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The catalase or catalase-peroxidase activity commonly exists in many pathogens and plays an important role in resisting the oxidative burst of phagocytes helping the pathogen persistently colonize in the host. Yersinia pestis is a facultative pathogen and the causative agent of plague. KatY has been identified as a thermosensing antigen with modest catalase activity in this pathogen. Here Y. pestis KatA and KatY were experimentally confirmed as a monofunctional catalase and bifunctional catalase-peroxidase, respectively. Their expression induced by H2O2 was proven to be mediated by the oxidative regulator, OxyR. Expression of KatA changed with growth phases and was crucial to its traditional physiological role in protecting Y. pestis cells against toxicity of exogenous H2O2. KatY was regulated by temperature and H2O2, two major elements of phagolysosomal microenvironments. Consistent with the above results, gene expression of katY increased significantly during intracellular growth of Y. pestis compared with that in vitro growth. However, a DeltakatY mutant was fully virulent to mice, suggesting that KatY is not required for Y. pestis virulence.

In colony collapse disorder (CCD), honey bee colonies inexplicably lose their workers. CCD has resulted in a loss of 50 to 90% of colonies in beekeeping operations across the United States. The observation that irradiated combs from affected colonies can be repopulated with naive bees suggests that infection may contribute to CCD. We used an unbiased metagenomic approach to survey microflora in CCD hives, normal hives, and imported royal jelly. Candidate pathogens were screened for significance of association with CCD by the examination of samples collected from several sites over a period of 3 years. One organism, Israeli acute paralysis virus of bees, was strongly correlated with CCD.

The FOB-3, anew type fiber optic biosensor, is designed to rapidly detect a variety of biological agents or analytes with better stability, sensitivity and specificity. In order to detect Y. Pestis, a sandwich immunoassay was developed by using the purified antibody against antigen FI immobilized on polystyrene probes as the capture antibody and the monoclonal antibody-Cy5 conjugate as the detector. After a series of optimization for the stability, sensitivity and specificity of the FOB-3, 50-1000 ng/ml of antigen FI and 6 x 10(1)-6 x 10(7) CFU/ml Y. pestis could be detected constantly in about 20 min, and Y pestis could be detected specifically from Y. pseudotuberculosis, Y. enterocolitica, B. anthracis and E. coli. Then, 39 blind samples, including 27 tissues of mice infected with Y pestis and 12 tissues of healthy mice as negative control, were detected with the FOB-3. 92.6% infected tissues were identified from the tissues of healthy mice and the tissues containing more than 100 CFU/ml bacteria could be detected by the biosensor. The results demonstrated the feasibility of the FOB-3 as an effective method to detect Y. pestis rapidly and directly from the infected animal specimens with the advantage of portability, simple-operation as well as high sensitivity and specificity. (c) 2006 Elsevier B.V. All rights reserved.

Polymerase chain reaction (PCR) is widely applied in clinical and environmental microbiology. Primer design is key to the development of successful assays and is often performed manually by using multiple nucleic acid alignments. Few public software tools exist that allow comprehensive design of degenerate primers for large groups of related targets based on complex multiple sequence alignments. Here we present a method for designing such primers based on tree building followed by application of a set covering algorithm, and demonstrate its utility in compiling Multiplex PCR primer panels for detection and differentiation of viral pathogens.

Using DNA microarray analysis, mRNA levels from wild-type Yersinia pestis cells treated with the iron chelator 2,2′-dipyridyl were compared with those supplemented with excessive iron, and subsequent to this, gene expression in the fur mutant was compared with that in the wild-type strain under iron rich conditions. The microarray analysis revealed many iron transport or storage systems that had been induced in response to the iron starvation, which is mediated by the Fur protein, using the iron as a co-repressor. The iron–Fur complex also affected some genes involved in various non-iron functions (ribonucleoside-diphosphate reductase, membrane proteins, electron transport and oxidative defense, etc.). The Fur protein still participated in the regulation of genes involved in broad cellular processes (virulence factors, pesticin activity, haemin storage and many proteins with unknown functions) that were not affected by iron depletion conditions. In addition to its classical negative regulatory activities, the Fur protein activates gene transcription. Using bioinformatics tools, we were able to predict the Y. pestis Fur box sequence that was clearly the over-presented motif in the promoter regions of members of the iron–Fur modulon.

Oligonuleotides (oligo) microarray is a promising method for virus genotyping. In this study, we developed an oligo microarray used for genotyping hepatitis B virus (HBV). It consists of 15 probes targeting HBV genotypes A-G and two genotypes of apes, and one conserved probe selected from the HBV pre-S region. To test a clinical sample, the gene targets of clinical samples were amplified and labelled with Cy5-dCTP in a PCR reaction using primers covering the flanking region of the HBV pre-S gene. Following purification, the labelled PCR products were hybridized to the microarray. In an analysis of 96 HBV patients' serum samples using our developed microarray, we identified 34 genotype B, 60 genotype C and 2 genotypes B/C coinfection samples. Sequencing results of 24 randomly selected samples agreed 100% with microarray data. Geographic distribution of genotypes C and B was also divergent, 95.7% (44/46) of genotype C and 64.0% (32/50) of genotype B samples were collected from Northern and Southern China, respectively. The accuracy of genotyping was confirmed by analyzing DNA sequences of 24 samples. It is demonstrated that low-density oligo microarray can serve as a reliable and time-saving method to HBV genotyping from patient's sera.

Yersinia pestis, the causative agent of bubonic and pneumonic plague, has been classified into four biovars: Antiqua, Mediaevalis, Orientalis and Microtus. Although the entire genome sequences of three Y. pestis strains, CO92, KIM and 91001, of biovar Orientalis, Mediaevalis and Microtus, respectively, have been decoded, the genome sequence of the biovar Antiqua strain is unknown. In an initial effort to find Antiqua-specific sequences, suppression subtractive hybridization (SSH) was performed and four different regions (DFRs) were identified. Among the four DFRs, only DFR4 was specific to the tester (strain 49006, biovar Antiqua). PCR demonstrated that DFR4 was present only in 57 of 60 Antiqua strains from the Marmota baibacina-Spermophilus undulates plague focus in the Tianshan Mountains (focus B) and in three strains of Y. pseudotuberculosis (serotypes I and II), showing that not all Antiqua strains had DFR4. Five DFR profiles were identified based on the presence or absence of these four DFRs in 636 strains of Y. pestis from 10 plague foci in China.

DNA microarray was used as a tool to investigate genome-wide transcriptional responses of Yersinia pestis to hyperosmotic and high-salinity stress. Hyperosmotic stress specifically upregulated genes responsible for ABC-type transport and the cytoplasmic accumulation of certain polysaccharides, while high-salinity stress induced the transcription of genes encoding partition proteins and several global transcriptional regulators. Genes whose transcription was enhanced by both kinds of stress comprised those encoding osmoprotectant transport systems and a set of virulence determinants. The number of genes downregulated by the two kinds of stress was much lower than that of upregulated genes, suggesting that neither kind of stress severely depresses cellular processes in general. Many differentially regulated genes still exist whose functions remain unknown. Y. pestis recognized high-salinity and hyperosmotic stress as different kinds of environmental stimuli, and different mechanisms enabled acclimation to these two kinds of stress, although Y. pestis still executed common mechanisms to accommodate both types of stress.

Plague is a natural focus-based disease, and for better understanding of this disease it is crucial to determine the molecular mechanisms of its pathogen, Yersinia pestis, for adapting to different foci. Gene inactivation, loss and acquisition are the main mechanisms that contribute to a pathogen's fitness. Determination of the whole-genome sequences of three Y. pestis strains, CO92, KIM and 91001, provided a good opportunity to probe into its genome in minute detail. Many genetic variations were found between the three strains. The present work focused on adaptive microevolutionary analysis of Y. pestis from different natural plague foci in China based on pseudogene profiles. Twenty-four mutations that led to inactivation in the corresponding genes were analysed, and a PCR-based screening method was employed to investigate the distribution of these mutations among Y. pestis isolates from different foci and also among seven strains of Yersinia pseudotuberculosis. It was found that Y. pestis isolates from the same focus had identical mutation profiles, and 260 isolates of Y. pestis were divided into eight genotypes, while Y. pseudotuberculosis harboured wild-type alleles for all the mutations. The isolates of three known biovars were grouped into distinct branches in the phylogenetic tree, which supports the proposition that biovars mediaevalis and orientalis directly arose from biovar antiqua individually. The constructed phylogenetic tree suggests that the isolates from focus B should be the oldest lineage of Y. pestis in China except for isolates from foci L and M, which might be a special lineage of Y. pestis and originated differently to the others.

A PCR-based screening method was used to study the genetic variations of the pgm locus among natural isolates of Yersinia pestis from China. Our results indicate that genetic variations in the pgm locus are well correlated with biovars of Y. pestis and plague foci, suggesting that the pgm locus plays a role in Y. pestis adaptation to its environment. The gene encoding two-component regulatory system sensor kinase became a pseudogene in all strains of biovar Orientalis due to a thymidine deletion, while it is intact in all the strains of the other biovars. Only strains from Foci H and L are the same as Yersinia pseudotuberculosis in that they have an intact transmembrane helix in the sensor kinase protein, which is lost in all the other strains because of the 18 bp in-frame deletion. The IS100 element that flanks the 39 terminus of the pgm locus was inserted into the chromosome during the within-species microevolution of Y. pestis, which is absent in strains from Foci G, H and L and also in Y. pseudotuberculosis. This fact indicates that the strains from these three foci are of an older lineage of Chinese Y. pestis. It is this IS100 element's absence that maintained high stability of the pgm locus in the Y. pestis strains from these three foci. The IS285 element insertion in the pigmentation segment and the IS100 element insertion in the downstream flanking region of the pgm locus are only present in strains from Foci H and L. The flanking region outside the 59 terminus of the upstream IS100 element is identical in the strains from these two foci, which is different in the other strains. All of these unique characteristics suggest that they are of a special lineage of Chinese Y. pestis.

Genomics research provides an unprecedented opportunity for us to probe into the pathogenicity and evolution of the world's most deadly pathogenic bacterium, Yersinia pestis , in minute detail. In our present work, extensive microarray analysis in conjunction with PCR validation revealed that there are considerable genome dynamics, due to gene acquisition and loss, in natural populations of Y. pestis . We established a genomotyping system to group homologous isolates of Y. pestis , based on profiling or gene acquisition and loss in their genomes, and then drew an outline of parallel microevolution of the Y. pestis genome. The acquisition of a number of genomic islands and plasmids most likely induced Y. pestis to evolve rapidly from Yersinia pseudotuberculosis to a new, deadly pathogen. Horizontal gene acquisition also plays a key role in the dramatic evolutionary segregation of Y. pestis lineages (biovars and genomovars). In contrast to selective genome expansion by gene acquisition, genome reduction occurs in Y. pestis through the loss of DNA regions. We also theorized about the links between niche adaptation and genome microevolution. The transmission, colonization, and expansion of Y. pestis in the natural foci of endemic plague are parallel and directional and involve gradual adaptation to the complex of interactions between the environment, the hosts, and the pathogen itself. These adaptations are based on the natural selections against the accumulation of genetic changes within genome. Our data strongly support that the modern plague originated from Yunnan Province in China, due to the arising of biovar orientalis from biovar antiqua rather than mediaevalis.

Yersinia pestis has been historically divided into three biovars: antiqua, mediaevalis, and orientalis. On the basis of this study, strains from Microtus -related plague foci are proposed to constitute a new biovar, microtus. Based on the ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis strains can be assigned to one of four biovars: antiqua (glycerol positive, arabinose positive, and nitrate positive), mediaevalis (glycerol positive, arabinose positive, and nitrate negative), orientalis (glycerol negative, arabinose positive, and nitrate positive), and microtus (glycerol positive, arabinose negative, and nitrate negative). A 93-bp in-frame deletion in glpD gene results in the glycerol-negative characteristic of biovar orientalis strains. Two kinds of point mutations in the napA gene may cause the nitrate reduction-negative characteristic in biovars mediaevalis and microtus, respectively. A 122-bp frameshift deletion in the araC gene may lead to the arabinose-negative phenotype of biovar microtus strains. Biovar microtus strains have a unique genomic profile of gene loss and pseudogene distribution, which most likely accounts for the human attenuation of this new biovar. Focused, hypothesis-based investigations on these specific genes will help delineate the determinants that enable this deadly pathogen to be virulent to humans and give insight into the evolution of Y. pestis and plague pathogenesis. Moreover, there may be the implications for development of biovar microtus strains as a potential vaccine.

During the 2003 severe acute respiratory syndrome (SARS) outbreak, a real-time quantitative polymerase chain reaction, which targets the nucleocapsid gene at the 3′-end of the viral genome, was established to detect and identify the SARS-associated coronavirus. We describe the use of this assay to screen >700 clinical samples.

Compared to advances in enzyme sensors, immunosensors, and microbial biosensors, relatively little work exists on DNA based biosensors. Here we review the DNA based biosensors that rely on nucleic acid hybridization. Major types DNA biosensors—electrochemical, optical, acoustic, and piezoelectric—are introduced and compared. The specificity and response characteristics of DNA biosensors are discussed. Overall, a promising future is foreseen for the DNA based sensor technology.

Background DFR (different region) analysis has been developed for typing Yesinia pestis in our previous study, and in this study, we extended this method by using 23 DFRs to investigate 909 Chinese Y. pestis strains for validating DFR-based genotyping method and better understanding adaptive microevolution of Y. pestis. Methodology/Principal Findings On the basis of PCR and Bionumerics data analysis, 909 Y. pestis strains were genotyped into 32 genomovars according to their DFR profiles. New terms, Major genomovar and Minor genomovar, were coined for illustrating evolutionary relationship between Y. pestis strains from different plague foci and different hosts. In silico DFR profiling of the completed or draft genomes shed lights on the evolutionary scenario of Y. pestis from Y. pseudotuberculosis. Notably, several sequenced Y. pestis strains share the same DFR profiles with Chinese strains, providing data for revealing the global plague foci expansion. Conclusions/significance Distribution of Y. pestis genomovars is plague focus-specific. Microevolution of biovar Orientalis was deduced according to DFR profiles. DFR analysis turns to be an efficient and inexpensive method to portrait the genome plasticity of Y. pestis based on horizontal gene transfer (HGT). DFR analysis can also be used as a tool in comparative and evolutionary genomic research for other bacteria with similar genome plasticity.

The ferric uptake regulator (Fur) is a predominant bacterial regulator controlling the iron assimilation functions in response to iron availability. Our previous microarray analysis on Yersinia pestis defined the iron-Fur modulon. In the present work, we reannotated the iron assimilation genes in Y. pestis , and the resulting genes in complementation with those disclosed by microarray constituted a total of 34 genome loci (putative operons) that represent the potential iron-responsive targets of Fur. The subsequent real-time reverse transcription-PCR (RT-PCR) in conjunction with the primer extension analysis showed that 32 of them were regulated by Fur in response to iron starvation. A previously predicted Fur box sequence was then used to search against the promoter regions of the 34 operons; the homologue of the above box could be predicted in each promoter tested. The subsequent electrophoretic mobility shift assay (EMSA) demonstrated that a purified His 6 tag-fused Fur protein was able to bind in vitro to each of these promoter regions. Therefore, Fur is a global regulator, both an activator and a repressor, and directly controls not only almost all of the iron assimilation functions but also a variety of genes involved in various non-iron functions for governing a complex regulatory cascade in Y. pestis . In addition, real-time RT-PCR, primer extension, EMSA, and DNase I footprinting assay were used to elucidate the Fur regulation of the ybt locus encoding a virulence-required iron uptake system. By combining the published data on the YbtA regulation of ybt , we constructed a concise Fur/YbtA regulatory network with a map of the Fur-promoter DNA interactions within the ybt locus. The data presented here give us an overview of the iron-responsive Fur regulon in Y. pestis .

Filoviruses have the capacity to cause lethal outbreaks of hemorrhagic fever in primates. Here we present a simple consensus reverse transcription-PCR method for filovirus recognition and characterization and demonstrate its utility with all known filovirus strains. Phylogenetic assignment is achieved by automated web-based sequence analysis of amplification products.

Using DNA microarray analysis, mRNA levels from wild-type Yersinia pestis cells treated with the iron chelator 2,2'-dipyridyl were compared with those supplemented with excessive iron, and subsequent to this, gene expression in the fur mutant was compared with that in the wild-type strain under iron rich conditions. The microarray analysis revealed many iron transport or storage systems that had been induced in response to the iron starvation, which is mediated by the Fur protein, using the iron as a co-repressor. The iron-Fur complex also affected some genes involved in various non-iron functions (ribonucleoside-diphosphate reductase, membrane proteins, electron transport and oxidative defense, etc.). The Fur protein still participated in the regulation of genes involved in broad cellular processes (virulence factors, pesticin activity, haemin storage and many proteins with unknown functions) that were not affected by iron depletion conditions. In addition to its classical negative regulatory activities, the Fur protein activates gene transcription. Using bioinformatics tools, we were able to predict the Y. pestis Fur box sequence that was clearly the over-presented motif in the promoter regions of members of the iron-Fur modulon.

A novel coronavirus, the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), was identified as the causative agent of SARS. The profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. In this study, 13 recombinant proteins associated with four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were prepared and used for screening and monitoring their specific IgG antibodies in SARS patient sera by protein microarray. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase SARS patients, whereas those to proteins E, M, 3b, 6 and 7a were undetected. In the detectable specific antibodies, anti-S and anti-N were dominant and could persist in the sera of SARS patients until week 30. Among the rabbit antisera to recombinant proteins S3, N, 3a and 9b, only anti-S3 serum showed significant neutralizing activity to the SARS-CoV infection in Vero E6 cells. The results suggest (1) that anti-S and anti-N antibodies are diagnostic markers and in particular that S3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle.

Yersinia pestis, the etiologic agent of plague, must acclimatize itself to temperature shifts between the temperature (26 C) for flea blockage and the body temperature (37 C) of warm-blooded hosts during its life cycle. Here a whole-genome DNA microarray was used to investigate transcriptional regulation upon the upshift of growth temperature from 26 to 37 C in a chemically defined medium. Four hundred and one genes were regulated differentially under the two temperatures. About 39% of these genes were up-regulated at 37 C, whereas 61% were down-regulated. Temperature-induced changes occurred at the level of transcription of genes encoding proven or predicted virulence factors, regulators, metabolism-associated proteins, prophages, and hypothetical proteins. Strikingly, many gene clusters displayed a co-transcription pattern in response to temperature upshift. Our data provided a genome-wide profile of gene transcription induced by temperature shift and should shed light on the pathogenicity and host-microbe interaction of this deadly pathogen.

Plague, caused by Y ersinia pestis, is one of the most dangerous diseases that impressed a horror onto human consciousness that persists to this day. Cases of plague can be normally controlled by timely antibiotic administration. Streptomycin is the first-line antibiotic for plague treatment. In this study, a DNA microarray was used to investigate the changes in the gene expression profile of Y. pestis upon exposure to streptomycin. A total of 345 genes were identified to be differentially regulated, 144 of which were up-regulated, and 201 down-regulated. Streptomycin-induced transcriptional changes occurred in genes responsible for heat shock response, drug/analogue sensitivity, biosynthesis of the branched-chain amino acids, chemotaxis and mobility and broad regulatory functions. A wide set of genes involved in energy metabolism, biosynthesis of small macromolecules, synthesis and modification of macromoclecules and degradation of small and macro molecules were among those down-regulated. The results reveal general changes in gene expression that are consistent with known mechanisms of action of streptomycin and many new genes that are likely to play important roles in the response to streptomycin treatment, providing useful candidates for investigating the specific mechanisms of streptomycin action.

DNA microarray was used as a tool to define the heat- and cold-shock stimulons in Yersinia pestis. Heat shock dramatically enhanced the transcription of genes encoding major heat-shock proteins (MHSPs) that are important for cell survival against the heat. Many other genes were also greatly up-regulated, but their roles in heat-shock response need to be elucidated. Meanwhile, heat shock retarded most of the metabolic processes, i.e. RNA transcription, protein translation, aerobic respiration, energy metabolism, small molecule metabolism, peptidoglycan biosynthesis, sulfate uptake and cysteine biosynthesis. In response to cold shock, Y. pestis has evolved complex adaptive mechanisms by elevating the transcription of a specific set of genes whose protein products are designed to prevent or eliminate cold-induced DNA or RNA structuring, to remodel cell membrane components for maintenance of normal functions, to elevate the energy generation for ensuring ATP-dependent responses during cold adaptation and to synthesize or transport compatible solutes such as cryoprotectants, and at the same time, by repressing the mRNA level of certain genes whose protein products are not needed for bacterial growth at low temperatures, such as the MHSPs. These results provide a set of new candidate genes for hypothesis-based investigations of their roles in stress response, host adaptation and pathogenicity of this deadly pathogen.

Polymerase chain reaction (PCR) amplification of DNA-based unique markers, the signature sequences, is ideal for rapid detection and identification of pathogens. We described the discovery of twenty-eight signature genes of Yersinia pestis by DNA microarray-based comparative genome hybridization in conjunction with PCR validation. Three pairs of Y. pestis-specific primers designed from signature genes were demonstrated to have the expected specificity to this target bacterium, without cross-reaction with the closely related Y. pseudotuberculosis or a large collection of genomic DNAs from other organisms.

Background: The widespread threat of severe acute respiratory syndrome (SARS) to human health has made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use. For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein. Methods: The cDNA for full-length N protein and its various regions from the SARS-CoV was cloned and expressed in Escherichia coli. After purification, all of the protein fragments were printed on glass slides to fabricate a protein microarray and then probed with the sera from SARS patients to determine the reactivity of these protein fragments. Results: The full-length protein and two other fragments reacted with all 52 sera tested. Four important regions with possible epitopes were identified and named as EP1 (amino acids 51–71), EP2 (134–208), EP3 (249–273), and EP4 (349–422), respectively. EP2 and EP4 possessed linear epitopes, whereas EP1 and EP2 were able to form conformational epitopes that could react with most (>80%) of the tested sera. EP3 and EP4 also formed conformational epitopes, and antibodies against these epitopes existed in all 52 of the sera tested. Conclusion: The N protein is a highly immunogenic protein of the SARS-CoV. Conformational epitopes are important for this protein, and antigenicity of the COOH terminus is higher than that of the NH2 terminus. The N protein is a potential diagnostic antigen and vaccine candidate for SARS-CoV.

Yersinia pestis whole-genome DNA microarrays were developed to perform genomic comparison of a collection of live plague vaccines. By using the genomic DNA to probe the DNA microarrays, we detected dozens of deletions and amplifications of the genomic regions in the 19 vaccine strains analyzed. The revealed genomic differences within the vaccine strains of different origins provide us an unprecedented opportunity to understand the molecular background of the variability of the immunogenic and protective potency of plague live vaccine. The whole-genome DNA microarray also provides an ideal tool to perform the pre-evaluation of a vaccine strain for its high throughput to determine the genomic features essential or unallowable for the live vaccines.

Background Although assays for detecting Yersinia pestis using TaqMan probe-based real-time PCR have been developed for years, little is reported on room-temperature-stable PCR reagents, which will be invaluable for field epidemic surveillance, immediate response to public health emergencies, counter-bioterrorism investigation, etc. In this work, a set of real-time PCR reagents for rapid detection of Y. pestis was developed with extraordinary stability at 37°C. Methods/Principal Findings TaqMan-based real-time PCR assays were developed using the primers and probes targeting the 3a sequence in the chromosome and the F1 antigen gene caf1 in the plasmid pMT1of Y. pestis, respectively. Then, carbohydrate mixtures were added to the PCR reagents, which were later vacuum-dried for stability evaluation. The vacuum-dried reagents were stable at 37°C for at least 49 days for a lower concentration of template DNA (10 copies/µl), and up to 79 days for higher concentrations (≥102 copies/µl). The reagents were used subsequently to detect soil samples spiked with Y. pestis vaccine strain EV76, and 5×104 CFU per gram of soil could be detected by both 3a- and caf1-based PCR reagents. In addition, a simple and efficient method for soil sample processing is presented here. Conclusions/Significance The vacuum-dried reagents for real-time PCR maintain accuracy and reproducibility for at least 49 days at 37°C, indicating that they can be easily transported at room temperature for field application if the machine for performing real-time PCR is available. This dry reagent is of great significance for routine plague surveillance., Author Summary Plague, caused by Yersinia pestis, is one of the oldest and most dangerous diseases in human history, and has claimed millions of lives in the three major historical pandemics. Although panic caused by the Black Death is fading, the threat of the reemergence of plague pandemics still exists, with the additional potential of misuse in biowarfare or bioterrorism. Rapid on-site detection and identification of the pathogen is of paramount significance for timely implementation of effective countermeasures. TaqMan probe-based real-time PCR assays can give quick and accurate identification; however, the need for cold delivery and storage prevents its potential on-site application. The objective of this study was to develop a stable PCR system for easy delivery and storage under room temperature, which is vital for conventional plague surveillance and for preparedness in public health emergencies. We present a solution to this particular issue, hoping that it is helpful to future applications.

Viral hemorrhagic fevers are associated with high rates of illness and death. Although therapeutic options are limited, early differential diagnosis has implications for containment and may aid in clinical management. We describe a diagnostic system for rapid, multiplex polymerase chain reaction identification of 10 different causes of viral hemorrhagic fevers.

Background The transcription regulator PhoP has been shown to be important for Y. pestis survival in macrophages and under various in vitro stresses. However, the mechanism by which PhoP promotes bacterial intracellular survival is not fully understood. Our previous microarray analysis suggested that PhoP governed a wide set of cellular pathways in Y. pestis. A series of biochemical experiments were done herein to study members of the PhoP regulon of Y. pestis biovar Microtus. Results By using gel mobility shift assay and quantitative RT-PCR, a total of 30 putative transcription units were characterized as direct PhoP targets. The primer extension assay was further used to determine the transcription start sites of 18 PhoP-dependent promoters and to localize the -10 and -35 elements. The DNase I footprinting was used to identify the PhoP-binding sites within 17 PhoP-dependent promoters, enabling the identification of PhoP box and matrix that both represented the conserved signals for PhoP recognition in Y. pestis. Data presented here providing a good basis for modeling PhoP-promoter DNA interactions that is crucial to the PhoP-mediated transcriptional regulation. Conclusion The proven direct PhoP targets include nine genes encoding regulators and 21 genes or operons with functions of detoxification, protection against DNA damages, resistance to antimicrobial peptides, and adaptation to magnesium limitation. We can presume that PhoP is a global regulator that controls a complex regulatory cascade by a mechanism of not only directly controlling the expression of specific genes, but also indirectly regulating various cellular pathways by acting on a set of dedicated regulators. These results help us gain insights into the PhoP-dependent mechanisms by which Y. pestis survives the antibacterial strategies employed by host macrophages.