啤酒生产过程中厌氧菌的危害与控制

过去,国内一些啤酒厂普遍不太重视厌氧菌对啤酒的危害,并且很少对啤酒酵母和发酵液以及成品啤酒进行厌氧菌数目的检测,只是基于食品卫生的要求对于啤酒生产过程中的冷麦汁、压缩空气、无菌水、发酵液、成品啤酒进行细菌总数和大肠菌群的检测,其实一些厌氧菌对啤酒有很大的危害,它不仅可以影响啤酒口味的纯正,还可以使啤酒异常发酵,酒液严重混浊、酸败、难以入口。因此,必须加强对啤酒有害菌的控制工作。啤酒有害菌是指厌氧条件下生长,革兰氏阳性,过氧化氢酶阴性的细菌,一般在麦汁发酵初期,由于氧的存在,厌氧菌在此时还不能生成…     

选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV

本研究通过单因素试验和响应面分析试验建立了能够选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV,采用平板计数法及三重荧光PCR技术验证了SVV的增菌效果。结果表明:SVV能同时富集以不同浓度比例混合的3种目标菌,37oC振荡培养18h后,菌体浓度达到105~108CFU/mL;SVV强烈抑制大部分的非目标菌;用荧光PCR方法检测经过37oC振荡培养18h的10份人工接种样品和608份实际样品,结果表明目标菌在SVV中增殖18h后菌量达到检测限以上,SVV联合荧光PCR检测方法的检出率为4.06%,比传统检测方法(3.78%)高,无假阴性和假阳性。SVV可望应用于水产品中沙门氏菌、副溶血弧菌和霍乱弧菌检测前的增菌处理,可简化检测过程,有效克服漏检,提高检出率。     

流感病毒亚单位疫苗中间体中沙门菌快速检测方法的建立

目的：建立一种能够快速、准确地检测流感病毒亚单位疫苗中间体样品中沙门菌污染的方法。方法：首先利用选择性增菌培养基对样品进行增菌培养,然后提取样品中的细菌基因组DNA,通过沙门茵特异性引物对基因组DNA进行PCR扩增,以琼脂糖凝胶电泳实现对目的片段的检测。结果：该PCR反应体系的扩增检测灵敏度可达1pg的沙门菌DNA,利用选择性增菌培养配合该PCR体系可在最快24h内实现对沙门菌的准确检测,测定结果与传统方法相符。结论：此方法应用于流感病毒亚单位疫苗中间体的沙门菌检查,较之传统的培养法结合生化鉴定的方法,大大缩短了检测周期,降低了结果判读的难度,在实际生产中有很好的应用前景。     

添加有扩增内标的沙门氏菌荧光定量PCR 检测体系的建立与评价

【目的】建立添加有扩增内标(IAC,Internal amplification control)的沙门氏菌EvaGreen荧光定量PCR检测体系,提高PCR检测可靠性。【方法】通过比较已有沙门氏菌属细菌的基因组序列,筛选沙门氏菌属特异检测靶点,设计特异引物;再用复合引物法构建扩增内标,优化参数,建立沙门氏菌内标PCR检测体系,利用特异性和灵敏度实验评价体系的检测性能。【结果】筛选得到的新特异靶点基因编码III型分泌系统蛋白(ssaQ)。针对该基因设计特异引物(SsaQ6),建立了添加有扩增内标的常规PCR和EvaGreen荧光定量PCR检测体系;二者对151株沙门氏菌和34株非沙门氏菌的检测符合率均达100%,对基因组DNA的检测下限达14.9拷贝/PCR和2.76拷贝/PCR;人工污染牛奶样品(初始染菌量:4-6 cfu/10 mL),増菌10 h和8 h后分别可检出沙门氏菌。【结论】本研究发掘的新靶点基因ssaQ特异性强,基于这一新靶点建立的添加有扩增内标的EvaGreen荧光定量PCR比常规内标PCR的检测限更低,重复性更好,快速方便,在12 h内即可得出检测结果,并且定量准确,有利于推进沙门氏菌PCR检测方法的标准化应用。     

利用色谱法快速检测分析啤酒腐败菌的新方法

啤酒在生产过程中很容易染菌,而传统的用于啤酒腐败菌检测的方法耗时长,不能满足实际需求.由于腐败菌污染的啤酒通过色谱检测,相应组分(生物胺、有机酸和风味物质)都会有特征峰的产生,所以本研究通过建立无腐败菌污染的啤酒中各组分的标准色谱图,再使啤酒强制染菌,对其组分进行色谱分析,并与标准色谱图进行比较,从而找出各组分对应的特征峰.未来,此方法可用于实际生产线上快速检测啤酒是否发生微生物污染.     

水稻白叶枯病菌和水稻细菌性条斑病菌的实时荧光PCR快速检测鉴定

成功建立了水稻白叶枯菌与水稻细菌性条斑病菌快速检测鉴定的实时荧光PCR方法。根据含铁细胞接受子基因设计两菌的通用引物PSRGF/PSRGR（扩增一个152bpDNA片段）和特异性探针（Baiprobe和Tiaoprobe）,并对13种细菌和1种植原体进行实时荧光PCR。结果表明,两个特异性探针能分别特异性检测到目标病原菌产生荧光信号而其它参考菌不产生荧光信号。检测的绝对灵敏度是30.6fg/μL质粒DNA和103CFU/mL的菌悬浮液,相当于1个细菌细胞的基因,比常规PCR电泳检测高约100倍,相对灵敏度为105CFU/mL。整个检测过程只需2h,完全闭管,降低了污染的机会,无需PCR后处理。 用这两个特异性探针分别对自然感染白叶枯菌和条斑菌的叶片DNA提取液和种子浸泡液进行实时荧光PCR,结果均可特异性检测到目标菌的存在并完全可将两种病原细菌区分开来,且只需03g叶片和10g种子。     

FTA滤膜用于PCR检测肉中的金黄色葡萄球菌

利用FTA滤膜,采用PCR技术可直接检测肉及肉制品中的金黄色葡萄球菌,无需增菌,灵敏度高。在采用浮选与溶剂萃取相结合方法的基础上,使用FTA膜可高效地从鲜肉中提取金黄色葡萄球菌的DNA,消除PCR反应的抑制因子。以金黄色葡萄球菌耐热核酸酶基因（nuc）为靶基因,经过PCR扩增得到279bp的产物。经过DNA测序证实该产物为目的扩增产物。使用FTA滤膜处理样品,再通过PCR方法检测金黄色葡萄球菌,猪肉、牛肉、羊肉、鸡肉匀浆液的检出限均为10cfu/mL,可在6h内完成对肉中金黄色葡萄球菌的检测,比目前普遍采用的先增菌再进行PCR检测的方法缩短了12～24h 。实际检测了72份样品,同时与GB 4789.10-94方法及两种快速检测致病性金黄色葡萄球菌检测方法做比较,PCR方法的检出率为72.2％,检出时间为6h,GB 4789.10-94方法检出率为70.8%,检出时间为5d,Prfilm RSA方法的检出率为61.1％, 检出时间为18h,BairdParker R.P.F方法的检出率为69.4％,检出时间为18h,结果表明FTA滤膜用于PCR检测肉中金黄色葡萄球菌检出率高,耗时短。使用FTA滤膜法制备模板DNA,为食品中的致病菌快速检测构建了一个技术平台。     

沙门菌、志贺菌、副溶血性弧菌多重PCR检测方法的研究

建立快速检测沙门菌、志贺菌和副溶血性弧菌的多重PCR方法[1-4].根据沙门菌hilA基因、志贺菌ipaH基因及副溶血性弧菌TDH基因设计特异性PCR引物[5-6],被检样品经4 h振荡培养后金属浴裂解制备DNA模板,使用全自动毛细管电泳核酸检测系统分析PCR扩增产物.在580、423和245 bp处分别出现预期的特异性DNA条带,且无非特异扩增条带出现.敏感性试验显示沙门菌在模拟标本中的检测灵敏度为101-2cfu/mL、志贺菌为101cfu/mL、副溶血性弧菌为102cfu/mL.该方法操作方便、分析时间短、特异性和灵敏度高,可用于公共卫生突发事件食源性病原菌的快速检测.     

扩增内标在沙门氏菌PCR检测方法中的应用*

在PCR反应体系中添加了一条人工构建的扩增内标片段,以指示沙门氏菌PCR快速检测中出现的假阴性。对9株沙门氏菌和15株非沙门氏菌进行PCR检测,结果显示所有沙门氏菌都能扩增到一条invA基因中的374bp特异性片段,而模板来源于非沙门氏菌时则只能扩增到一条513bp扩增内标片段。灵敏度试验显示,该PCR检测体系对猪霍乱沙门氏菌纯DNA模板的检测灵敏度为12·8fg/μL,如果将增菌时间确定为8h,则该检测体系对人工染菌牛乳中沙门氏菌的检测灵敏度可以达到起始浓度为8cfu/25mL。采用上述方法检测了80份     

鼠伤寒沙门氏菌多重PCR检测方法的研究

分别根据沙门氏菌16S rRNA、质粒毒力基因spvC、致病基因invB、fimA序列设计4对引物,对沙门氏菌株及非沙门氏株菌基因组DNA进行多重PCR检测。结果该方法能检测出6.3×10² 个cfu/ml纯培养的沙门氏菌,人工染菌食品模拟检测结果显示,熟鸡肉初始含菌量为17cfu/g、全脂奶粉为11cfu/g、生牛肉为13.6cfu/g,经过8h增菌,PCR检测为阳性。该体系能鉴定产生多种毒力因子的沙门氏菌,特异性强、敏感性高,为检测和鉴定沙门氏菌株提供了一个新方法。     

Modified multiplex PCR methods for comprehensive detection of Pectinatus and beer-spoilage cocci

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.     

Modified Multiplex PCR Methods for Comprehensive Detection of Pectinatus and Beer-Spoilage Cocci

Specific PCR primers were designed based on the 16S rRNA genes of recently proposed beer-spoilage species, Pectinatus haikarae, Megasphaera sueciensis, and M. paucivorans, and two sets of our previously reported multiplex PCR methods for Pectinatus spp. and beer-spoilage cocci were reconstructed. Each modified multiplex PCR method was found specifically to detect beer-spoilage species of Pectinatus and cocci, including new species.     

Random amplified polymorphic DNA-PCR based cloning of markers to identify the beer-spoilage strains of Lactobacillus brevis, Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis

AIMS: Beer-spoilage ability of lactic acid bacteria such as Lactobacillus brevis is a strain-dependent phenomenon in which the mechanism has not yet been completely clarified. In order to systematically identify genes that contribute to beer-spoilage, large-scale random amplified polymorphic DNA (RAPD)-based cloning methods was carried out. METHODS AND RESULTS: A systematic RAPD polymerase chain reaction (PCR) analysis using 600 primers was performed on beer-spoilage and on nonspoilage strains of L. brevis. Among 600 primers, three were found to amplify a single locus highly specific to beer-spoilage strains. DNA sequencing of this locus revealed a three-part operon encoding a putative glycosyl transferase, membrane protein and teichoic acid glycosylation protein. PCR analysis of typical beer-spoilage lactic acid bacteria suggested that this locus is highly specific to beer-spoilage strains. CONCLUSION: The cloned markers are highly specific to identify the beer-spoilage strains not only in L. brevis but also in Pediococcus damnosus, Lactobacillus collinoides and Lactobacillus coryniformis. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper proves that RAPD-PCR is an efficient method for cloning the strain-specific genes from bacteria. The markers described here is one of the most useful tools to identify the beer-spoilage strains of lactic acid bacteria.     

Genetic characterization and specific detection of beer-spoilage Lactobacillus sp. LA2 and related strains

AIMS: Lactobacillus sp. LA2 (DSM15502) and related strains (LA2 group) possess strong beer-spoilage ability. The 16S rDNA sequence of LA2 strain is virtually indistinguishable from that of L. collinoides, generally considered to be nonbeer-spoilage bacteria. The aim of this study was to identify the genetic marker to distinguish between Lactobacillus sp. LA2 group and L. collinoides and to provide a rapid means of identifying beer-spoilage strains belonging to Lactobacillus sp. LA2 group. METHODS AND RESULTS: The 16-23S rDNA intergenic spacer (ITS) regions of Lactobacillus sp. LA2 and L. collinoides JCM1123T were sequenced to identify a genetic marker to distinguish between the two groups. As a result, 300 and 500 bp ITS regions of Lactobacillus sp. LA2 were found to be almost identical with those of L. collinoides JCM1123T. Sequence comparison analysis between Lactobacillus sp. LA2 and L. collinoides JCM1123T revealed that the two contiguously located nucleotides are absent in both ITS regions of Lactobacillus sp. LA2. Based on the sequence difference, we have designed specific PCR primers with a minor modification to the primer sequence that can differentiate between beer-spoilage Lactobacillus sp. LA2 group and nonbeer-spoilage L. collinoides. CONCLUSIONS: The PCR-based method has been developed to identify Lactobacillus sp. LA2 group, providing a rapid and sensitive means of determining the beer-spoilage ability of detected bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The substitution of one nucleotide, located at the third position to the 3'-end in the primer sequence, enhanced the specificity of the PCR method while retaining sufficient sensitivity. The nucleotide gap identified in this study appeared to serve as a useful genetic marker that can differentiate 12 beer-spoilage Lactobacillus sp. LA2 group strains from its close relatives that exhibit no beer-spoilage ability.     

Characterization of horA and its flanking regions of Pediococcus damnosus ABBC478 and development of more specific and sensitive horA PCR method

AIMS: To characterize horA and its flanking regions of Pediococcus damnosus ABBC478 and, on the basis of this insight, to develop a more specific and sensitive horA PCR method. METHODS AND RESULTS: A plasmid harbouring the homologue of a hop-resistance gene, horA, was sequenced and designated pRH478. The nucleotide sequence and open reading frame structure of horA and its flanking regions of pRH478 were found to be highly similar to those of pRH45, a horA-harbouring plasmid previously identified in Lactobacillus brevis ABBC45. The nucleotide sequence of the horA homologue of P. damnosus ABBC478 was 99.6% identical with that of horA. Based on this insight, new primers specific to horA were designed and compared with the previously reported specific primer pair. As a consequence, it was demonstrated that the new primer pair is superior in specificity and sensitivity. CONCLUSIONS: The newly developed horA PCR method allows more specific and sensitive determination of the beer-spoilage ability of lactic acid bacteria (LAB). SIGNIFICANCE AND IMPACT OF THE STUDY: The nucleotide sequences of the horA homologues were found to be essentially identical among distinct species of LAB, indicating that horA-specific primers can be designed from almost any region of the horA gene.     

Application of Rep-PCR fingerprint in rapid identification of beer-spoilage bacteria

The application potential of rep-PCR in typing beer-spoilage isolates was studied. The effects of different factors, including DNA templates and primers, on the quality and reproducibility of fingerprints were investigated. The CATB protocol was shown to be the feasible method for DNA extraction. Primers BOXA1R and (GTG)₅ were used in rep-PCR, and the PCR products were sequenced to identify strains isolated from two breweries. Rep-PCR fingerprint profiles were obtained using GelCompar II software. Cluster analysis showed that the isolates belonging to Lactobacillus brevis, L. buchneri, L. casei/paracasei, and L. plantarum are divided into 2 or 3 subgroups. In addition, the two rep-PCR fingerprint profiles complemented each other in typing these isolates. By combining the similarity coefficient cut-off (SCC) of species, 9 unknown isolates were rapidly identified using both fingerprint databases. The results indicate that rep-PCR is a simple, reliable, and promising method for the rapid identification of beer-spoilage bacteria.     

脂蛋白脂酶基因的克隆、序列测定及定点突变

 以人的脂肪组织总RNA为模板 ,参考已报道的脂蛋白脂酶 (lipoproteinlipase ,LPL)cDNA设计引物 ,利用RT PCR方法扩增得到了LPLcDNA ,并经序列测定证实其序列是正确的 .在冠心病患者LPL基因第 5外显子的 830位碱基处发现了G→A的转换 ,该变异导致LPL基因第 192位的密码子CGA被CAA取代 ,使LPL第 192位精氨酸改变为谷氨酰胺 .在变异碱基附近设计合成两条引物 ,其中一条包含所要改变的碱基 ,利用基于PCR的定点突变技术和体外重组的方法获得了G830A变异的LPLcDNA     

A rapid sex-identification test for the forest musk deer (Moschus berezovskii) based on the ZFX/ZFY gene

We describe a rapid sex-identification method for the forest musk deer (Moschus berezovskii) using PCR based on zinc-finger protein-encoding genes (ZFX/ZFY) located on the X and Y chromosomes. Fragments of the ZFX and ZFY genes were amplified and sequenced. The ZFX and ZFY fragments were identical in length and 94% similar in nucleotide sequence. Specific primers for forest musk deer sex identification were designed on the basis of sequence differences between ZFX and ZFY. All the primers were multiplexed in single-tube PCR. Both male and female forest musk deer showed amplification bands of 447 bp and 212 bp separated in agarose gels. A sex-specific 278-bp band was amplified only from males. These results show that testing by PCR for the presence of the 278-bp sequence is a rapid and reliable method for sex identification.     

Rep-PCR应用于快速鉴定啤酒污染菌的研究

为评价rep-PCR在快速鉴定啤酒污染菌中的应用,首先比较了DNA提取方法,确定CTAB法作为制备rep-PCR的DNA模板的方法。并通过PCR产物直接测序的方法,从分离菌中鉴定得到11种常见的啤酒污染菌。用BOXA1R和(GTG)_5引物扩增分离菌,采用Gel ComparⅡ软件处理电泳图,构建污染菌的标准指纹图库。经过聚类分析表明,BOXA1R和(GTG)5对Lactobacillusbrevis、L.buchneri、L.casei/paracasei、L.plantarum和L.fermentum的聚类效果具有互补性,并首次提出指纹比对快速鉴定的相似系数阈值的概念。对来自三个不同来源的9株乳酸菌的快速鉴定结果表明,rep-PCR鉴定技术简单、快速、可靠,在快速鉴定方面将具有重要的应用价值。     

Cloning the swamp buffalo SRY gene for embryo sexing with multiplex-nested PCR

The polymerase chain reaction (PCR) is an efficient method for sexing embryos. The objective of this study was to develop an accurate and reliable method for sexing swamp buffalo (Bubalus bubalis) embryos. The SRY gene from swamp buffalo genomic DNA was amplified by PCR, using primers based on the sequence of the Holstein SRY gene. This fragment was sequenced based on a BLAST search; the SRY gene was highly conserved. Using a Southern blot, there was a strong signal in genomic DNA only from male swamp buffalo. Two pairs of nested primers, targeted to amplify the swamp buffalo SRY conserved region, were designed for sex identification. Simultaneously, the G3PDH gene was co-amplified to serve as an internal control. A multiplex-nested PCR system was optimized by varying the following individually: concentrations of Mg(2+) and dNTPs, ratio of concentrations of primers and numbers of cycles. Biopsies of 27 IVF-derived embryos and 24 embryos fertilized with Y-chromosome-bearing sperm were examined. Using optimized procedures, clear signals following PCR amplification were obtained from all embryo samples; PCR amplification accuracy was further verified by comparing PCR and dot blots. We concluded that this PCR technique was highly reliable for sexing swamp buffalo embryos.