啤酒污染乳酸菌PCR引物的设计

根据细菌16s rDNA序列的特点，通过对啤酒污染菌16s rDNA序列进行分析，设计合成了针对啤酒污染乳酸菌的引物，在16srDNA基因水平证明了该引物对乳酸菌的特异性，该引物的特异性是PCR检测技术在啤酒厂推广应用的前提，同时反映了16s rDNA在微生物鉴定中所起的重要作用。     

啤酒腐败菌的分离鉴定及对啤酒的危害

从啤酒厂分离到31株啤酒腐败菌。对其进行了AP150CHL试剂条生理生化反应试验。并测定了这3l株菌的16SrDNA部分序列，与Genebank中已知序列进行了比较，并以此为依据进行了系统进化关系分析。结果表明，31株啤酒腐败菌分属于5个种，部分腐败菌对啤酒风味有不良影响。     

啤酒污染乳酸菌PCR引物的设计与验证

根据细菌16SrDNA序列的特点,对啤酒中常见污染乳酸菌16SrDNA序列进行分析,设计合成了针对啤酒污染乳酸菌的特征引物。并用该引物对从啤酒厂分离到的7种乳酸菌进行了检测,PCR结果表明该引物能够准确检测到啤酒中常见的乳酸菌。     

脆肉鲩鱼在冷藏条件下的特定腐败菌分析

利用16S rDNA克隆文库及限制性内切酶片段长度多态性(Restriction Fragment Length Polymorphism,RFLP)技术分析脆肉鲩鱼在冷藏条件下的特定腐败菌。分别提取新鲜鱼肉及腐败的脆肉鲩鱼肉样品中的细菌总DNA并利用通用引物27F/1492R进行PCR扩增,所得PCR产物用于构建克隆文库,阳性克隆子用RFLP进行分析。结果表明:新鲜鱼肉的克隆文库中细菌种类比较丰富,达15种类型,而腐败鱼肉的细菌种类只有7种,且有2种分型的细菌占优势,共占克隆子总数的88.4%。序列比对分析表明,这2种优势菌的16S rDNA序列与假单孢菌属(Pseudomonas)细菌的核苷酸序列的相似性高达99%,说明脆肉鲩鱼在冷藏条件下的特定腐败菌是假单孢菌(Pseudomonassp.)。     

中国东部啤酒厂腐败细菌和野生酵母的检测

本文根据分离的100株啤酒腐败细菌和22株野生酵母,研究了中国东部7家啤酒厂的污染程度评价和质量控制工艺。从每家啤酒厂的五个主要点取样：酿造水和麦汁;发酵液：种酵母;设备、空气和周围环境以及成品啤酒。通过比较分离微生物菌株的16S rDNA,把菌株鉴定到属或种水平,野生酵母鉴定到非酵母属（non-Saccharomyces）和酵母属（Saccharomyces）。在市售脱气lager啤酒中测定微生物生长,表明80％的细菌可引起啤酒污染。中国东部啤酒厂污染情况的研究表明,有害微生物检测对提高这些啤酒厂的卫生质量控制十分重要。     

啤酒废水处理系统中活性污泥微型生物群落结构分析

为了分析啤酒厂废水处理系统活性污泥运行状态,选取了啤酒厂污水处理系统中不同阶段的微型生物群落结构作为研究对象。本文利用16S rDNA及18S rDNA特异性引物作为分子标记,通过PCR扩增污水处理系统中的环境生物群落DNA,以变性梯度凝胶电泳(DGGE)技术分离检测PCR产物获得微生物群落的DNA指纹图谱。研究结果显示,经过活性污泥处理的啤酒厂废水中细菌群落和真核生物群落结构发生了明显的改变。其中在细菌群落DGGE图谱中检测到21条特异性条带,而在真核生物DGGE图谱检测到10条。UPGMA聚类分析显示,进水与活性污泥中的细菌及真核群落结构十分相似(相似性>0.67),而与出水的差异较大(相似性<0.41),这表明了进水对活性污泥生物群落结构的重要影响。     

应用16S rDNA全序列测序技术鉴定生鲜乳中的细菌种类

采用玻璃珠法提取生鲜乳中分离的34株细菌基因组DNA,用细菌通用引物(27f/1492r)PCR扩增16S r DNA片段并测序,与Gen Bank数据库同源序列比对,系统发育树聚类分析,并结合形态学和生理学特征确定了34株菌的种属地位。结果表明,共鉴定出16个种属。3个生鲜乳样中细菌种类及其分布特点不同。其中1号样有7种细菌,以Acinetobacter sp.(不动杆菌属)为主,2号样有6种细菌,以Klebsiella sp.(克雷贝氏杆菌属)为主,3号样有6种细菌,分布较为分散。从致病性来看,分离菌株主要为条件致病菌或腐败菌,无烈性致病菌。     

浓香型白酒窖泥中细菌多样性的免培养技术分析

采用免培养(culture independent)技术直接从浓香型白酒窖泥中提取细菌微生物混合基因组DNA(也叫元基因组DNA),利用细菌16S rDNA通用引物扩增窖泥细菌的序列,根据16S rDNA序列对细菌多样性进行初步分析。采用PCR扩增技术、分子克隆技术以及序列同源性分析等方法测定细菌的16S rDNA,通过与基因数据库中相似菌群序列同源性的比较,得到样品菌种多样性,分别构建系统发育树。结果显示,细菌含有几大类群,表现出高度的细菌多样性。共分为Uncultured bacterium、Clostridium、Lactobacillus、Eubacterium和Syntroph-omonas五个细菌分类。     

低温熏煮香肠中败菌的分离及鉴定

研究低温熏煮香肠中腐败菌相,分离出一株主要腐败菌,命名为NO.IA.利用生理生化检验和16S rDNA序列分析对该菌进行鉴定,结合生理生化检验结果和系统进化树分析结果,确定该菌为枯草芽孢杆菌.     

细菌型豆豉发酵菌株的鉴定

为了明确细菌型豆豉发酵菌株的身份,首先采用形态及生理生化方法进行鉴定,接着提取该菌株的基因组DNA,并根据芽孢杆菌属16S rDNA序列两端的保守性片段设计引物,然后用PCR方法扩增出16S rDNA部分序列,纯化、测序,最后经BLAST搜索进行序列比对和构建系统进化树.结果表明,菌株BBDC3为革兰氏阳性芽孢杆菌,生理生化特征与枯草芽孢杆菌相符;扩增出的16S rDNA部分序列长为1344bp,与序列号为EF488171的枯草芽孢杆菌16SrDNA序列的相似度最高,为99%;系统进化树中,菌株BBDC3与枯草芽孢杆菌AB018487在同一分支.因此,菌株BBI)C3属于枯草芽孢杆菌.     

An improved PCR primer pair based on 16S rDNA for the specific detection of Salmonella serovars in food samples

Salmonella serovars are some of the major bacterial pathogens that can cause sporadic cases and outbreaks of foodborne illness. Based on the sequence data in the V3 region of the 16S rRNA gene, two PCR primer pairs have been designed for the detection of all serovars of Salmonella. However, none of these primers were specific for Salmonella because complete sequence homology with certain non-Salmonella strains has been found within each of them. Thus, the specificities of these two primer pairs could not rely on only one of the two primers. In this study, we modified our previous 16SFI primer by extending one base at the 5' end and three bases at the 3' end. The modified primer, 16S-Sal, was designed with one or more mismatched bases near the 3' end of the primer annealing to the corresponding sequences of non-Salmonella strains. Such modification eliminates interference from Citrobacter freundii and Enterobacter cloacae as occurs with the 16SFI primer. When 16S-Sal and a degenerate primer, 16S-CCR, were used as a primer pair, detection specificity of Salmonella serovars was achieved. Because this primer pair was used for PCR detection of the salmonellae in food samples, such as whole milk and chicken meat, as low as 1 to 9 CFU/g (ml) of the food sample could be detected when a 8-h preculture step was performed prior to the PCR. For chicken meat, the endogenous microflora did not interfere with the PCR results.     

Real-Time PCR Quantification of Protease-Producing Bacteria in Traditional Chinese Fish Sauce

This study aims to isolate, identify, and quantify protease-producing bacteria in traditional Chinese fish sauce. Fifty-five protease-producing bacteria were isolated from 10 fermented fish sauce samples in five growth media based on their morphology and caseinolytic activity. These isolates were identified through their 16S rDNA gene sequences. BLAST analysis of 16S rDNA sequences revealed that 46 and 9 strains belonged to Bacillus sp. and Virgibacillus halodenitrificans, respectively. We used EvaGreen dye in real-time PCR assay to target the partial bacterial 16S rDNA gene sequences of the 55 strains from fish sauce. Two primer pairs (A and B) were designed. Primer pair B was more suitable than primer pair A for real-time PCR assay, in which the optimum annealing temperature was 60 °C. The significance of the developed method was proven by the highly linear characteristic of the standard curve that relates lower threshold cycle (Ct) values and 16S rDNA copy numbers of the standard DNA sample. This method was used to calculate the number of protease-producing bacteria in fish sauce. The minimum level of detection (1.44?×?10³ copies/μL) was also verified. The concentration of protease-producing bacteria in fish sauce was estimated to be (1.47?±?0.73)?×?10³ colony-forming units (cfu)/mL by real-time PCR assay and showed a percentage of positive results correctly assigned of 91.8 % when compared to the plate culture method used as reference. The results may serve as a foundation for future studies on the microbial succession and variation of microorganisms during fish sauce fermentation.     

Species-specific identification of penicillium linked to patulin contamination

Certain species of Penicillium have been reported to produce the mycotoxin patulin, and research was undertaken to identify these with the use of oligonucleotide primer pairs. Species examined were found in food, plants, and soil and were reported to produce patulin. Penicillium expansum is the most commonly detected species linked to the presence of patulin in apple juice. At least 10 different enzymes are involved in the patulin biosynthetic pathway, including the isoepoxydon dehydrogenase (idh) gene. Based on nucleotide sequences previously determined for the idh gene in Penicillium species, PCR primers were designed for the species-specific detection of patulin-producing species. The 5' primers were based on differences in the second intron of the idh gene. To ensure that the primer pairs produced a PCR product restricted to the species for which it was designed, and not to unrelated species, all of the primer pairs were tested against all of the Penicillium species. With one exception, it was possible to detect a reaction only with the organism of interest. The primer pair for Penicillium griseofulvum also amplified DNA from Penicillium dipodomyicola, a closely related species; however, it was possible to distinguish between these two species by doing a second amplification, with a different primer pair specific only for P. dipodomyicola. Consequently, with different primer sets, it was possible to identify individual patulin-producing species of Penicillium.     

Detection method for genetically modified papaya using duplex PCR

A simple and rapid method for the identification of genetically modified (GM) papaya, derived from Line 55-1, was developed by modifying the Japanese official PCR method. Genomic DNA was directly extracted from the fresh fruit without the lyophilization step, using a commercial silica-based kit. To develop a duplex PCR method which simultaneously detects the GM papaya-specific gene and the intrinsic papain gene, the papain 2-5'/3' (amplicon size; 184 bp) primer pair for the detection of the papain gene was newly designed within the region of the products (211 bp) amplified using the papain 1-5'/-3' primer pair adopted in the Japanese official PCR method. To detect the GM papaya-specific gene, the primer pair Nos C-5'/CaM N-3' described in the Japanese official method was used. The DNA sequences of the GM papaya gene and the intrinsic papain gene were co-amplified using the PCR method in a single tube. The developed duplex PCR method allows the simultaneous detection of the products by means of agarose gel electrophoresis or microchip electrophoresis. The proposed method for GM papaya identification is simple and rapid.     

The sequence heterogenicities among 16S rRNA genes of Salmonella serovars and the effects on the specificity of the primers designed

Previously, we have reported a 16S rDNA targeted polymerase chain reaction (PCR) method for the specific detection of Salmonella serovars [J. Appl. Bacteriol. 80 (1996) 659]. The target sites of its primers, i.e. 16SFI and 16SIII, according to the data in GenBank, were found mismatched to the corresponding sequences of some Salmonella serovars, such as those of S. Houten, S. Chingola, S. Bareilly, and S. Weltevreden. Accordingly, a PCR method using a nonspecific primer MINf combined with a primer modified from our 16SFI primer, i.e. the primer MINr, was developed and displayed better detection specificity [Int. J. Food Microbiol. 80 (2003) 67]. In this study, we show the sequence heterogenicity at the primer 16SFI targeting sites for some Salmonella serovars. Thus, the sequence used for designing of PCR primers might be just one of the several possible sequences. Such a situation may lead to the misjudgment on evaluation of the specificity of the primers if this was only based on the data in GenBank. Strains of the above described Salmonella serovars with target sequences from GenBank mismatched to the primer 16SF1 were reidentified and their PCR results were confirmed. Meanwhile, their 16SFI/16SIII primer annealing sites were sequenced and the sequences obtained were found completely and highly homologous to those of 16SFI and complementary to those of 16SIII primer, respectively.     

Investigation of false-positive reactions for CBH351 maize in screening PCR analysis

Examination for CBH351 maize was conducted by the qualitative polymerase chain reaction (PCR) method in maize grain and maize processed foods obtained in the Tokyo area. The numbers of samples possibly positive in the screening test were 7 of 22 (31.8%) for maize grain samples, 4 of 14 (28.6%) for semi-processed foods, 11 of 30 (36.7%) for canned products, 3 of 30 (10.0%) for maize snacks, 3 of 4 (75%) for tacos and 1 of 3 (33.3%) for tortillas. However, CBH351 maize was not detected in the confirmation test. Therefore, the results of the screening test were false-positive. Since the reaction might have been caused by the base sequences of the 3'-end of primers CaM03-5' and CBH02-3' used in the screening test, a new primer pair was designed. The PCR products obtained with the new primer pair TMC2-5'--TMS2-3' were specific for CBH351 and were not obtained with barley, wheat, rice, RRS, Bt11, or Event176. Thus, the new primer pair shows high specificity. CBH351 maize was detected from samples containing at least 0.05% CBH 351 maize DNA by using this primer pair.     

A single nucleotide polymorphism in the sheep kappa-casein coding region

Genetic polymorphisms in CSN3 gene in Pag (Croatia), Sarda (Italy) and Pramenka (Serbia) sheep breeds were investigated. A single nucleotide polymorphism (SNP) was localized by sequence analysis (sequence submitted to GenBank under accession AY237637) relying on an original primer pair. Primers for sequencing (kappa-casF and kappa-casR) were designed on the available CSN3 sequences to amplify the genomic region encoding the major part of the mature protein (exon 4). An SNP was detected at position 237 of the sheep kappa-casein mRNA (reference sequence: GenBank X51822), where a thymine was substituted for a cytosine. The SNP was typed by conventional PCR and SYBR Green I-based real-time PCR. C and T alleles were discriminated using a dedicated set of primers that consisted of one common forward primer (SNP-TC) and two reverse primers (SNP-T and SNP-C), the latter two differing in the 3' end base and in the presence of a 12 bp poly-G tail in SNP-C. The SNP was found in both the heterozygous and the homozygous state in Sarda and Pramenka breeds, and in the heterozygous state only in the Pag breed. The observed allelic frequencies of the SNP were 0.12 in Pag, 0.27 in Sarda and 0.45 in Pramenka.     

用于食源性疾病病原菌诊断的基因芯片研究及评价

目的：建立应用基因芯片结合酪胺信号放大法检测食源性疾病病原菌的方法。方法：针对细菌16S和23S rDNA基因设计各种病原菌诊断的特异性探针和通用引物。被检测的病原菌经过生物素标记的引物进行PCR扩增,然后与制备的基因芯片探针区杂交,再用酪胺-Cy3进行显色。最后通过荧光扫描仪扫描杂交图像。结果：建立的基因芯片检测方法用于诊断沙门菌、志贺菌、副溶血性弧菌、空肠弯曲菌、肠出血性大肠杆菌O157:H7、单增李斯特菌、霍乱弧菌、腊样芽孢杆菌等8种细菌性传染病病原菌。纯培养物的检测灵敏性达到5×102CFU/mL,对20例模拟双盲样本进行检测,结果与预期的完全相符。结论：本方法特异性强、灵敏度高,可以用于传染病防控和临床诊断等多个领域。     

Detection of allergenic substances in foods by a multiplex PCR method

A multiplex PCR (M-PCR) method was developed for the detection of DNAs of plant and three allergenic substances (wheat, buckwheat, and peanut) in foods. Genomic DNAs were extracted from allergenic substances with a commercial ion-exchange type kit. Four primer pairs suitable for the specific detection of plant DNA were designed to establish a M-PCR method detecting simultaneously the specific DNAs of plant and allergenic substances. Our four designed primer pairs and the primer pair described in the Japanese official method were applied to the specific detection of plant DNA. A primer pair of Plant01-5' and Plant01-3' (amplicon size; 161 bp) was the most suitable for the specific detection of plant DNA. M-PCR was performed to detect the specific DNAs of allergenic substances using four primer pairs, a pair of Plant01-5' and Plant01-3', and three pairs for allergenic components described in the Japanese official method. The four specific PCR bands were simultaneously amplified from genomic DNAs of allergenic substances. The proposed method is simple, rapid and inexpensive.