多响应曲面优化-毛细管电泳-激光诱导荧光快速检测食源性致病菌

建立了食品中常见致病菌: 沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR产物-毛细管电泳快速检测方法. 根据沙门菌、大肠杆菌O157∶H7、志贺菌及副溶血性弧菌的特异性基因保守序列设计出多重PCR引物, 优化PCR 扩增反应体系. 采用多响应曲面法优化毛细管电泳的分离条件, 以含有DNA荧光染料SYBR Green Ⅰ的1.0％甲基纤维素为筛分介质, 通过毛细管电泳-激光诱导荧光同时检测4种常见致病菌的PCR 扩增产物. 在优化的多重PCR反应体系和毛细管筛分电泳条件下, 此方法可以同时检测出沙门菌的invA基因、大肠杆菌O157∶H7的rfbO157基因、志贺菌的ipaH基因及副溶血性弧菌Vpara(16S-23S rDNA IGS)基因的多重PCR扩增产物, 25 min内即可完成检测. 迁移时间的日内相对标准偏差为0.92%~1.58%. 通过多响应曲面的优化, 有效改善了毛细管电泳对DNA分子的分离能力.     

Phylogenetic analysis of picoplankton in Lake Biwa and application to legal medicine

Three strains of picoplankton designated as brown, green, and pink belonging to the Synechococcus genus in cyanobacteria (approximately 1 microm in size) are found ubiquitously in Lake Biwa, Japan. However, they could not be morphologically discriminated from other bacteria such as Proteobacteria and Bacillus by microscopy. In this study, we attempted to use the polymerase chain reaction (PCR) analysis of the 16S ribosomal DNA (rDNA) from picoplankton for the diagnosis of death by drowning. A segment of 16S rDNA was sequenced in order to investigate their phylogenetic relationships and to design the specific primers. The PCR products from three picoplanktons were compared with those from five other cyanobacteria, Melosira (diatom), Staurastrum (green alga), bacteria from Lake Baikal, and humans. The picogram order of template DNA from picoplankton was specifically amplified by the primers. When the template of picoplankton was mixed with human tissue, at least 10 ng of template DNA was needed to obtain a PCR product. The efficiency of PCR was increased more than hundredfold by isolating the picoplankton from human lung tissue. The specific PCR products of the picoplankton were obtained from a formalin-fixed drowning body (lung and liver) that was found in a downstream river and Lake Biwa. The PCR analysis of the picoplanktion 16S rDNA is considered useful for the diagnosis of death by drowning.     

A novel, universal and sensitive lateral-flow based method for the detection of multiple bacterial contamination in platelet concentrations

In the present study, we aimed to develop a nucleic acid lateral-flow method for the rapid and sensitive detection of multiple bacteria that contaminate platelet concentrations (PCs). Polymerase chain reaction (PCR) amplicons were produced by a set of board-range primers that recognize the conserved region of bacteria 16S rDNA, followed by hybridization with both an FITC (fluorescein isothiocyanate)-labelled probe and biotin-labelled probe, and then a nucleic acid lateral-flow dipstick (LFD) assay. The LFD accurately identified 7 species of bacteria, but had no cross-reactivity with human genomic DNA. The limit of detection (LOD) of the LFD assay was as low as 10(1) copies/μL of 16S rDNA for plasmid. In the case of spiked PCs without enrichment, the detection limit of LFD for K. pneumonia was 5 CFU/mL, 6.5 × 10(4) CFU/mL for the S. epidermidis and 35 CFU/mL for P. aeruginosa.     

Species identification of Crassostrea and Ostrea oysters by polymerase chain reaction amplification of the 5S rRNA gene

A specific multiplex polymerase chain reaction (PCR) was developed for the identification of Crassostrea angulata, C. gigas, Ostrea edulis, and O. stentina oyster species. Universal primers were used for the amplification of complete repetition units of 5S rDNA in each of the 4 species. The alignment of the obtained sequences was the basis for the specific design of species-specific primers (ED1, ED2, ST1, ST2, CR1, and CR2) located in the nontranscribed spacer regions. The different sizes of the species-specific amplicons, separated by agarose gel electrophoresis, allowed identification of Crassostrea and Ostrea species. A multiplex PCR with a set of the 6 designed primers showed that they did not interfere with each other and bound specifically to the DNA target. This genetic marker can be very useful for traceability of the species, application in the management of oyster cultures, and conservation of the genetic resources of the species.     

Identification of lactic acid bacteria within the consortium of a kefir grain by sequencing 16S rDNA variable regions

The microflora of a kefir grain was identified using a polymerase chain reaction-based strategy combined with 16S rRNA gene sequencing. DNA was extracted from the kefir grain and amplified in its 16S rDNA V1 and V2 regions. To guarantee a good representation of the overall lactic acid bacteria populations, DNA amplification was performed separately with primers specific either to the dominant or to the less abundant bacterial groups. The amplified fragments were cloned in Escherichia coli and then sequenced. Sequences of the V1 region were gathered into 5 groups of similarity and identified by aligning with the sequences of a public library. The V1 region allowed the identification of Lactobacillus kefiranofaciens, L. kefir, L. parakefir, and Lactococcus lactis but was inappropriate for the identification of leuconostocs at species level. Among 16S rDNA variable regions, the V2 region showed the highest variability between Leuconostoc species. Nevertheless, even in the V2 region, differences were too tenuous for effective identification of L. mesenteroides. The methodology described here allowed detection of the dominant species within each targeted microbial group.     

多重聚合酶链反应-毛细管电泳-激光诱导荧光法检测三种食源性致病菌

建立了食品中常见致病菌大肠杆菌O157:H7的uidA基因、沙门菌的invA基因和志贺菌的ipaH基因的多重聚合酶链反应（PCR）产物的毛细管电泳快速检测方法。根据这3种致病菌的特异性基因序列设计多重PCR引物,优化PCR扩增反应体系,采用7.0 g/L 甲基纤维素为筛分介质,毛细管电泳-激光诱导荧光检测法同时检测了3种常见致病菌的PCR扩增产物。在优化的多重PCR反应和毛细管筛分电泳条件下,该方法可以同时检测沙门菌、志贺菌和大肠杆菌O157:H7基因的多重PCR扩增产物,22 min内即可完成3种常见致病菌的毛细管电泳检测。迁移时间的相对标准偏差为1.47%~2.07%。与凝胶电泳法比较,该法简便快速,灵敏度高,可用于多种致病菌脱氧核糖核酸的检测,为食品安全提供了一种可靠的快速检测方法。     

Application of horizontal staircase electrophoresis in agarose minigels to the random intergenic spacer analysis of clinical samples

The random intergenic spacer analysis is a recently developed technique for the study of microbial populations. The bacterial intergenic spacer (ITS) is located between 16S rRNA and 23S rRNA genes and presents different length and sequence among bacterial species. Therefore, the amplicons can be separated by electrophoresis commonly performed at low voltage during several hours. Although this technique is especially useful for unculturable microorganisms, it has not been applied before to clinical sample analysis. As these samples have a limited number of bacterial species, the size of the gels may be reduced to facilitate their handling and to reduce the running time. To obtain maximum separation among the ITS bands, we analysed in this work different electrophoretical conditions including staircase electrophoresis, a technique based on the application of several voltage steps. The results obtained showed a different behaviour of the electrical resistance during the performance of submarine horizontal and vertical staircase electrophoresis. In the first case the resistance decreased during most of the running time whereas in the second case it increased. Here, we show that the performance of horizontal staircase electrophoresis reduces the running time more than 80% with respect to conventional electrophoresis at low voltages. This procedure was applied to the separation of ITS bands from bacterial DNA present in a tissue from a vocal cord biopsy. The sequencing of these bands allowed their identification. This new procedure may be very useful in the rapid diagnosis of bacteria present in human, animal and plant tissues.     

Efficient DNA Extraction and Amplification of Samples from the Archeological Site of Pompei (Italy)

Due to their growth and metabolism, microorganisms can cause severe damage to Cultural Heritage, through mechanical and chemical processes. In this paper we used molecular approaches to study bacterial communities on samples from the archeological site of Pompei (Italy). In particular four DNA extraction methods without prior cultivation of the microflora were compared. In terms of efficiency, the DNA extraction method based on the Tri-Reagent™ kit gave the best results. All extraction protocols coupled with 16S rDNA fragments PCR amplification were satisfactory; but the best results were obtained by Klentaq DNA LA Polymerase™; this enzyme can be considered an effective tool for amplification of DNA from Cultural Heritage and possibly other environmental samples.     

Molecular diagnosis of microbial contamination in cosmetic and pharmaceutical products: a review.

Molecular methodologies such as adenosine triphosphate (ATP) bioluminescence and polymerase chain reaction (PCR)-based assays provide rapid quality control analysis of cosmetic and pharmaceutical finished products and raw materials. Using a single enrichment broth for bacteria, yeast, and mold, ATP bioluminescence detected microbial contamination within 27 h. Samples were automatically lysed to release microbial ATP and light production was quantitated using the Celsis Optocomp. However, to maintain the detection time to within 27 h, different enrichment broths were required for neutralization of antimicrobial ingredients in finished products and to provide specific nutrients for growth optimization. To perform the PCR reaction, bacterial DNA was extracted using a Tris-EDTA-Tween 20-proteinase K buffer at 35 degrees C while yeast and mold DNA were extracted using a Tris-EDTA-SDS buffer at 95 degrees C. Extracted microbial DNA was added to Ready-To-Go PCR beads and specific DNA primers. The primers were targeted to amplify specific regions within Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Burkholderia cepacia, Candida albicans, and Aspergillus niger. Furthermore, conserved bacterial ribosomal DNA sequences have also been used for sterility testing of samples. The results from these studies indicate that both ATP bioluminescence and PCR assays provide rapid, reliable, and cost effective methods for quality evaluation. This will ultimately result in faster product release and production optimization.     

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.     

Development of a multiplex system to assess DNA persistence in taphonomic studies

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single‐copy nuclear recombination activation gene (RAG‐1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature—24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.     

Real-time polymerase chain reaction detection of fishmeal in feedstuffs

A SYBR Green PCR system was developed for detection of fishmeal in feedstuffs. The real-time PCR method combines the use of fish-specific primers that amplify an 87 base pair (bp) fragment of the mitochondrial 12S ribosomal RNA gene from fish species, and a positive control primer pair that amplifies a 99 bp fragment of the nuclear 18S ribosomal RNA gene in all eukaryotic organisms. The specificity of the primers was tested against 52 animal species and six plant species. Reference feedstuff samples were successfully tested for the presence of fishmeal, demonstrating the applicability of the assay to feedstuffs.     

Application of polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) to identification of flatfish species.

A method of DNA analysis based on polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) was developed to verify the authenticity of labeled raw and frozen fillets of some flatfish species. PCR was used to amplify a short fragment (201 bp) of the mitochondrial cytochrome b gene, which was denatured and analyzed by polyacrylamide gel electrophoresis for detection of SSCPs. Species-specific patterns of DNA bands were obtained for sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides).     

Rapid identification and classification of Mycobacterium spp. using whole-cell protein barcodes with matrix assisted laser desorption ionization time of flight mass spectrometry in comparison with multigene phylogenetic analysis

The need of quick diagnostics and increasing number of bacterial species isolated necessitate development of a rapid and effective phenotypic identification method. Mass spectrometry (MS) profiling of whole cell proteins has potential to satisfy the requirements. The genus Mycobacterium contains more than 154 species that are taxonomically very close and require use of multiple genes including 16S rDNA for phylogenetic identification and classification. Six strains of five Mycobacterium species were selected as model bacteria in the present study because of their 16S rDNA similarity (98.4–99.8%) and the high similarity of the concatenated 16S rDNA, rpoB and hsp65 gene sequences (95.9–99.9%), requiring high identification resolution. The classification of the six strains by MALDI TOF MS protein barcodes was consistent with, but at much higher resolution than, that of the multi-locus sequence analysis of using 16S rDNA, rpoB and hsp65. The species were well differentiated using MALDI TOF MS and MALDI BioTyper™ software after quick preparation of whole-cell proteins. Several proteins were selected as diagnostic markers for species confirmation. An integration of MALDI TOF MS, MALDI BioTyper™ software and diagnostic protein fragments provides a robust phenotypic approach for bacterial identification and classification.     

Determination of Yersinia enterocolitica in Food by Capillary Electrophoresis with Laser Induced Fluorescence Detection

A duplex polymerase chain reaction (PCR)-capillary electrophoresis-laser induced fluorescence (CE-LIF) method was developed to determine Yersinia enterocolitica in food sensitively, rapidly, and reliably. Two sets of primers were selected to amplify the genus-specific 16 S ribosomal RNA gene and ail gene associated with the pathogenicity of Yersinia enterocolitica. The parameters of duplex PCR and the conditions for CE-LIF were optimized. Under the optimum conditions, the PCR products of Yersinia enterocolitica were determined within twenty minutes. Alignment analysis showed favorable agreement with published sequences from GenBank, indicating that the primers were specific and the PCR results were reliable. The method detected 16 colony forming units per milliliter pathogenic Yersinia enterocolitica. The intraday precision of migration time of the DNA marker and the PCR products were between 1.13 and 1.81 percent. In summary, a new method combining duplex PCR and CE-LIF is reported for specific, sensitive, and reproducible detection of Yersinia enterocolitica in food with low sample consumption and cost.     

Real-time PCR for quantitative detection of chamois (Rupicapra rupicapra) and pyrenean ibex (Capra pyrenaica) in meat mixtures

A real-time quantitative polymerase chain reaction (PCR) technique was developed for the quantification of chamois and pyrenean ibex DNAs in meat mixtures by using a SYBR green detection platform. Two species-specific systems and a eukaryotic endogenous system were combined in the real-time PCR approach to quantify the target species. In the specific systems, a 133 base pair (bp) fragment of the 12S rRNA gene was amplified from chamois DNA, and an 88 bp fragment from the D-loop region was amplified from pyrenean ibex DNA. In the endogenous system, universal primers amplified a 141 bp fragment on the nuclear 18S rRNA gene from eukaryotic DNA. The threshold cycle values obtained with the 18S rRNA primers were used to normalize those obtained from chamois- or pyrenean ibex-specific systems, serving as endogenous control for the total content of PCR-amplifiable DNA in the sample. Analysis of experimental raw and heat-treated binary mixtures of chamois and pyrenean ibex meat in a swine meat matrix demonstrated the suitability of the assay for the detection and quantification of the target DNAs in the range of 0.1-0.8%, depending on the species and treatment of the meat samples.     

Identification of Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus) fillets by PCR amplification of the 5S rDNA gene.

Nile perch (Lates niloticus), grouper (Epinephelus guaza), and wreck fish (Polyprion americanus) were differentiated by polymerase chain reaction (PCR) amplification of the nuclear 5S rDNA gene. The design of 3 species-specific primers complementary to the nontranscribed intergenic spacer region from the 5S rDNA molecule allowed amplification of clearly distinguishable gene fragments in each fish species. This approach is rapid and reliable and offers the potential to detect fraudulent or unintentional mislabeling of these species in routine seafood authentication analysis.     

Capillary and microchip gel electrophoresis for simultaneous detection of Salmonella pullorum and Salmonella gallinarum by rfbS allele-specific PCR

We report the use of capillary gel electrophoresis (CGE) based on a rfbS allele-specific polymerase chain reaction (PCR) for the analysis and simultaneous detection of Salmonella pullorum and Salmonella gallinarum, which are the major bacterial pathogens in poultry. rfbS allele-specific PCR was used to concurrently amplify two specific 147- and 187-bp DNA fragments for the simultaneous detection of S. pullorum and S. gallinarum at an annealing temperature of 54 ± 1 °C and an MgCl₂ concentration of 2.8-5.6 mM. Under an electric field of 333.3 V/cm and a sieving matrix of 1.0% poly(ethyleneoxide) (M_r 600 000), the amplified PCR products were analyzed within 6 min by CGE separation. This CGE assay could be translated to microchip format using programmed field strength gradients (PFSG). In the microchip gel electrophoresis with PFSG, both of the Salmonella analyses were completed within 30 s, without decreasing the resolution efficiency. rfbS allele-specific PCR-microchip gel electrophoresis with the PFSG technique might be a new tool for the simultaneous detection of both S. pullorum and S. gallinarum, due to its ultra-speed and high efficiency.     

Simultaneous Identification of Pork and Poultry Origins in Pet Foods by a Quick Multiplex Real-Time PCR Assay Using EvaGreen Florescence Dye

EvaGreen multiplex real-time polymerase chain reaction (EMRT-PCR) was designed for an assay that can join the advantages of multiplex PCR and real-time PCR to recognize animal genes more quickly in pet foods. EMRT-PCR based on melting temperatures discrimination by using EvaGreen fluorescence dye was developed for the analysis of pork and poultry in pet food. The method combines the use of poultry- and pork-specific primers that amplify small fragments of 12S rRNA and mitochondrial DNA genes. Appropriate mixtures of poultry and pork meat in reference samples were used to develop the assay. Gene yields of poultry and pork were represented in two melting peaks generated simultaneously at temperatures of 80.5 and 87.2 °C, respectively. Based upon the assay results, it has been concluded that EMRT-PCR assay might be an efficient tool for the verification of species origin in pet foods.     

Selection of aptamers by systematic evolution of ligands by exponential enrichment: addressing the polymerase chain reaction issue

Aptamers are DNA oligonucleotides capable of binding different classes of targets with high affinity and selectivity. They are particularly attractive as affinity probes in multiplexed quantitative analysis of proteins. Aptamers are typically selected from large libraries of random DNA sequences in a general approach termed systematic evolution of ligands by exponential enrichment (SELEX). SELEX involves repetitive rounds of two processes: (i) partitioning of aptamers from non-aptamers by an affinity method and (ii) amplification of aptamers by the polymerase chain reaction (PCR). New partitioning methods, which are characterized by exceptionally high efficiency of partitioning, have been recently introduced. For the overall SELEX procedure to be efficient, the high efficiency of new partitioning methods has to be matched by high efficiency of PCR. Here we present the first detailed study of PCR amplification of random DNA libraries used in aptamer selection. With capillary electrophoresis as an analytical tool, we found fundamental differences between PCR amplification of homogeneous DNA templates and that of large libraries of random DNA sequences. Product formation for a homogeneous DNA template proceeds until primers are exhausted. For a random DNA library as a template, product accumulation stops when PCR primers are still in excess of the products. The products then rapidly convert to by-products and virtually disappear after only 5 additional cycles of PCR. The yield of the products decreases with the increasing length of DNA molecules in the library. We also proved that the initial number of DNA molecules in PCR mixture has no effect on the by-products formation. While the increase of the Taq DNA polymerase concentration in PCR mixture selectively increases the yield of PCR products. Our findings suggest that standard procedures of PCR amplification of homogeneous DNA samples cannot be transferred to PCR amplification of random DNA libraries: to ensure efficient SELEX, PCR has to be optimized for the amplification of random DNA libraries.