Molecular subtyping methods for Listeria monocytogenes

Conventional, phenotypic, and DNA-based subtyping methods allow differentiation of Listeria monocytogenes beyond the species and subspecies level. Bacterial subtyping methods not only improve our ability to detect and track human listeriosis outbreaks, but also provide tools to track sources of L. monocytogenes contamination throughout the food system. The use of subtyping methods also provides an opportunity to better understand the population genetics, epidemiology, and ecology of L. monocytogenes. The last 5 years have seen tremendous advancements in the development of sensitive, rapid, automated, and increasingly easy-to-use molecular subtyping methods for L. monocytogenes. This review highlights key aspects of different L. monocytogenes subtyping methods and provides examples of their application in public health, food safety, population genetics, and epidemiology. A significant focus is on the application of subtyping methods to define L. monocytogenes subtypes and clonal groups, which may differ in phenotypic characteristics and pathogenic potential.     

The use of genetic typing methods to discriminate among strains of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus

This review article summarizes the findings of recent typing studies conducted on Vibrio cholerae, V. parahaemolyticus, and V. vulnificus. The DNA-based methods used to type the Vibrio spp. include whole genome approaches, such as pulsed field gel electrophoresis (PFGE), ribotyping, and repetitive extragenic palindromic (REP)-PCR, single gene targets, and multiple gene targets (multilocus approaches). The goals of these studies include establishing the relatedness of isolates from disease epidemics, discriminating among strains with more or less potential to cause disease or epidemics, and exploring the population biology of these waterborne pathogens. PFGE was consistently among the more discriminatory of the typing methods for all three Vibrio spp., and was useful for tracing the temporal and geographic relatedness of epidemic strains of V. cholerae and V. parahaemolyticus. However, PFGE did not group V. vulnificus strains according to the genotypes that have been proposed as markers of virulence potential. Typing methods that target repetitive elements distributed throughout the genome, such as BOX-PCR and REP-PCR, and DNA sequence-based methods, such as multilocus sequence typing, were also highly discriminatory and, in some cases, superior to PFGE for phylogenetic analysis and identification of strains with high epidemic or virulence potential. As typing methods and strategies are refined and used, the epidemiology, virulence potential, and ecology of these pathogenic Vibrio spp. will become better understood.     

Nanopore label‐free detection of single‐nucleotide deletion in Baxα/BaxΔ2

Baxα, a key tumor suppressor gene, will not be expressed correctly as a result of single nucleotide mutation in its microsatellite region; Instead, BaxΔ2, an isoform of Baxα, is often produced. In addition, lack of the exon 2 due to an alternative splicing, BaxΔ2 has the same sequence as Baxα except single base deletion from eight continuous guanines (G8) to G7. Most of the currently available methods for Bax∆2 detection are inefficient and time‐consuming, and/or require the use of labels or dyes. In this work, we reported a label‐free nanopore sensing strategy to differentiate between Baxα and BaxΔ2 with a DNA polymer as a molecular probe based on alternative spliced sequences. Two DNA molecules were designed to selectively detect Baxα and BaxΔ2, respectively. The method was rapid, accurate, and highly sensitive: picomolar concentrations of target nucleic acids could be detected in minutes. Our developed simple and fast nanopore‐based detection strategy is not only useful for distinguishing between Baxα and Bax∆2, but also provides a useful tool for detection of other single‐base mutations in genetic diagnosis.     

Virulence testing of Listeria monocytogenes

A major problem in understanding foodborne listeriosis from both the basic science and regulatory perspectives revolves around the role played by virulence factors of Listeria monocytogenes and how these interact with host susceptibility to result in the observed incidence of disease. From a mechanistic perspective, this problem has been well investigated, and many virulence components of L. monocytogenes have been discovered. Deletion of these genes results in large reductions in virulence functions in vitro and in vivo. The clonal bacteria and genetically identical hosts necessary to solve the riddles associated with virulence mechanisms are not likely to reflect the natural diversity found among wild populations of L. monocytogenes, including those associated with food. These factors contribute to a major dilemma in risk assessment and risk management of foodborne listeriosis: Although low-level L. monocytogenes contamination of certain foods is relatively common, suggesting widespread exposure, illness is overwhelmingly associated with only a relatively small subpopulation (3 of the 13 L. monocytogenes serotypes) and occurs in only a small proportion of susceptible individuals. Virulence testing based on DNA probes for virulence genes is confounded by the widespread distribution of these genes in food isolates. In terms of the distribution of virulence factors among food isolates of L. monocytogenes, only listeriolysin is well characterized, because beta-hemolysis is often used to confirm the presence of L. monocytogenes in foods. The presence of other virulence genes such as those involved in host cell invasion and cell-to-cell spread (inlA and actA) among food isolates has not been extensively investigated. How the presence of these components translates into functional virulence as measured in vivo and in vitro is also unknown. Animal studies and cell culture systems show a range of virulence among food isolates of L. monocytogenes. However, clinical isolates included in such studies are not consistently more virulent than food isolates with no known human disease association. Where multiple serotypes or ribotypes are compared, it has been difficult to demonstrate a consistent pattern of increased virulence associated with any subtype(s) in animal or in vitro studies. Development of model systems that adequately reflect the complexity of the host-pathogen relationship remains a challenge.     

Arbitrary single primer amplification of trace DNA substrates yields sequence content profiles that are discriminatory and reproducible

Single primer amplification is shown to yield a DNA profile that is reproducible when based on the sequence content of the amplicons rather than on the pattern of length polymorphism. The sequence-based profile increases in reliability with increasing numbers of cycles of amplification. This process uses an arbitrarily chosen primer and a low initial annealing temperature in order to amplify sequences from the whole metagenome present in a sample that may contain only trace DNA, and a large number of cycles to select subsets of sequences based on variable amplification efficiency. Using arrays, we demonstrate the utility and limitations of this approach for profiling the large metagenomes typical of soils and the trace DNA present in drug seizures. We suggest that this type of profiling will be most effective once next-generation sequencing and advanced sequence analysis becomes routine.     

Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes detection kit in combination with ShortPrep foodproof II Kit. Performance-Tested Method 070401

A method was developed for the detection of L. monocytogenes in food based on real-time polymerase chain reaction (PCR). This advanced PCR method was designed to reduce the time needed to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the Roche/BIOTECON Diagnostics ShortPrep foodproof II Kit (formerly called Listeria ShortPrep Kit) designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is performed by using the Roche/BIOTECON Diagnostics LightCycler foodproof L. monocytogenes Detection Kit. This kit provides primers and hybridization probes for sequence-specific detection, convenient premixed reagents, and different controls for reliable interpretation of results. For repeatability studies, 20 different foods, covering the 15 food groups recommended from the AOAC Research Institute (AOAC RI) for L. monocytogenes detection were analyzed: raw meats, fresh produce/vegetables, processed meats, seafood, egg and egg products, dairy (cultured/noncultured), spices, dry foods, fruit/juices, uncooked pasta, nuts, confectionery, pet food, food dyes and colorings, and miscellaneous. From each food 20, samples were inoculated with a low level (1-10 colony-forming units (CFU)/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, 5 uninoculated samples were prepared from each food. The food samples were examined with the test kits and in correlation with the cultural methods according to U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) or U.S. Department of Agriculture (USDA)/Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook. After 48 h of incubation, the PCR method in all cases showed equal or better results than the reference cultural FDA/BAM or USDA/FSIS methods. Fifteen out of 20 tested food types gave exactly the same amount of positive samples for both methods in both inoculation levels. For 5 out of 20 foodstuffs, the PCR method resulted in more positives than the reference method after 48 h of incubation. Following AOAC RI definition, these were false positives because they were not confirmed by the reference method (false-positive rate for low inoculated foodstuffs: 5.4%; for high inoculated foodstuffs: 7.1%). Without calculating these unconfirmed positives, the PCR method showed equal sensitivity results compared to the alternative method. With the unconfirmed PCR-positives included into the calculations, the alternative PCR method showed a higher sensitivity than the microbiological methods (low inoculation level: 100 vs 98.0%; sensitivity rate: 1; high inoculation level: 99.7 vs 97.7%; sensitivity rate, 1). All in-house and independently tested uninoculated food samples were negative for L. monocytogenes. The ruggedness testing of both ShortPrep foodproof II Kit and Roche/BIOTECON LightCycler foodproof L. monocytogenes Detection Kit showed no noteworthy influences to any variation of the parameters component concentration, apparatus comparison, tester comparison, and sample volumes. In total, 102 L. monocytogenes isolates (cultures and pure DNA) were tested and detected for the inclusivity study, including all isolates claimed by the AOAC RI. The exclusivity study included 60 non-L. monocytogenes bacteria. None of the tested isolates gave a false-positive result; specificity was 100%. Three different lots were tested in the lot-to-lot study. All 3 lots gave equal results. The stability study was subdivided into 3 parts: long-term study, stress test, and freeze-defrost test. Three lots were tested in 4 time intervals within a period of 13 months. They all gave comparable results for all test intervals. For the stress test, LightCycler L. monocytogenes detection mixes were stored at different temperatures and tested at different time points during 1 month. Stable results were produced at all storage temperatures. The freeze-defrost analysis showed no noteworthy aggravation of test results. The independent validation study examined by Campden and Chorleywood Food Research Association Group (CCFRA) demonstrated again that the LightCycler L. monocytogenes detection system shows a comparable sensitivity to reference methods. With both the LightCycler PCR and BAM methods, 19 out of 20 inoculated food samples were detected. The 24 h PCR results generated by the LightCycler system corresponded directly with the FDA/BAM culture results. However, the 48 h PCR results did not relate exactly to the FDA/BAM results, as one sample found to be positive by the 48 h PCR could not be culturally confirmed and another sample which was negative by the 48 h PCR was culturally positive.     

Evaluation of a chromogenic medium for identification and differentiation of Listeria monocytogenes in selected foods

Listeria monocytogenes continues to be a threat to food safety in the United States despite a "zero tolerance" policy. When Listeria species are identified by standard cultural methods, confirmation of L. monocytogenes takes days to complete. RAPID'L.Mono agar, developed by Bio-Rad Laboratories, is a chromogenic medium that differentiates L. monocytogenes from other species of Listeria by a simple color change reaction. Differentiation is based on the specific detection of phosphatidylinositol phospholipase C activity, resulting in a blue colony, and the inability of L. monocytogenes to metabolize xylose, resulting in the absence of a yellow halo. Detection principles of standard method agars, Oxford and PALCAM, are based on the ability of all species of Listeria to hydrolyze esculin. Thus, all species of Listeria have similar colony morphology on these agars, making differentiation of pathogenic L. monocytogenes from other nonhuman pathogens difficult. RAPID'L.Mono agar has been validated with surimi, mixed salad, brie, and processed deli turkey because of the prevalence of L. monocytogenes in these foods. Sensitivity and specificity for this medium was determined to be 99.4 and 100%, respectively. Overall method agreement of RAPID'L.Mono with standard culture methods (U.S. Department of Agriculture/Food Safety and Inspection Service; U.S. Food and Drug Administration/Bacteriological Analytical Manual; and AOAC INTERNATIONAL) was excellent, with enrichment protocols 24 h shorter than those of standard methods.     

BIOTECON diagnostics foodproof Listeria monocytogenes Detection Kit, 5' nuclease in combination with the foodproof ShortPrep II Kit

A method was developed for the detection of Listeria monocytogenes in food. The method is based on real-time PCR using hydrolysis probes (5' Nuclease). This advanced PCR method was designed to reduce the time necessary to achieve results from PCR reactions and to enable the user to monitor the amplification of the PCR product simultaneously, in real-time. After DNA isolation using the BIOTECON foodproof ShortPrep II Kit designed for the rapid preparation of L. monocytogenes DNA for direct use in PCR, the real-time detection of L. monocytogenes DNA is carried out using the foodproof Listeria monocytogenes Detection Kit. The kit provides primers and hydrolysis probes for sequence-specific detection, convenient premixed reagents, and controls for reliable interpretation of results. For the internal comparison study, three different foods (soft cheese, coalfish, and smoked ham) were analyzed, chosen from the 15 food groups recommended by the AOAC Research Institute for detection of L. monocytogenes. From each food, 20 samples were inoculated with a low level (1-10 CFU/25 g) and 20 samples with a high level (10-50 CFU/25 g) of L. monocytogenes. Additionally, five nonspiked samples were prepared from each food. Depending on the matrix, the food samples were examined with the test kits and compared with the cultural methods according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook.     

Incorporation of guanosine gels into sieving matrices for length- and sequence-based separation of DNA in capillary electrophoresis

Sieving gels are used in capillary gel electrophoresis to resolve DNA strands of different lengths. For complex samples, however, such as those encountered in metagenomic analysis of microbial communities or biofilms, length-based separation may mask the true genetic diversity of the community since different organisms may contribute same-length DNA with different sequences. There is a need, therefore, for DNA separations based on both the length and sequence. Previous work has demonstrated the ability of guanosine gels (G-gels) to separate four single-stranded DNA 76-mers that differ by only a few A/G base substitutions. The goal of the present work is to determine whether G-gels could be combined with commercial sieving gels in order to simultaneously separate DNA based on both length and sequence. The results are given for the four 76-mers and for a standard dsDNA ladder. Commercial sieving gels were used alone and in combination with G-gels. For the 76-mers, the combined medium was less efficient than the G-gel alone but was able to achieve partial resolution. The combined medium was at least as effective as the sieving gel alone at resolving the denatured DNA ladder and showed indications of sequence-based resolution as well, as supported by MALDI-MS. The results show that the combined sieving gel/G-gel medium retains the selectivity of the individual media, providing a promising approach to simultaneous length- and sequence-based DNA separation for metagenomic analysis of complex systems.     

Single Conducting Polymer Nanowire Based Sequence‐Specific,Base‐Pair‐Length Dependant Label‐free DNA Sensor

We have fabricated a highly sensitive, simple and label‐free single polypyrrole (Ppy) nanowire based conductometric/chemiresistive DNA sensor. The fabrication was optimized in terms of probe DNA sequence immobilization using a linker molecule and using gold‐thiol interaction. Two resultant sensor designs working on two different sensing mechanisms (gating effect and work function based sensors) were tested to establish reliable sensor architecture with higher sensitivity and device‐to‐device reproducibility. The utility of the work function based configuration was demonstrated by detecting 19 base pair (bp) long breast cancer gene sequence with single nucleotide polymorphism (SNP) discrimination with high sensitivity, lower detection limit of ∼10⁻¹⁶ M and wide dynamic range (∼10⁻¹⁶ to 10⁻¹¹ M) in a small sample volume (30 µL). To further demonstrate the utility of the DNA sensor for detection of target sequences with different number of bases, targets with 21 and 36 bases were detected. These sequences have implications in environmental sample analysis or metagenomics. Sensor response showed increase with the number of bases in the target sequence. For long sequence (with 36 bases), effect of DNA alignment on sensor performance was studied.     

Phylogenetic implications and secondary structure analyses of Vigna mungo (L.) Hepper genotypes based on nrDNA ITS2 sequences

The internal transcribed spacers are highly preferred nuclear markers for the phylogenetic assessment of most eukaryotes, including plants. More recently, ITS2 has shown to possess equivalent phylogenetic significance as the entire ITS region. Vigna mungo L. Hepper is comparatively less explored from the molecular aspects as compared to the other species of the Vigna genus. The study presents the intra-individual characterization of 24 distinct genotypes Vigna mungo L. Hepper, using morphological as well as nrDNA ITS2 sequences and secondary structural data. The morphological characterization has been carried out using nine important agro-morphological traits. The molecular phylogeny of the sequence data, using the maximum parsimony and neighbor joining methods, shows the significant distinction based on the haplotypic variations amongst blackgram genotypes. The ITS2 secondary structures predicted using the homology modeling approach were compatible with the eukaryote-universal ITS2 secondary structure. The sequence-structure phylogeny reconstructed using the profile neighbour joining approach, also showed the presence of haplotypic variations in form of clusters on the phylogenetic tree. Further, the high GC content in the sequence data and highly negative ΔG values of the folded secondary structures ruled out the possibilities of the presence of any pseudogenes in the data set. Our analysis recommends the use of ITS2 sequence and secondary structure data at the intraspecific levels of plant taxonomical classification. Moreover, this study for the very first time reports the combined use morphological, and molecular data (using ITS2 sequence and secondary structural information) for the characterization of plants at the varietal level of taxonomical classification.     

Nonenzymatic protocol for Pseudomonas aeruginosa DNA preparation and rapid subtyping by mini pulsed-field gel electrophoresis.

The fastest protocol for Pseudomonas aeruginosa subtyping by contour clamped homogeneous electric field (CHEF) electrophoresis takes around 20 h. It includes enzymatic sample preparation, DNA restriction and fragment separation. Here, P. aeruginosa cells embedded in agarose miniplugs were lysed and deproteinized by incubating the miniplugs for 30 min in a single nonenzymatic solution. DNA molecules were digested for 2 h with 5 U of XbaI, and fragments were separated in 4.96 h by miniCHEF electrophoresis at 10 V/cm. Total time for P. aeruginosa subtyping was 8 h. Control experiments included DNA preparation by enzymatic or nonenzymatic protocols, different times of DNA restriction and comparisons of DNA separations done by miniCHEF or CHEF electrophoresis. Both methods and chambers gave similar results, but the rapid nonenzymatic method and the miniCHEF gave them in less time. Cells grown in broth or on plates were useful for nonenzymatic DNA preparation. Thirteen P. aeruginosa isolates were successfully fingerprinted using the protocol described here.     

Sensing Picomolar Concentrations of RNA Using Switchable Plasmonic Chirality

Detecting small sequences of RNA in biological samples such as microRNA or viral RNA demands highly sensitive and specific methods. Here, a reconfigurable DNA origami template has been used where a chiral arrangement of gold nanorods on the structure can lead to the generation of strong circular dichroism (CD). Switching of the cross‐like DNA structure is achieved by the addition of nucleic acid sequences, which arrests the structure in one of the possible chiral states by specific molecular recognition. A specific sequence can thus be detected through the resulting changes in the plasmonic CD spectrum. We show the sensitive and selective detection of a target RNA sequence from the hepatitis C virus genome. The RNA binds to a complementary sequence that is part of the lock mechanism, which leads to the formation of a defined state of the plasmonic system with a distinct optical response. With this approach, we were able to detect this specific RNA sequence at concentrations as low as 100 pm .     

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.     

Combining ligation reaction and capillary gel electrophoresis to obtain reliable long DNA probes

New DNA amplification methods are continuously developed for sensitive detection and quantification of specific DNA target sequences for, e.g. clinical, environmental or food applications. These new applications often require the use of long DNA oligonucleotides as probes for target sequences hybridization. Depending on the molecular technique, the length of DNA probes ranges from 40 to 450 nucleotides, solid-phase chemical synthesis being the strategy generally used for their production. However, the fidelity of chemical synthesis of DNA decreases for larger DNA probes. Defects in the oligonucleotide sequence result in the loss of hybridization efficiency, affecting the sensitivity and selectivity of the amplification method. In this work, an enzymatic procedure has been developed as an alternative to solid-phase chemical synthesis for the production of long oligonucleotides. The enzymatic procedure for probe production was based on ligation of short DNA sequences. Long DNA probes were obtained from smaller oligonucleotides together with a short sequence that acts as bridge stabilizing the molecular complex for DNA ligation. The ligation reactions were monitored by capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF) using a bare fused-silica capillary. The capillary gel electrophoresis-LIF method demonstrated to be very useful and informative for the characterization of the ligation reaction, providing important information about the nature of some impurities, as well as for the fine optimization of the ligation conditions (i.e. ligation cycles, oligonucleotide and enzyme concentration). As a result, the yield and quality of the ligation product were highly improved. The in-lab prepared DNA probes were used in a novel multiplex ligation-dependent genome amplification (MLGA) method for the detection of genetically modified maize in samples. The great possibilities of the whole approach were demonstrated by the specific and sensitive detection of transgenic maize at percentages lower than 1%.     

High affinity, paralog-specific recognition of the Mena EVH1 domain by a miniature protein

Many protein domains involved in cell signaling contain or interact with proline-rich sequences, and the design of molecules that perturb signaling pathways represents a foremost goal of chemical biology. Previously we described a protein design strategy in which the well-folded alpha-helix in avian pancreatic polypeptide (aPP) presents short alpha-helical recognition epitopes. The miniature proteins designed in this way recognize even shallow protein clefts with high affinity and specificity. Here we show that the well-folded type-II polyproline helix in aPP can present the short PPII-helical recognition epitope within the ActA protein of Listeria monocytogenes. Like miniature proteins that use an alpha-helix for protein recognition, the miniature protein designed in this way displays high affinity for a natural ActA target, the EVH1 domain Mena1-112, and achieves the elusive goal of paralog specificity, discriminating well between EVH1 domains Mena1-112, VASP1-115, and Evl1-112. Most importantly, the miniature protein competed with ActA in Xenopus laevis egg cytoplasmic extracts, decreasing actin-dependent motility of L. monocytogenes and causing extreme speed variations and discontinuous tail formation. Our results suggest that miniature proteins based on aPP may represent an excellent framework for the design of ligands that differentiate the roles of EVH1 domains in vitro and in vivo.     

Reading Polymers: Sequencing of Natural and Synthetic Macromolecules

The sequencing of biopolymers such as proteins and DNA is among the most significant scientific achievements of the 20th century. Indeed, modern chemical methods for sequence analysis allow reading and understanding the codes of life. Thus, sequencing methods currently play a major role in applications as diverse as genomics, gene therapy, biotechnology, and data storage. However, in terms of fundamental science, sequencing is not really a question of molecular biology but rather a more general topic in macromolecular chemistry. Broadly speaking, it can be defined as the analysis of comonomer sequences in copolymers. However, relatively different approaches have been used in the past to study monomer sequences in biological and manmade polymers. Yet, these “cultural” differences are slowly fading away with the recent development of synthetic sequence‐controlled polymers. In this context, the aim of this Minireview is to present an overview of the tools that are currently available for sequence analysis in macromolecular science.     

Rapid determination of Listeria monocytogenes by automated enzyme-linked immunoassay and nonradioactive DNA probe

A rapid and reliable analytical method was developed to detect and confirm the presence of Listeria monocytogenes in raw and partially processed foods. Forty-nine food samples (25 mixed cut vegetable salad, 12 smoked salmon, and 12 sterile smoked salmon) were individually inoculated with high levels [10-100 colony forming units (cfu)/25 g sample] and low levels (1-10 cfu/25 g sample) of L. monocytogenes, and were screened using the Vitek Immuno Diagnostic Assay (VIDAS) Listeria monocytogenes (VIDAS LMO)]. Positive test results were confirmed as L. monocytogenes by nonradioactive DNA probe. All samples inoculated with high levels of L. monocytogenes were detected by VIDAS and 96% were confirmed as L. monocytogenes by DNA probe. VIDAS LMO detected 89% of samples inoculated with low levels of L. monocytogenes, and 87% of these were confirmed as positive by DNA probe. In addition, 12 other samples (4 from each of mixed cut vegetable salad, smoked salmon, and sterile smoked salmon) were inoculated with high levels of L. ivanovii, L. seeligeri, L. welshimeri, L. innocua, L. grayi, and L. murrayi. Samples were assayed by the same protocol and all gave negative results. Compared with the cultural method, the VIDAS LMO nonradioactive DNA probe combination is highly specific, discriminates between L. monocytogenes and all other Listeria species, and reduces analytical time.     

Predicting protein folding rates using the concept of Chou's pseudo amino acid composition

One of the most important challenges in computational and molecular biology is to understand the relationship between amino acid sequences and the folding rates of proteins. Recent works suggest that topological parameters, amino acid properties, chain length and the composition index relate well with protein folding rates, however, sequence order information has seldom been considered as a property for predicting protein folding rates. In this study, amino acid sequence order was used to derive an effective method, based on an extended version of the pseudo-amino acid composition, for predicting protein folding rates without any explicit structural information. Using the jackknife cross validation test, the method was demonstrated on the largest dataset (99 proteins) reported. The method was found to provide a good correlation between the predicted and experimental folding rates. The correlation coefficient is 0.81 (with a highly significant level) and the standard error is 2.46. The reported algorithm was found to perform better than several representative sequence-based approaches using the same dataset. The results indicate that sequence order information is an important determinant of protein folding rates.     

Evaluation of applied biosystems MicroSeq real-time PCR system for detection of Listeria monocytogenes in food. Performance Tested Method 011002

Increasingly, more food companies are relying on molecular methods, such as PCR, for pathogen detection due to their improved simplicity, sensitivity, and rapid time to results. This report describes the validation of a new Real-Time PCR method to detect Listeria monocytogenes in nine different food matrixes. The complete system consists of the MicroSEQ L. monocytogenes Detection Kit, sample preparation, the Applied Biosystems 7500 Fast Real-Time PCR instrument, and RapidFinder Express software. Two sample preparation methods were validated: the PrepSEQ Nucleic Acid extraction kit and the PrepSEQ Rapid Spin sample preparation kit. The test method was compared to the ISO 11290-1 reference method using an unpaired-study design to detect L. monocytogenes in roast beef, cured bacon, lox (smoked salmon), lettuce, whole cow's milk, dry infant formula, ice cream, salad dressing, and mayonnaise. The MicroSEQ L. monocytogenes Detection Kit and the ISO 11290-1 reference method showed equivalent detection based on Chi-square analysis for all food matrixes when the samples were prepared using either of the two sample preparation methods. An independent validation confirmed these findings on smoked salmon and whole cow's milk. The MicroSEQ kit detected all 50 L. monocytogenes strains tested, and none of the 30 nontargeted bacteria strains.