A Color Image Analysis Method for Assessment of Germination Based on Differential Fluorescence Staining of Bacterial Spores and Vegetative Cells Using Acridine Orange

Color fluorescence image analysis of acridine orange (AO) stained germinating Bacillus subtilis var. niger bacteria revealed a cell population initially dominated by small green spores followed by the emergence of at least three additional discernible subpopulations in response to stimulation with D-glucose. These subpopulations were small, round or oblong red cells; intermediate to large metachromatic cells; and large red rods. Large green rods were rarely observed. An increase in red emissions (i.e., putative RNA synthesis) was sometimes seen as early as 90 min after exposure to D-glucose and uptake of AO at room temperature. This may represent either metabolic recovery from quiescence or RNA synthesis associated with germination. In the absence of D-glucose, or using autoclaved bacteria in the presence of glucose, no relative increase in the red signal was observed despite hours of observation. Digital image analysis was used for relative measurement of red, green and blue signals and to correlate the size of various subpopulations with their fluorescence color emissions over time. Image analysis demonstrated a trend toward increasing size and red emission in the presence of glucose. The average red emission was found to be a good discriminator of the various subpopulations, while the average green emission was approximately equal among the subpopulations making it a poor discriminator. These data suggest that AO staining might be used for rapid computer-assisted discrimination of spores vs. vegetative cells.     

Protection of Bacterial Spores in Space, a Contribution to the Discussion on Panspermia

Spores of Bacillus subtilis were exposed to space in theBIOPAN facility of the European Space Agency onboard of the Russian Earth-orbiting FOTON satellite. The spores were exposed either in dry layers without any protecting agent, or mixed withclay, red sandstone, Martian analogue soil or meteorite powder,in dry layers as well as in so-called `artificial meteorites', i.e. cubes filled with clay and spores in naturally occurring concentrations. After about 2 weeks in space, their survival was tested from the number of colony formers. Unprotected spores in layers open to space or behind a quartz window were completely or nearly completely inactivated (survival rates in most cases10^-6). The same low survival was obtained behind a thin layer of clay acting as an optical filter. The survival rate was increased by 5 orders of magnitude and more, if the spores in the dry layer were directly mixed with powder of clay,rock or meteorites, and up to 100% survival was reached in soilmixtures with spores comparable to the natural soil to spore ratio. These data confirm the deleterious effects of extraterrestrial solar UV radiation. Thin layers of clay, rock or meteorite are only successful in UV-shielding, if they are indirect contact with the spores. The data suggest that in a scenario of interplanetary transfer of life, small rock ejecta ofa few cm in diameter could be sufficiently large to protectbacterial spores against the intense insolation; however, micron-sized grains, as originally requested by Panspermia, may notprovide sufficient protection for spores to survive. The data arealso pertinent to search for life on Mars and planetaryprotection considerations for future missions to Mars.     

Rapid Identification of ESKAPE Bacterial Strains Using an Autonomous Microfluidic Device

This article describes Bacteria ID Chips ('BacChips'): an inexpensive, portable, and autonomous microfluidic platform for identifying pathogenic strains of bacteria. BacChips consist of a set of microchambers and channels molded in the elastomeric polymer, poly(dimethylsiloxane) (PDMS). Each microchamber is preloaded with mono-, di-, or trisaccharides and dried. Pressing the layer of PDMS into contact with a glass coverslip forms the device; the footprint of the device in this article is ～6 cm(2). After assembly, BacChips are degased under large negative pressure and are stored in vacuum-sealed plastic bags. To use the device, the bag is opened, a sample containing bacteria is introduced at the inlet of the device, and the degased PDMS draws the sample into the central channel and chambers. After the liquid at the inlet is consumed, air is drawn into the BacChip via the inlet and provides a physical barrier that separates the liquid samples in adjacent microchambers. A pH indicator is admixed with the samples prior to their loading, enabling the metabolism of the dissolved saccharides in the microchambers to be visualized. Importantly, BacChips operate without external equipment or instruments. By visually detecting the growth of bacteria using ambient light after ～4 h, we demonstrate that BacChips with ten microchambers containing different saccharides can reproducibly detect the ESKAPE panel of pathogens, including strains of: Enterococcus faecalis, Enteroccocus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter aerogenes, and Enterobacter cloacae. This article describes a BacChip for point-of-care detection of ESKAPE pathogens and a starting point for designing multiplexed assays that identify bacterial strains from clinical samples and simultaneously determine their susceptibility to antibiotics.     

Development of a New, Combined Rapid Method Using Phage and PCR for Detection and Identification of Viable Mycobacterium paratuberculosis Bacteria within 48 Hours

The FASTPlaqueTB assay is an established diagnostic aid for the rapid detection of Mycobacterium tuberculosis from human sputum samples. Using the FASTPlaqueTB assay reagents, viable Mycobacterium avium subsp. paratuberculosis cells were detected as phage plaques in just 24 h. The bacteriophage used does not infect M. avium subsp. paratuberculosis alone, so to add specificity to this assay, a PCR-based identification method was introduced to amplify M. avium subsp. paratuberculosis-specific sequences from the DNA of the mycobacterial cell detected by the phage. To give further diagnostic information, a multiplex PCR method was developed to allow simultaneous amplification of either M. avium subsp. paratuberculosis or M. tuberculosis complex-specific sequences from plaque samples. Combining the plaque PCR technique with the phage-based detection assay allowed the rapid and specific detection of viable M. avium subsp. paratuberculosis in milk samples in just 48 h.     

赤芝孢子粉中一个葡聚糖的分离纯化与结构鉴定

从破壁的赤芝 (Ganodermalucidum (Fr.)Karst)孢子粉中分离纯化到一个水溶性的、以 1,4连接的D_葡萄糖为主链、在 6位接 1,6连接葡萄糖的α_D 葡聚糖。高效分子排阻色谱测定 ,重均分子量为 9.3× 10 3 ,[α]2 1D = 174.2° (c 0 .87,H2 O)。通过糖组成分析 ,甲基化反应 ,乙酰解及一维和二维核磁等光谱解析 ,确定其结构如下 :　　　　　　　　　　　　　　　　α_D_Glcp_(1→ 6 )_α_D_Glcp_(1→ 6 )_α_D_Glcp　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 1　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　↓　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　 6　　　　　　　〔→ 4)_α_D_Glcp_(1→ 4)_α_D_Glcp_(1→ 4)_α_D_Glcp_(1→ 4)_α_D_Glcp_(1→ 4_)_α_D_Glcp_(1→〕n     

Structural Characterization and Immunomodulating Activity of a Complex Glucan from Spores of Ganoderma lucidum

A polysaccharide with a molecular weight of 1.26×10⁵, obtained from the sporoderm-broken spores of Ganoderma lucidum, was purified by anion-exchange and gel-permeation chromatography. This polysaccharide had a strong effect on suppressing the antibody production and the Con A or LPS induced lymphocyte proliferation in mice. Chemically, the structure of the polysaccharide was identified by methylation analysis, 1D, 2D NMR and ESI-MS spectroscopic studies of the native one and of the oligosaccharide fragments generated by partial acid hydrolysis, Smith degradation, and acetolysis. It was concluded that the intact polysaccharide was a complex β-D-glucan consisting of a (1→6)-linked backbone chain, in which every other glucosyl residue was substituted at C-3 or C-4 with mono-, di- and trisaccharide branches.     

Spores of a New Microsporidan Species Parasitizing Molluscan Neurons

Synopsis.
A new species of Microsporida was observed in neurons of the marine mollusc Aplysia californica and the ultrastructure of its spores was investigated. The spores were ˜ 1.3 m long and had a thick 3-layered coat. An anchoring disc and polar aperture were present. The polaroplast occupied most of the anterior half of the spore. The vesicular portion included lengths of loosely packed lamellar structures as well as the usual vesicular profiles embedded in fibrillar material; it was entirely surrounded by tightly packed, electron-dense lamellae. The polar tube had 5 or 6 coils. In this stage of the life cycle (mature spores), the parasites did not appear to be disturbing the host cells. The organism was named Microsporidium aplysiae sp. n.     

Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.     

2-Heptanone Production by Spores of Penicillium roquefortii in a Water-Organic Solvent Two-Phase System

The production of 2-heptanone from octanoic acid may be performed by free and entrapped spores of Penicillium roquefortii in a water-organic solvent two phase system.

An industrial, isoparafflnic solvent, i.e. Hydrosol IP 230 O.S., which may be considered as tetradecane, is well suited for the process. Activities nearly double those achieved with aqueous systems are observed using an initial fatty acid content in the organic layer close to 100 mM and a ratio of the volume of the organic phase to the total volume of the medium of 0.88. The presence of the solvent allows a better recovery of the metabolite by lowering its activity coefficient.

Fed-batch experiments performed in an aerated, stirred reactor show that the bioconversion may proceed in the two-phase system for at least 300 h. These conditions allow conversion of 750 mM (108 g · 1^-1) fatty acid, and production of 600 mM (68.5 g · 1^-1) 2-heptanone.     

银耳碱提孢子多糖A-BTF的分离与结构研究

银耳孢子发酵粉(Tremella fuciformisBerk),用热水煮提后,除去水溶性多糖。其沉淀用1M NaOH提取,Sevage法除去蛋白,用乙醇沉淀得到粗多糖。粗多糖经DEAE-32-cellulose和sephadex G-200反复分离后,纯化得到分布均一的多糖A-BTF。HPGPC测定A-BTF的分子量为67000,糖组成分析显示主要由葡萄糖组成。多糖A-BTF的甲基化产物,经水解、还原、乙酰化,通过GC-MS分析表明,主要含有1,6连接的葡萄糖和1,3,6连接的甘露糖,另外还有1,4连接的葡萄糖少量的半乳糖和1-NH2-来苏糖,末端为端基连接的葡萄糖。     

Rapid Identification of Candidate Genes for Seed Weight Using the SLAF-Seq Method in Brassica napus

Seed weight is a critical and direct trait for oilseed crop seed yield. Understanding its genetic mechanism is of great importance for yield improvement in Brassica napus breeding. Two hundred and fifty doubled haploid lines derived by microspore culture were developed from a cross between a large-seed line G-42 and a small-seed line 7–9. According to the 1000-seed weight (TSW) data, the individual DNA of the heaviest 46 lines and the lightest 47 lines were respectively selected to establish two bulked DNA pools. A new high-throughput sequencing technology, Specific Locus Amplified Fragment Sequencing (SLAF-seq), was used to identify candidate genes of TSW in association analysis combined with bulked segregant analysis (BSA). A total of 1,933 high quality polymorphic SLAF markers were developed and 4 associated markers of TSW were procured. A hot region of ~0.58 Mb at nucleotides 25,401,885–25,985,931 on ChrA09 containing 91 candidate genes was identified as tightly associated with the TSW trait. From annotation information, four genes (GSBRNA2T00037136001, GSBRNA2T00037157001, GSBRNA2T00037129001 and GSBRNA2T00069389001) might be interesting candidate genes that are highly related to seed weight.     

Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 10⁹ ± 5 × 10⁸ CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 10⁷ ± 5 × 10⁶ CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4′,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.Salmonella spp. are enteropathogenic bacteria that cause diseases that range from a mild gastroenteritis to systemic infections (5, 18) The disease severity is determined by the virulence characteristics of the Salmonella strain, host species, and host health condition. Phylogenetic analysis has demonstrated that the genus Salmonella includes two species: Salmonella bongori and Salmonella enterica. Salmonella strains are conventionally identified and classified according to the Kauffmann-White serotyping scheme, which is based on antigenic variation in the outer membrane (23). To date, more than 2,500 Salmonella serovars have been identified, and most of them are capable of infecting a wide variety of animal species and humans (33). Salmonella can be transmitted directly by person to person via the fecal-oral route or by contact with external reservoirs if fecal contamination of soil, water, and foods occurs. It is therefore necessary to develop robust detection methods for all of these sample types.The diagnostic method currently used for Salmonella detection is bacterial culture (International Organization for Standardization [ISO] method 6579:2002), a time-consuming and laborious process (40). A rapid and reliable tool to assist disease control management should aim to reduce salmonellosis in both people and animals. For this purpose a number of assays, such as the enzyme-linked immunosorbent assay (ELISA), PCR, and fluorescence in situ hybridization (FISH), have been developed to decrease the time required to identify Salmonella in food, feces, water, and other clinical samples (8, 10, 14, 15, 25, 26, 31, 41).Several authors have compared some of these approaches, especially culture-based, ELISA, and PCR methods, for Salmonella detection. Some authors found that PCR and ELISA-based methods failed to detect some samples that were positive by culture method (12, 13, 36, 39, 40). Even so, PCR-based methods have proved to be more accurate. Other work showed that when a selective enrichment step was performed before PCR, all Salmonella samples recovered by the culture method were detected. Moreover, the presence of Salmonella that was not recovered by the culture method could be detected by PCR (13, 35). These studies revealed that the enrichment step could increase the molecular assay sensitivity by eliminating problems such as the low numbers of bacteria and the presence of inhibitory substances in certain types of samples, such as food and fecal matter (11, 28, 36). However, PCR-based methods usually require a DNA extraction step, and none of the methods referred to above allows a direct, in situ visualization of the bacterium within the sample.FISH is a molecular assay widely applied for bacterial identification and localization within samples (2, 3). The method is usually based on the specific binding of nucleic acid probes to particular RNAs, due to their higher numbers of copies in the cells. There are already some studies reporting Salmonella detection by FISH using DNA probes (21, 29). A recently developed synthetic DNA analogue, named peptide nucleic acid (PNA), capable of hybridizing to complementary nucleic acid targets, has made FISH procedures easier and more efficient (38, 42). PNA-FISH methods have been successfully applied to the detection of several pathogenic microorganisms (6, 16, 17, 19, 22, 30, 34, 37, 42). For Salmonella, a PNA probe, designated Sal23S10, that targets the 23S rRNA of both Salmonella species has been already developed (31). However, the probe is also complementary to Actinobacillus actinomycetemcomitans, Buchnera aphidicola, and Haemophilus influenzae 23S rRNAs.In this paper, we identify and describe the design of a new fluorescently labeled PNA probe for the specific identification of the Salmonella genus. A novel, rapid, and reliable PNA-FISH method that can be easily applied to a great variety of sample types, either clinical or environmental, has consequently been developed and optimized.     

Rapid Identification of Rice Samples Using an Electronic Nose

Four rice samples of long grain type were tested using an electronic nose (Cyranose-320).Samples of 5 g of each variety ofrice were placed individually in vials and were analyzed with the electronic nose unit consisting of 32 polymer sensors.TheCyranose-320 was able to differentiate between varieties of rice.The chemical composition of the rice odors for differentiatingrice samples needs to be investigated.The optimum parameter settings should be considered during the Cyranose-320 trainingprocess especially for multiple samples,which are helpful for obtaining an accurate training model to improve identificationcapability.Further,it is necessary to investigate the E-nose sensor selection for obtaining better classification accuracy.A re-duced number of sensors could potentially shorten the data processing time,and could be used to establish an application pro-cedure and reduce the cost for a specific electronic nose.Further research is needed for developing analytical procedures thatadapt the Cyranose-320 as a tool for testing rice quality.     

Rapid Identification of Major-Effect Genes Using the Collaborative Cross

The Collaborative Cross (CC) was designed to facilitate rapid gene mapping and consists of hundreds of recombinant inbred lines descended from eight diverse inbred founder strains. A decade in production, it can now be applied to mapping projects. Here, we provide a proof of principle for rapid identification of major-effect genes using the CC. To do so, we chose coat color traits since the location and identity of many relevant genes are known. We ascertained in 110 CC lines six different coat phenotypes: albino, agouti, black, cinnamon, and chocolate coat colors and the white-belly trait. We developed a pipeline employing modifications of existing mapping tools suitable for analyzing the complex genetic architecture of the CC. Together with analysis of the founders’ genome sequences, mapping was successfully achieved with sufficient resolution to identify the causative genes for five traits. Anticipating the application of the CC to complex traits, we also developed strategies to detect interacting genes, testing joint effects of three loci. Our results illustrate the power of the CC and provide confidence that this resource can be applied to complex traits for detection of both qualitative and quantitative trait loci.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.     

蓝粒小麦易位系的荧光原位杂交鉴定

普通小麦（Triticum aestivum L.)和长穗偃麦草（Agropyron elongatum (Host)Beauv=Elytriga elongatum(Host)Nevski=Thinopyrum ponticum (Host)Barkworth and Dewey,2n=10x=70)杂交后选育出的蓝粒小麦异代换系（蓝５８）,２n=42其中９９０６中被易位蓝粒片段的相对长度约占易位小麦染色体短臂的１／３,而９９０２中被易位蓝粒片段的相对长度约占易位小麦染色体长臂的１／２,并将９９０２的蓝粒易位片段定位在小麦Ｄ组染色体上;（２）９９１５易位附加和９９０４易位－易位附加,其体细胞染色体数均为４４,其中９９１５的体细胞染色体只有一对发生了易位,另外队了两条长穗偃麦草染色体;而９９０４有两对染色体发生了易位,并易位系中控制蓝粒性状的长穗偃麦草染色体片段的定位和蓝粒小麦易位系的应用进行了讨论。     

Heterodimerization,Altered Subcellular Localization,and Function of Multiple Zinc Transporters in Viable Cells Using Bimolecular Fluorescence Complementation

Zinc plays a crucial role in numerous key physiological functions. Zinc transporters (ZnTs) mediate zinc efflux and compartmentalization in intracellular organelles; thus, ZnTs play a central role in zinc homeostasis. We have recently shown the in situ dimerization and function of multiple normal and mutant ZnTs using bimolecular fluorescence complementation (BiFC). Prompted by these findings, we here uncovered the heterodimerization, altered subcellular localization, and function of multiple ZnTs in live cells using this sensitive BiFC technique. We show that ZnT1, -2, -3, and -4 form stable heterodimers at distinct intracellular compartments, some of which are completely different from their homodimer localization. Specifically, unlike the plasma membrane (PM) localization of ZnT1 homodimers, ZnT1-ZnT3 heterodimers localized at intracellular vesicles. Furthermore, upon heterodimerization with ZnT1, the zinc transporters ZnT2 and ZnT4 surprisingly localized at the PM, as opposed to their vesicular homodimer localization. We further demonstrate the deleterious effect that the G87R-ZnT2 mutation, associated with transient neonatal zinc deficiency, has on ZnT1, ZnT3, and ZnT4 upon heterodimerization. The functionality of the various ZnTs was assessed by the dual BiFC-Zinquin assay. We also undertook a novel transfection competition assay with ZnT cDNAs to confirm that the driving force for heterodimer formation is the core structure of ZnTs and not the BiFC tags. These findings uncover a novel network of homo- and heterodimers of ZnTs with distinct subcellular localizations and function, hence highlighting their possible role in zinc homeostasis under physiological and pathological conditions.     

Viability PCR,a Culture-Independent Method for Rapid and Selective Quantification of Viable Legionella pneumophila Cells in Environmental Water Samples

PCR-based methods have been developed to rapidly screen for Legionella pneumophila in water as an alternative to time-consuming culture techniques. However, these methods fail to discriminate between live and dead bacteria. Here, we report a viability assay (viability PCR [v-PCR]) for L. pneumophila that combines ethidium monoazide bromide with quantitative real-time PCR (qPCR). The ability of v-PCR to differentiate viable from nonviable L. pneumophila cells was confirmed with permeabilizing agents, toluene, or isopropanol. v-PCR suppressed more than 99.9% of the L. pneumophila PCR signal in nonviable cultures and was able to discriminate viable cells in mixed samples. A wide range of physiological states, from culturable to dead cells, was observed with 64 domestic hot-water samples after simultaneous quantification of L. pneumophila cells by v-PCR, conventional qPCR, and culture methods. v-PCR counts were equal to or higher than those obtained by culture and lower than or equal to conventional qPCR counts. v-PCR was used to successfully monitor in vitro the disinfection efficacy of heating to 70°C and glutaraldehyde and chlorine curative treatments. The v-PCR method appears to be a promising and rapid technique for enumerating L. pneumophila bacteria in water and, in comparison with conventional qPCR techniques used to monitor Legionella, has the advantage of selectively amplifying only viable cells.Legionella organisms are ubiquitous bacteria found in many types of water sources in the environment. Their growth is especially favored in human-made warm water systems, including cooling towers, hot tubs, showerheads, and spas (3, 14, 15, 38). Legionella bacteria replicate as intracellular parasites of amoebae and persist in the environment as free-living microbes or in biofilms. In aerosol form, they enter the lungs and can cause an acute form of pneumonia known as Legionnaires'' disease or a milder form of pulmonary infection called Pontiac fever. The species Legionella pneumophila is responsible for the vast majority of the most severe form of this atypical pneumonia (52, 70). Legionellosis outbreaks are associated with high mortality rates (15 to 20%) (15, 16, 38, 46), which can reach up to 50% for people with weakened immune systems (immunocompromised patients) (69). Legionella surveillance programs include regular monitoring of environmental water samples (9, 13, 66). It is generally acknowledged that Legionella represents a health risk to humans when cell densities are greater than 10⁴ to 10⁵ CFU per liter of water, and epidemiological data show that outbreaks of legionellosis occur at these concentrations (36, 47).The evaluation of the risk associated with Legionella has traditionally been performed using culture-based methods (1, 24). Culture is essential for identifying and typing Legionella strains during epidemics. However, Legionella culture requires long incubation times (up to 10 days) before results can be scored. This problem makes culture unsuitable for preventive actions and rapid response in emergency situations. Moreover, under certain conditions (i.e., low-nutrient environments, oxidative or osmotic stress, etc.), Legionella cells can lose the ability to be cultured, although they are still viable (7, 17, 20, 22, 39, 45, 67). These viable but nonculturable (VBNC) Legionella cells may still represent a public health hazard because they can regain their ability to grow in new, more favorable conditions (12, 19, 23, 61).Molecular approaches, such as quantitative real-time PCR (qPCR), are faster and can mitigate the main drawbacks of culture-based methods. qPCR is an alternative tool that offers rapid, sensitive, and specific detection of Legionella bacteria in environmental water samples (4, 5, 12, 26, 65, 68). PCR results can be obtained in hours instead of days, and VBNC Legionella cells can also be detected (12, 26). However, the major disadvantage of qPCR lies in its inability to evaluate viability due to the persistence of DNA in cells after death (27, 34). The monitoring of Legionella contamination levels by conventional qPCR may thus result in an overestimation of the risk of infection because false-positive results can be scored. However, the real risk from Legionella is limited to the live fraction of the total Legionella population. Only live or viable Legionella cells are able to replicate in pulmonary macrophages and cause severe pneumonia (14, 15). The development of more rapid, culture-independent methods capable of discriminating between live and dead cells is of major interest for measuring Legionella infection risks and preventing legionellosis. The nucleic acid-binding dye ethidium monoazide bromide (EMA), used in combination with qPCR, is an attractive alternative for selectively detecting and enumerating viable bacteria. EMA is particularly useful because it selectively penetrates cells with damaged membranes and covalently binds to DNA after photoactivation (21, 53). DNA-bound EMA molecules prevent PCR amplification and thereby lead to a strong signal reduction during qPCR. DNA from viable cells with intact cell membranes prevents EMA molecules from entering the cell and therefore can be amplified and quantified (56). Nocker et al. (41, 42) suggested that the signal reduction was due to a selective loss of genomic DNA from dead cells (rendered insoluble after cross-linkage) during the DNA extraction procedure rather than to PCR inhibition. However, Soejima et al. (59, 60) recently reported that treatment with EMA followed by visible light irradiation directly cleaves the chromosomal DNA of dead bacteria.In this study we optimized the EMA-staining procedure in conjunction with qPCR with pure cultures of L. pneumophila. We analyzed the potential for the EMA-qPCR method to discriminate Legionella cells with compromised or intact cell membranes. We optimized this EMA-qPCR technique, viability PCR, hereafter named v-PCR, and used it to quantify viable Legionella cells in environmental water samples. We compared our results with those obtained by conventional qPCR and culture methods. In addition, we evaluated the ability of v-PCR to monitor the efficacy of different disinfection strategies.     

实时荧光定量PCR方法快速检测转基因大豆

针对转基因大豆中普遍含有的35S启动子进行引物设计,以双链DNA染料SYBR GreenⅠ为荧光标记物,利用实时荧光定量PCR方法对大豆样品进行检测。该法检测转基因大豆的检测低限为0.005 nmol/L的35S启动子,线性范围达3个数量级,可快速区分转基因大豆和非转基因大豆,具有快速、简便、灵敏、安全、高通量、低成本等优点,可推广用于转基因植物产品的快速定量检测。