Sequencing of an internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. in food

DNA sequences of an internal transcribed spacer (ITS) region for 40 Salmonella serovars were determined and compared with ITS sequences of Salmonella spp., and non-Salmonella spp. already available on the GenBank database. From such comparison, two Salmonella-specific ITS based PCR primers, ITSF and ITSR, were designed. When Salmonella strains with various serotypes were PCR assayed with primers ITSF/ITSR, all generated PCR products with molecular weight bands equal to 312 bp. On the other hand, 48 non-Salmonella isolates, including strains of Enterobacteriaceae and other food pathogens generated negative results. Detection limits of this PCR method was 1-9 CFU per assay. These PCR primers were used for the detection of Salmonella cells in artificially contaminated foods, including chicken meat and whole milk. The detection limit was 1-9 x 10(3) CFU per assay. With an 8-h enrichment step performed prior to the PCR assay, however, the detection limit became 1-9 CFU per gram of the food sample.     

创伤弧菌16S-23S rDNA间区变形梯度凝胶电泳多态性分析

目的 建立创伤弧菌基因分型的新方法,了解创伤弧菌的分子特征.方法 应用PCR-变形梯度凝胶电泳(PCR-DGGE)技术对创伤弧菌的16S-23S rDNA间区(16S-23S rDNA intergenic spacer regions,ISR)多态性进行分析比较,结合药敏试验,以进一步验证分离菌株之间的亲缘关系.结果 ISR-PCR将创伤弧菌菌株扩增出4条约900、750、650、550 bp大小不同的条带;同时,创伤弧菌的ISR-DGGE序列表现出明显的株间差异,18株共产生了16种不同的指纹图谱;聚类分析将所有的细菌分为两大类,相似性分别为0.65和0.71;亲缘关系较近的菌株具有不同于关系较远的菌株的耐药谱,与ISR-DGGE指纹图谱分析相一致.结论 这种分子操作技术完全能够用于创伤弧菌株型的调查与鉴定,为创伤弧菌的基因分型提供了一种新的方法.     

Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants

Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n = 40) and from udder tissue (n = 7) and foremilk (n = 24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology.     

PCR detection of Klebsiella pneumoniae in infant formula based on 16S-23S internal transcribed spacer

A PCR detection based on 16S-23S rDNA internal transcribed spacer (ITS) of Klebsiella pneumoniae was developed in the present study. Nineteen different ITS sequences were amplified from 6 strains of K. pneumoniae by universal primers. By sequencing and alignment of these sequences to the other homologous in GenBank, species-specific primers of K. pneumoniae, Pf/Pr1 and Pf/Pr2, were designed for amplification of the ITS sequence from the operon containing tDNA(Ala) and tDNA(Ile). Ten type strains and 21 isolates of K. pneumoniae were positive to the PCR detection, and all of the non-K. pneumoniae reference strains (79 strains) were negative. The enrichment was performed in this procedure with a modified growth media to enrich K. pneumoniae from 1.5 CFU/100 g infant formula to about 10(5) CFU/ml in 900 ml of the media. Combination of the enrichment, with the PCR assay can detect 1.5 CFU/100 g infant formula of K. pneumoniae within 48 h. Furthermore, K. pneumoniae strains KPE050803 and KPE 050830 were identified by this method in 63 infant formula samples.     

Characterization of the gene encoding the 16S rRNA of Enterobacter sakazakii and development of a species-specific PCR method

The gene encoding the 16S rRNA of Enterobacter (E.) sakazakii (ATCC 29544, plus four strains isolated from powdered infant formula) was studied, and the sequence compared with those of other Enterobacteriaceae in aspects of genetic variability. Sequence differences between E. sakazakii and other Enterobacteriaceae within the hypervariable regions V1, V2, and V3, respectively, were used to develop two PCR methods for E. sakazakii. PCR1 employed a primer pair located in V1/V2, while PCR2 utilized a primer pair located in V1/V3, respectively. The two PCR methods were tested against a set of 57 E. sakazakii and 148 non-E. sakazakii isolates. PCR1 gave an amplicon with a size of 406 bp and resulted in 100% positive results for E. sakazakii, but also detected Citrobacter koseri/amalonaticus and all nine tested Salmonella enterica serovars. In contrast, PCR2 (amplicon size of 952 bp) gave positive results only for E. sakazakii, thus allowing specific identification of this species.     

Identification of Acetobacter strains from Thai fermented rice products based on the 16S rRNA gene sequence and 16S-23S rRNA gene internal transcribed spacer restriction analyses

BACKGROUND: Fermented rice flour (khao‐khab, a non‐glutinous rice) and related products are Thai traditional products. The types of acetic acid bacteria (AAB) microflora in khao‐khab have not been reported. In this study, Acetobacter strains were isolated and identified based on the phenotypic and chemotaxonomic characteristics and molecular aspects. RESULTS: Twenty‐five acetic acid bacteria isolated from fermented rice products and a starter for sweetened rice in Thailand by an enrichment culture approach, were assigned to the genus Acetobacter by phenotypic and chemotaxonomic characterisations. On the basis of the 16S rRNA gene sequence and 16S–23S rRNA gene ITS restriction analyses, 25 isolates were divided into six groups and identified at the specific level: (1) Group 1 included five isolates, which were identified as A. indonesiensis; (2) Group 2 included two isolates, which were identified as A. lovaniensis; (3) Group 3 included one isolate, which was identified as A. orientalis; (4) Group 4 included eleven isolates, which were identified as A. pasteurianus; (5) Group 5 included three isolates, which were identified as A. syzygii and (6) Group 6 included three isolates, which were unidentified and considered to constitute a new species. CONCLUSION: Results revealed that various Acetobacter species were distributed in Thai fermented rice flour and related products. A novel Acetobacter species was isolated from the product. Copyright © 2011 Society of Chemical Industry     

Differentiation of acetic acid bacteria based on sequence analysis of 16S-23S rRNA gene internal transcribed spacer sequences

The 16S-23S gene internal transcribed spacer sequence of sixty-four strains belonging to different acetic acid bacteria genera were analyzed, and phylogenetic trees were generated for each genera. The topologies of the different trees were in accordance with the 16S rRNA gene trees, although the similarity percentages obtained between the species was shown to be much lower. These values suggest the usefulness of including the 16S-23S gene internal transcribed spacer region as a part of the polyphasic approach required for the further classification of acetic acid bacteria. Furthermore, the region could be a good target for primer and probe design. It has also been validated for use in the identification of unknown samples of this bacterial group from wine vinegar and fruit condiments.     

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

In the present study, 44 Fusarium spp. isolates (5 Fusarium culmorum, 7 Fusarium graminearum, 1 Fusarium cerealis, 1 Fusarium poae, 26 Fusarium oxysporum, and 4 Gibberella fujikuroi species complex) were characterized morphologically, physiologically and genetically. All except one (Dutch Collection: CBS 620.72) were isolated from different hosts grown in various Spanish localizations. Morphological characterization was made according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). ZEA was determined by liquid chromatography and trichothecenes by gas chromatography. Confirmation was carried out by liquid chromatography-ion trap-mass spectrometry (ZEA) or gas chromatography-mass spectrometry (trichothecenes). Molecular characterization of isolates was performed using an optimized, simple and low-cost method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rRNA gene (rDNA). The results indicate that F. graminearum, F. culmorum and F. cerealis isolates were high ZEA and type B trichothecene producers, the F. poae isolate produced very low level of nivalenol while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. Restriction patterns of the IGS region did not show any relationship with the host, geographic origin of the isolate and mycotoxin-producing capacity. However, the haplotypes obtained with six restriction enzymes (CfoI, AluI, HapII, XhoI, EcoRI and PstI) permitted to discern the six assayed Fusarium species. Therefore, this is a rapid and suitable methodology that allows closely related strains to group and to estimate the genetic relationships between the groups.     

基于16S-23S rRNA ITS AFLP对米酒发酵过程中原核微生物的演替分析

利用16S-23S rRNA ITS AFLP指纹图谱技术监控四川传统米酒发酵过程中原核微生物的演替,并结合米酒发酵过程中理化因子的动态变化对其演替过程进行了分析。研究结果表明,米酒发酵过程中,随着酒曲的接入,米酒醅中原核微生物伴随米酒理化因子的动态变化而发生群落演替。在适应期和发酵期,米酒醅中的原核微生物群落分别聚成群Ⅰ和群Ⅱ,二者相关系数为0．49。群Ⅱ中,在相关系数0．638水平上又分为ⅡA和ⅡB两个分支。分支ⅡA又进一步分为ⅡA1和ⅡA2两簇,相关系数为0．73。簇ⅡA2中主要发生的是发酵旺盛期微生物的演替;簇ⅡA1中,ⅡA1—1和ⅡA1—2亚簇分别表示发酵前期、发酵后期米酒醅中的原核微生物群落演替,二者聚集于相关系数0．80。其中,ⅡA1—2中,发酵52h和55h的米酒醅中原核微生物群落几乎相似,二者群落相关系数为1．0。     

不同来源泡菜中乳酸菌的16S-23S rDNA间隔序列扩增及种群多样性分析

对不同来源的三种泡菜进行乳酸菌分离,并应用16S-23SrDNA间隔序列扩增指纹图谱结合16SrDNA测序对分离株进行种群多样性分析。结果显示在368株革兰氏阳性、接触酶阴性的产酸细菌中存在7个不同的分子指纹图谱(A～G型),其中杆菌指纹图谱为A、B和D型,球菌的指纹图谱为C、E、F和G型,不同来源的泡菜样品中乳酸菌谱型构成具有明显差异,其中52.3%(193/368)菌株扩增指纹图谱属于E型,显示了E型菌株是泡菜中的优势菌。从不同指纹图谱的菌株中随机挑取25株分离株进行16SrDNA扩增和测序分析,比对结果表明A型指纹图谱菌株属于Lactobacillus acidophilus;B和D型指纹图谱菌株属于Lactobacillus casei;E和G型指纹图谱菌株属于Lactococcus lactis;C和F型指纹图谱菌株属于Enterococcus sp.。在三种泡菜样品(a、b、c)中存在不同的乳酸菌种群构成,其中样品a和b菌群构成较为接近,而样品c中的菌群与a和b在构成上具有显著差异。本研究结果为泡菜发酵剂研究提供了基础数据及分析方法。     

16S-23S rRNA ITS-AFLP指纹图谱分析在白酒窖泥原核微生物多样性分析中的应用

利用非培养的分子生物学方法对位于多粮原料生产的窖壁和窖底的新窖和老窖窖泥中原核微生物的16S-23S rRNA ITS进行AFLP（amplified fragment length polymorphism）分析,利用NTSYSpc2．10e软件构建聚类图,分析其关系。结果表明,来自不同窖池的不同部位的原核微生物在16S-23SrRNAITS水平上呈现出差异,其中老窖窖底和新窖窖底的原核微生物多样性存在着明显差异,其相关系数为0．47;而老窖窖壁和新窖窖壁的原核微生物多样性却差异不大,其相关系数为1．00。     

Direct application to dairy foods of a Listeria-specific oligonucleotide probe to 16S rRNA.

A potentially Listeria-specific 28 base oligonucleotide probe was designed from 16S rRNA sequence data. Using either 32P or non-radioactive (alkaline phosphatase) labels, the probe was shown to be highly specific as it hybridised to RNA extracted from all of the species of Listeria but not to any of the other bacteria tested. Both probe methods were highly sensitive and ca 10(2) cfu/ml Listeria could be detected in pure cultures. A rapid procedure for extracting RNA from milk, Camembert and cottage cheese was developed. This allowed the direct application of the probe to these foods and gave a rapid and specific method of detecting > 10(2) cfu/g or ml Listeria in these foods.     

rDNA间区序列测定在乳杆菌鉴定中的应用

利用16S-23S rDNA Short ISR区间序列分析方法,对分离自酸马奶中的12株杆菌进行鉴定。通过对Short ISR序列测序并进行BLAST比对,结合系统发育树分析,可将ZL3-1、XM2-1和XF5-2判定为Lacto-bacillus.caseisubsp.casei,MG2-1、A6-2、B10-2和XB2-3归入L.helveticus,C56、D73和E96暂定为L.ferintoshensis,无法准确判定E91和E112的归属。16S-23S rDNA Short ISR区间序列可以识别L.rhamnosus和L.casei,而对于L.ferintoshensis、L.kefir、L.buchneri和L.hilgardii无法加以区分,但是仍有助于将未知杆菌归入这一群。     

0癌症进展Oncology Progress5R-5E0546E

ieved in the study of gynecological cancers,early diagnosis,early treatment and valid recognition of precancerous stage are still the priority of therapy.The standardization of treatment on gynecological cancers remains to be generalized and the surgeries are prone to be humanize and individuate.The technique of clinical treatment should be based on evidence-based medicine and cooperation of multi-ceneri和L.hilgardii无法加以区分,但是仍有助于将未知杆菌归入这一群。     

Rapid detection and identification of Streptococcus macedonicus by species-specific PCR and DNA hybridisation

The aim of this study was to develop a simple and specific method for the rapid detection and identification of Streptococcus macedonicus. The method was based on polymerase chain reaction (PCR) using species-specific primers derived from the 16S rRNA gene. Specific identification was proven on seven S. macedonicus strains, while 16 strains belonging to different lactic acid bacteria species were tested negative. The PCR assay was capable of detecting 100 pg of S. macedonicus DNA, and it was also efficient on single colonies of the bacterium. Furthermore, the same bacterial strains were used for the specificity evaluation of a S. macedonicus species-specific probe. Neither species-specific PCR nor DNA hybridisation experiments could differentiate Streptococcus waius from S. macedonicus, due to the identity of the 16S rRNA gene of the two species, indicating high phylogenetical relatedness. This was further confirmed by the comparative sequence analysis of the 16S-23S rRNA intergenic regions. It was thus clearly demonstrated that S. waius, recently described as a novel Streptococcus species, is phylogenetically identical to S. macedonicus.     

An improved 16S rRNA based PCR method for the specific detection of Salmonella enterica

A molecular method for the detection of Salmonella enterica strains based on 16S rRNA sequence analysis was developed by a modification of the previously described PCR primer 16SFI [J. Appl. Bacteriol. 80 (1996) 659], which was combined with a newly developed primer annealing at the position 66-82. Only approximately two thirds of now determined Salmonella 16S rRNA sequences contained a region identical to the 16SFI primer sequence and the reverse primer 16SIII was also not specific. Combined, these two primers have been claimed to allow the specific detection of all Salmonella; however, in this study, they did not recognize S. bongori and 3 out of 78 tested S. enterica strains. They also identified some of the tested Enterobacter cloacae strains as Salmonella. On the contrary, the new primer pair, MINf and MINr, made it possible to recognize correctly all of the 78 tested S. enterica strains, representing 31 different Salmonella serovars. None of the 23 non-Salmonella strains from the related gamma-proteobacterial genera was incorrectly recognized as belonging to S. enterica.     

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.     

Application of 16S-ARDRA and RFLP-PFGE for improved genotypic characterisation of dairy propionibacteria and combination with characteristic phenotypes

Propionic acid bacteria (PAB) are important for the manufacture of Swiss-type cheeses since they are involved in the formation of eyes and are responsible for the characteristic flavour and aroma of these cheeses. Propionibacterium freudenreichii is most commonly encountered in Swiss-type cheeses and is the sole Propionibacterium species used as a cheese starter. Aspartase activity determined in PAB is strain-dependent and it is important to find a usable way to identify the specific strains involved in cheese making. In this work, 16S-amplified ribosomal DNA restriction analysis and pulsed-field gel electrophoresis (PFGE) were used to differentiate 107 PAB strains. Consequently, the presence and persistence of inoculated Propionibacterium starter strains can be monitored during cheese ripening. The studied metabolic features of lactose utilisation, nitrate reductase activity and aspartase activity could not be directly associated with particular genotypes, but principal component analysis (PCA) suggested a linkage between related PFGE patterns and aspartase activity.     

Characterization of Fusarium verticillioides strains by PCR‐RFLP analysis of the intergenic spacer region of the rDNA

Thirty‐three Fusarium verticillioides strains from diverse origins and hosts have been analysed for fumonisin production and characterized in order (i) to detect the variability present in this species and (ii) to discriminate among isolates. The method used was a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) generated by restriction endonucleases applied to the IGS region (intergenic spacer of rDNA). All the F. verticillioides strains associated with crops produced fumonisins B₁ and B₂ except those isolated from banana. Analysis of the IGS region by PCR‐RFLP proved to be useful to detect variability within F. verticillioides and allowed discrimination of two related groups of isolates belonging to distinct lineages differing in fumonisin production and host preferences: the fumonisin‐producing group associated with cereals and the fumonisin non‐producing group associated with banana. The method used facilitates early detection and characterization of F. verticillioides strains required to control both types of pathogens and to evaluate plant exposure to the toxin, quality of the raw material to be processed and the potential fumonisin contamination in order to prevent fumonisins entering the food chain. Copyright © 2005 Society of Chemical Industry     

Molecular Characterization and Identification of Bioactive Peptides Producing Lactobacillus sps. Based on 16S rRNA Gene Sequencing

In the present study, 3 bacterial cultures were isolated from faecal samples of human infant. The biochemical traits showed similarity with Lactobacillus sps and 16S rRNA sequence analyses, confirmed as Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus rhamnosus. The cultures were screened for their proteolytic activity and good ability to release peptides from milk proteins was found. Hence, these bacteria were used as a proteolytic starter culture for the fermentation of skim milk and whey for the liberation of small peptides. Bioactive nature of the peptides released from whey and skim milk was tested, and results demonstrated that peptides obtained after fermentation of whey and skim milk by Lactobacillus strains showed antimicrobial activity against all the pathogens causing food borne infections in humans. These peptides also indicated antioxidant as well as ACE (angiotensin-converting enzymes) inhibitory activity.