis, Lactobacillus plantarum, and Enterococcus faecalis survived to the final cheese and were detected both in the core and the rind. Using PCR denaturing gradient gel electrophoresis analysis of the cheese process samples, Staphylococcus equorum and Enterococcus durans were found in the rind of prepiercing samples but not in the core and veins; after piercing, these species were found in all parts of the cheese but survived only in the rind when the cheese was fully ripened. Brevibacterium sp., Halomonas sp., Acinetobacter sp., Alkalibacterium sp., and Corynebacterium casei were identified only by PCR denaturing gradient gel electrophoresis and not cultured from the samples. Brevibacterium sp. was initially identified in the cheese postpiercing (core and veins), Halomonas sp. was found in the matured cheese (rind), and Acinetobacter sp., Alkalibacterium sp., and Corynebacterium casei were also found in the prepiercing samples (rind) and then found through the subsequent process stages. The work suggests that in this raw milk cheese, a limited community from the milk survive to the final cheese, with salt addition and handling contributing to the final cheese consortium.     

鲜切果蔬中4种病原微生物多重PCR检测技术

研发可同时检测鲜切果蔬中的单核细胞性李斯特菌、鼠伤寒沙门菌、金黄色葡萄球菌和大肠杆菌O157:H7的多重聚合酶链式反应(polymerase chain reaction,PCR)检测方法。根据单核细胞性李斯特菌inl A基因、鼠伤寒沙门菌inv A基因、大肠杆菌O157:H7 wzy基因、金黄色葡萄球菌nuc基因设计及筛选出4对引物。对多重PCR体系及条件进行优化。该方法对单核细胞性李斯特菌、鼠伤寒沙门菌、金黄色葡萄球菌和大肠杆菌O157:H7的检出限分别为3.5×10~6、1.6×10~5、2.4×10~5、4.8×10~5 CFU/m L。将优化的多重PCR方法对不同接种量富集后验证,结果表明,经过9 h富集后,该方法检出限为1 CFU/m L。该方法在鲜切莴苣、鲜切黄瓜、鲜切木瓜、鲜切哈密瓜中应用同样可扩增出4条目标菌。因此,利用所建立的多重PCR方法对鲜切果蔬中侵染的病原菌检出限可达到1 CFU/g。该方法相较于传统的培养检测方法具有节约大量的劳力、试剂、时间等优点,检测时间也由原来的5~7 d缩短至9~11 h,对于企业或分析检验中心大批量样品的监测具有指导意义。     

多重PCR检测4种常见食源性致病菌

目的 建立一种多重聚合酶链式反应法(multiplex polymerase chain reaction, MPCR)快速检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌的分析方法。方法 选取金黄色葡萄球菌nuc基因、沙门氏菌SipB基因、志贺氏菌ipaH基因、单增李斯特菌inlA基因作为目标基因, 设计4对PCR引物, 建立并优化多重PCR反应体系, 评价该体系的特异性和灵敏度, 并对人工污染的熟肉样品进行检测。结果 构建的多重PCR方法特异性强、灵敏度高, 人工污染熟肉匀浆中4种致病菌的检出限为103 CFU/mL。结论 构建的多重PCR检测方法能够快速、准确、高效地检测肉制品中金黄色葡萄球菌、沙门氏菌、志贺氏菌和单增李斯特氏菌, 为食源性疾病菌的快速检测提供参考依据。     

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。     

3种快速检测沙门氏菌方法的比较分析

目的 比较PCR法、胶体金法和VITEK-2 Compact法检测沙门氏菌的检测效果。方法 使用PCR法、胶体金法和VITEK-2 Compact法和国标法分别对待测样品进行检测, 并将3种快速检测方法的检测结果与国标法检测结果进行对比分析。结果 3种快速检测法的检测结果和国标法检测结果符合率分别为100%、92.9%和100%, PCR法和VITEK-2 Compact法符合率略高于胶体金法, 但3种快速检测方法总检出率与国标法比较差异均无统计学意义(P>0.05), PCR法、胶体金法和VITEK-2 Compact法对35个沙门氏菌阳性样本的检出率分别为100%、85.7%和100%。针对阳性样品, PCR法和VITEK-2 Compact法高于胶体金法, 且差异有统计学意义(P<0.05)。PCR法和VITEK-2 Compact法对沙门氏菌的最低检出限为1×101 CFU/mL, 而胶体金法为1×102 CFU/mL。结论 3种快速检测方法均可作为沙门氏菌国标检测方法的有效补充。     

实时荧光PCR法与环介导等温扩增法检测食品中动物源性成分的比较

目的比较实时荧光PCR(real-time PCR)法及环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测食品中动物源性成分,为食品安全监管部门动物源性成分鉴定提供准确、高效的检测方法。方法采用实时荧光PCR法及LAMP法分别对市售肉及其制品进行牛、猪和鸡源性成分鉴定,并与标签明示的肉源成分比对,以准确性和时效性2个指标对上述2个方法进行评价。结果 Real-time PCR法检出4份样品与标签明示肉源不符,LAMP法检出5份样品与标签明示肉源不符,而16份样品中仅有1份样品2种方法检测结果不同。Real-time PCR法检测用时1.5 h,提取检测用DNA用时1.5 h,总用时3 h;而LAMP法检测用时45 min,提取检测用DNA用时20 min,总用时65 min。结论 LAMP法与Real-time PCR均具有较好的特异性、准确性,但LAMP法耗时短、成本低、操作简单,便于现场快速监督抽检。