基于内转录间隔区序列鉴定浓香型大曲发酵表面霉菌

浓香型大曲生产时曲块表面生长的霉菌很难通过形态学观察进行鉴定,为了确定大曲表面菌丝的生物学分类,该研究借助 内转录间隔区（ITS）克隆文库的构建,对大曲表面霉菌进行了分子生物学鉴定。 结果表明,曲块表面生长的菌丝体为米根霉（Rhizopus oryzae）,是一种非常重要的酿酒微生物。     

基于内转录间隔区序列对云南12株鹅膏菌的分子分析

目的 基于内转录间隔区(internal transcribed spacer, ITS)序列对云南省12株鹅膏菌进行分类鉴定。方法 采用十六烷基三甲基溴化铵法(cetyltrimethylammonium Ammonium Bromide, CTAB)法提取12株鹅膏菌DNA, 以ITS4和ITS5为引物进行PCR扩增, 完成DNA序列测序, 对序列进行分析并对发育树进行构建。 结果 12个样品的ITS序列长度574~755 bp, GC含量39%~47%, 平均遗传距离为0.329, YN05、YN03与其他10株鹅膏亲缘关系较远。结论 ITS序列高度保守, 在真菌的科属种上能够初步实现对物种的鉴定和系统 发育分析, 为建立云南省鹅膏属分子数据库提供基础数据。     

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.     

PCR detection of Alternaria spp. in processed foods, based on the internal transcribed spacer genetic marker

The genus Alternaria is considered one of the most important fungal contaminants of vegetables, fruits, and cereals, producing several mycotoxins that can withstand food processing methods. Conventional methods for Alternaria identification and enumeration are laborious and time-consuming, and they might not detect toxigenic molds inactivated by food processing. In this study, a PCR method has been developed for the rapid identification of Alternaria spp. DNA in foodstuffs, based on oligonucleotide primers targeting the internal transcribed spacer (ITS) 1 and ITS2 regions of the rRNA gene. The specificity of the Alternaria-specific primer pair designed (Dir1ITSAlt-Inv1ITSAlt) was verified by PCR analysis of DNA from various Alternaria spp., and also from several fungal, bacterial, yeast, animal, and plant species. The detection limit of the method was 10(2) CFU/ml in viable culture, heated culture, or experimentally inoculated tomato pulp. The applicability of the method for detection of Alternaria spp. DNA in foodstuffs was assessed by testing several commercial samples. Alternaria DNA was detected in 100% of spoiled tomato samples, 8% of tomato products, and 36.4% of cereal-based infant food samples analyzed.     

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.     

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     

Distribution of Candida in the oral cavity and its differentiation based on the internally transcribed spacer (ITS) regions of rDNA

This study aimed to determine the distribution of Candida species in the oral cavity and differentiate the species based on PCR amplification, including HinfI and MspI digestion, in order to assess the effectiveness of using the rDNA region for species identification. Samples from saliva as well as palate, tongue and cheek mucosa surfaces were collected from 45 individuals, consisting of three groups: periodontal disease patients; denture‐wearers; and the control group. The samples were serially diluted, spread on BHI and YPD agar plates and scored for colony‐forming units (CFUs). Fifteen random candidal colonies were isolated and subjected to genomic DNA extraction, based on glass beads disruption. Four primers were used to amplify regions in the rDNA, and the ITSI‐5.8S‐ITSII PCR product was digested by HinfI and MspI restriction enzymes. The microbial loads on all sites of the denture‐wearers were found to be significantly higher than control, while in the periodontal disease group only the microbial loads on the tongue were significantly higher than control. Meanwhile, there was no significant difference at other sites. The restriction fragment lengths of the clinical samples were compared to those of seven control species, allowing the differentiation of all seven species and the identification of 14 species from the clinical samples. The MspI restriction digest was not able to distinguish between C. albicans and C. dubliniensis, whereas the HinfI digest could not distinguish between C. tropicalis and C. parapsilosis. It was concluded that PCR–RFLP of the candidal rDNA region has potential for species identification. This study demonstrates the potential use of candidal rDNA as a means for identifying Candida species, based on genotype. The results also indicate the possibility of constructing genetic probes that target specific restriction fragments in the ITSI‐5.8S‐ITSII region, enabling swift and precise identification of Candida species. Copyright © 2012 John Wiley & Sons, Ltd.     

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.     

Modeling the effect of time and temperature on respiration rate of pomegranate arils (cv. "Acco" and "Herskawitz")

Understanding the effect of time and temperature on the respiration rate (RR) of fresh-cut produce, towards the design of a suitable modified atmosphere packaging (MAP) system, requires an adequate mathematical model for prediction of RR as a function of both time and temperature. This study investigated the effect of temperature (5, 10, and 15 °C) and storage time (1 to 5 d) on the RR (R(O2) and R(CO2)) of 2 pomegranate cultivars (cv. "Acco" and "Herskawitz") fresh arils. R(O2) and R(CO2) were 3 to 4 folds significantly higher with increased temperature from 5 to 15 °C and were within the range of 2.51 to 7.59 mL/kg h and 2.72 to 9.01 mL/kg h, respectively, for both cultivars. At 15 °C R(CO2) increased significantly from 8.4 to 25.96 mL/kg h from day 1 to 5, respectively, while at 5 °C R(CO2) changed from 2.9 to 2.05 mL/kg h from day 1 to 5. Temperature had the greatest influence on RR and the interaction of time and temperature also significantly affected R(O2) and R(CO2). The respiratory quotient (RQ) estimated by linear regression was 0.98 at 95% significant level. The dependence of RR on temperature and time was accurately described with a combination of an Arrhenius-type and power equation model for and of fresh pomegranate arils.     

基于调控DNA序列变化的MphR(A)蛋白类ELISA系统性能提升研究

利用MphR(A) 蛋白和启动子mph(A) 序列的结合-解离特性可以构建大环内酯类抗生素的体外类ELISA检测系统。该系统的关键是蛋白和其结合DNA序列的特异性相互作用,通过调控蛋白或者DNA序列可以调控系统性能。本研究目的是考察DNA序列对该系统性能的影响。通过MphR(A)蛋白与3段野生型DNA序列及其它突变序列构建类ELISA检测系统,获得与红霉素反应的IC50值。结果表明MphR(A)蛋白可以和不同DNA序列结合,从而获得不同的检测性能。采用突变序列AC4-DNA建立的类ELISA系统IC50值为2.33±0.70 ng/mL,低于野生型DNA所得IC50,系统灵敏度提升。通过DNA调控该类ELISA系统是一种可行的手段。此外,在野生型A-DNA、B-DNA、C-DNA均发现一段22 bp伪回文结构,该伪回文结构符合Tet蛋白家族的DNA序列特征,认为是该伪回文结构是与蛋白结合的核心序列。研究为提升以MphR(A)蛋白为基础的检测系统构建提供了思路。     

The DNA sequence analysis of the HAP4–LAP4 region on chromosome XI of Saccharomyces cerevisiae suggests the presence of a second aspartate aminotransferase gene in yeast

The nucleotide sequence of a 19 000 base pair region from the left arm of chromosome XI of Saccharomyces cerevisiae has been determined and analysed. It covers the HAP4–GFA1–LAP4 loci already described. As expected HAP4, GFA1 and LAP4 genes have been found and six new open reading frames (ORFs) with a coding capacity of more than 100 amino acid residues have been identified. One of them (YKL461) shows a high degree of identity with an aspartate aminotransferase gene. This raises the question of a second aspartate aminotransferase gene in yeast. A second ORF (YKL462) shows features compatible with a membranous localization. The other ORFs do not show a similarity with any known gene. A member of the highly repetitive ‘CAT’ DNA sequence is present.     

Discrimination of the cultivar,growing region,and geographical origin of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis>) using a mass spectrometer-based electronic nose

The objective of this study was to discrimination the cultivar, growing region, and geographical origin of rice (Oryza sativa) using a mass spectrometry-based electronic nose (MSE-nose). The inside-needle dynamic extraction (INDEX) system was used to concentrate the samples for MSEnose, following which the ion fragment data obtained were used to perform discriminant function analysis. Discriminant functions 1 and 2 readily separated all 16 cultivars of rice sampled. It was also confirmed that MSE-nose could distinguish the region in which rice cv. Chucheong and Koshihikari were grown, likely due to variation in environmental factors, such as soil and climate. Finally, it was confirmed that MSE-nose could be used to detect the geographical origin of rice, discrimination Korea rice from Japanese rice. Therefore, this simple and rapid technique is of value for discriminating the cultivar, growing region, and geographical origin of rice.