黄芩苷铝胁迫下产肠毒素大肠杆菌内参基因的筛选

[背景]产肠毒素大肠杆菌(Enterotoxigenic Escherichia coli,ETEC)是导致仔猪腹泻的主要致病菌之一,黄芩苷铝对ETEC诱导的仔猪腹泻具有良好的治疗效果,但作用机制尚不明了.[目的]筛选出黄芩苷铝胁迫下ETEC中表达水平最为稳定的内参基因.[方法]采用qPCR技术测定了 12个内参基因 ...     

Rapid enumeration of Escherichia coli in marine bathing waters: potential interference of nontarget bacteria

Aims: To compare the Escherichia coli quantification given by the ‘Coliplage^®’ assay, based on the direct measurement of the β‐d ‐glucuronidase (GLUase) activity and the reference Most Probable Number (MPN) method from seawater sites and investigate the possible interference of non‐E. coli strains in the GLUase activity measurement. Methods and Results: Comparison performed from 69 French coastal bathing sites (1401 samples) showed nonconcordance between both methods, only for 8% of samples. Non‐E. coli 4‐methylumbelliferyl‐β‐d ‐glucuronide (MUG+) were isolated from nonconcordant samples. Phylogenetic analysis showed that Gammaproteobacteria were dominants and mainly represented by Vibrio species, which displayed GLUase activities on the same order of magnitude and sometimes much higher as E. coli reference strains. Conclusions: The‘Coliplage^®’ assay is a rapid method for the quantification of E. coli showed few discordances with the standard MPN method. Some Vibrio species could interfere on the direct GLUase activity measurement of E. coli. Significance and Impact of the Study: Data present the first qualitative investigation on disagreement between Coliplage^® and the MPN results. If the interference of Vibrio species is confirmed in situ, appropriate treatments should be developed to remove the interfering signal.     

Comparison of a culture‐based and a PCR‐based methods for estimating bacterial abundance on eggshells,with comments on statistical analyses

Field ornithologists have used traditional culture‐based techniques to determine the presence and abundance of microbes on surfaces such as eggshells, but culture‐independent PCR‐based methods have recently been introduced. We compared the traditional culture‐based and the real‐time PCR‐based methods for detecting and quantifying Escherichia coli on the eggshells of Eurasian Magpies (Pica pica). PCR estimates of bacterial abundance were ～10 times higher than culture‐based estimates, and the culture‐based technique failed to detect bacteria at lower densities. When both methods detected bacteria, bacterial densities determined by the two methods were positively correlated, indicating that both methods can be used to study factors affecting bacterial densities. The difference between the two methods is consistent with generally acknowledged higher sensitivity of the PCR method, but the extent of the difference in our study (10×) may have been influenced by both a PCR‐based overestimation and culture‐based underestimation of bacterial densities. Our results also illustrate that bacterial counts may sometimes produce left‐censored data (i.e., we did not detect E. coli in 62% of our samples using the culture‐based method). Specific statistical methods have been developed for analyzed left‐censored data, but, to our knowledge, have not been used by ornithologists. In future studies, investigators studying bacterial loads should provide information about the possible degree of left censoring and should justify their choice of statistical methods from the broad set of available methods, including those explicitly designed for censored data.     

Comparison of TEMPO® EC and TBX medium for the enumeration of Escherichia coli in cheese

Aims: TEMPO^® EC (Escherichia coli) is based on glucuronidase activity and is a test for use with the TEMPO system for the automated 24 h enumeration of E. coli in food products. In this study, TEMPO EC was compared with TBX medium, the current standard plate method for the enumeration of E. coli in cheese. Methods and Results: For comparative purposes, both naturally (92) and artificially contaminated (31) cheese samples were studied. Pearson correlation coefficients were determined as 0.954 and 0.978 between the two methods for naturally and artificially contaminated samples, respectively. Regression analysis yielded the following equations: log₁₀ TEMPO EC = 0.340 + 0.889 log₁₀ TBX medium and log₁₀ TEMPO EC = 0.174 + 0.899 log₁₀ TBX medium for naturally and artificially contaminated samples, respectively. In general, absolute differences did not exceed one log between results obtained by the two methods. Conclusions: Statistical analysis of the results showed good agreement between the two enumeration methods. Significance and Impact of the Study: TEMPO EC is a practical and reliable alternative to the current standard plate method for the enumeration of E. coli in foods.     

Development and comparison of SYBR Green quantitative real‐time PCR assays for detection and enumeration of sulfate‐reducing bacteria in stored swine manure

Aims: To develop and evaluate primer sets targeted to the dissimilatory sulfite reductase gene (dsrA) for use in quantitative real‐time PCR detection of sulfate‐reducing bacteria (SRB) in stored swine manure. Methods and Results: Degenerate primer sets were developed to detect SRB in stored swine manure. These were compared with a previously reported primer set, DSR1F+ and DSR‐R, for their coverage and ability to detect SRB communities in stored swine manure. Sequenced clones were most similar to Desulfovibrio sp. and Desulfobulbus sp., and these SRB populations differed within different manure ecosystems. Sulfur content of swine diets was shown to affect the population of Desulfobulbus‐like Group 1 SRB in manure. Conclusions: The newly developed assays were able to enumerate and discern different groups of SRB, and suggest a richly diverse and as yet undescribed population of SRB in swine manure. Significance and Impact of the Study: The PCR assays described here provide improved and efficient molecular tools for quantitative detection of SRB populations. This is the first study to show population shifts of SRB in swine manure, which are a result of either the effects of swine diets or the maturity of the manure ecosystem.     

Development and application of a spiral plating method for the enumeration of Escherichia coli O157 in bovine faeces

AIM: To develop and validate a direct plating method applicable to epidemiological studies for enumerating Escherichia coli O157 in cattle faeces. METHODS AND RESULTS: The spiral plate count method was used to enumerate E. coli O157 in faecal samples. The accuracy and variation of counts was then assessed using faecal samples inoculated with E. coli O157. There was good agreement between inoculated levels of E. coli O157 and those recovered from faeces, particularly when counts were > 10(2) CFU g(-1) of faeces. The method was applied to a small study assessing short-term survival of E. coli O157 in naturally infected cattle faeces. E. coli O157 was found to survive in faeces for over 10 days at concentrations above 10(3) CFU g(-1) of faeces. Populations of E. coli O157 were also found to increase 100-fold in the first few hours after defecation. CONCLUSIONS: The enumeration method is easy to implement and enables a quick throughput of large numbers of samples. The method is accurate and reliable and enables the inherent variation in count data to be explored but needs to be used in combination with a more sensitive method for samples containing < 10(2) CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is appropriate for enumeration of E. coli O157 in cattle faeces in large-scale epidemiological studies.     

Genome size estimation with quantitative real‐time PCR in two Tephritidae species: Ceratitis capitata and Bactrocera oleae

The medfly Ceratitis capitata and the olive fruit fly Bactrocera oleae belong to the Tephritidae family of Diptera, a family whose members cause severe damages in agriculture worldwide. For such insect pests, the utmost concern is their population control. The sterile insect technique (SIT) has been used in the Tephritidae family with varying degrees of success. Its efficient use usually depends on the development of genetic sexing strains and the release of only male flies. However, such advances are based on modern genetic, molecular and genomic tools. The medfly is clearly the prototype of the family, since such tools have advanced considerably, which has resulted in effective SIT efforts around the world. A whole‐genome sequencing project of this insect is already underway. In contrast, similar tools in the olive fly lag behind, even though the insect is considered a promising candidate for a next SIT target. An accurate estimate of genome size provides a preliminary view of genome complexity and indicates possible difficulties in genome assembly in whole‐genome projects. Taking advantage of a quantitative real‐time PCR approach, we determined the genome size of these two species C. capitata and B. oleae as 591 Mb (CI range: 577–605 Mb) and 322 Mb (CI range: 310–334 Mb) respectively.     

Evaluation of a new medium for the enumeration of total coliforms and Escherichia coli in Japanese surface waters

Aim: A new medium, EC‐Blue‐10, containing chromogenic and fluorogenic substrates, KNO₃ and sodium pyruvate has been developed for the rapid simultaneous detection and enumeration of total coliforms and Escherichia coli in water. Methods and Results:  Two evaluations of EC‐Blue‐10 were carried out. Firstly, EC‐Blue‐10 was compared with Colilert‐MPN for 96 water samples using MPN for total coliforms and E. coli. Secondly, the detection of coliforms and E. coli were compared using 2400 tubes of EC‐Blue‐10 and Colilert‐MPN. The regression coefficients between EC‐Blue‐10 and Colilert‐MPN for total coliforms and E. coli were 0·91 and 0·89, respectively. For the detection results, the Cohen’s kappa values between the two media were 0·79 for coliforms and 0·72 for E. coli. Conclusions: EC‐Blue‐10 is almost same as Colilert‐MPN for the detection of coliforms and E. coli in surface waters. Further evaluation for EC‐Blue‐10 is needed to verify in different geographical areas. Significance and Impact of the Study: EC‐Blue‐10 is useful method for the rapid and simultaneous detection of total coliforms and E. coli in water sample.     

Evaluation of Legionella pneumophila contamination in Italian hotel water systems by quantitative real‐time PCR and culture methods

Aims: This study was designed to define the extent of water contamination by Legionella pneumophila of certain Italian hotels and to compare quantitative real‐time PCR with the conventional culture method. Methods and Results: Nineteen Italian hotels of different sizes were investigated. In each hotel three hot water samples (boiler, room showers, recycling) and one cold water sample (inlet) were collected. Physico‐chemical parameters were also analysed. Legionella pneumophila was detected in 42% and 74% of the hotels investigated by the culture method and by real‐time PCR, respectively. In 21% of samples analysed by the culture method, a concentration of >10⁴ CFU l^?1 was found, and Leg. pneumophila serogroup 1 was isolated from 10·5% of the hotels. The presence of Leg. pneumophila was significantly influenced by water sample temperature, while no association with water hardness or residual‐free chlorine was found. Conclusions: This study showed a high percentage of buildings colonized by Leg. pneumophila. Moreover, real‐time PCR proved to be sensitive enough to detect lower levels of contamination than the culture method. Significance and Impact of the Study: This study indicates that the Italian hotels represent a possible source of risk for Legionnaires’ disease and confirms the sensitivity of the molecular method. To our knowledge, this is the first report to demonstrate Legionella contamination in Italian hotels using real‐time PCR and culture methods.     

Ultrasensitive detection of Shiga toxin 2 and its variants in Shiga toxin‐producing Escherichia coli strains by a time‐resolved fluorescence immunoassay

A rapid and sensitive two‐step time‐resolved fluorescence immunoassay (TRFIA) was developed for the detection of Shiga toxin 2 (Stx2) and its variants in Shiga toxin‐producing Escherichia coli (STEC) strains. In sandwich mode, a monoclonal antibody against Stx2 was coated on a microtiter plate as a capture antibody. A tracer antibody against Stx2 labeled with europium(III) (Eu³⁺) chelate was then used as a detector, followed by fluorescence measurements using time‐resolved fluorescence. The sensitivity of Stx2 detection was 0.038 ng/ml (dynamic range, 0.1–1000 ng/ml). The intra‐ and inter‐assay coefficients of variation of the assay were 3.2% and 3.6%, respectively. The performance of the established assay was evaluated using culture supernatants of STEC strains, and the results were compared to those of a common HRP (horseradish peroxidase) labeling immunosorbent assay. A polymerase chain reaction (PCR) for the detection of genes encoding Stx1 and Stx2 was used as the reference for comparison. Correlation between the Stx2‐specific TRFIA and PCR was calculated by the use of kappa statics, exhibiting a perfect level of agreement. The availability of the sensitive and reliable Stx2‐specific TRFIA method for quantifying Stx2 and its variants in STEC strains will complement bacteria isolation‐based platform and aid in the accurate and prompt diagnosis of STEC infections.     

Hepatitis E virus‐based evaluation of a virion concentration method and detection of enteric viruses in environmental samples by multiplex nested RT‐PCR

Aims: The prevalence of enteric viruses in drinking and river water samples collected from Pune, India was assessed. During an outbreak of HEV in a small town near pune, water samples were screened for enteric viruses. Methods and Results: The water samples were subjected to adsorption–elution‐based virus concentration protocol followed by multiplex nested PCR. Among 64 Mutha river samples, 49 (76·56%) were positive for Hepatitis A Virus, 36 (56·25%) were positive for Rotavirus, 33 (51·56%) were positive for Enterovirus and 16 (25%) were positive for Hepatitis E Virus RNA. Only enterovirus RNA was detected in 2/662 (0·3%) drinking water samples, and the samples from the city’s water reservoir tested negative for all four viruses. HEV RNA was detected in three out of four river water samples during HEV outbreak and partial sequences from patients and water sample were identical. Conclusions: The study suggests absence of enteric viruses both in the source and in the purified water samples from Pune city, not allowing evaluation of the purification system and documents high prevalence of enteric viruses in river water, posing threat to the community. Significance and Impact of the Study: The rapid, sensitive and relatively inexpensive protocol developed for virological evaluation of water seems extremely useful and should be adapted for evaluating viral contamination of water for human consumption. This will lead to development of adequate control measures thereby reducing disease burden because of enteric viruses.     

Evaluation of real‐time PCR methods for quantification of Acanthamoeba in anthropogenic water and biofilms

Aims: To assess two real‐time PCR methods (the Riviere and Qvarnstrom assays) for environmental Acanthamoeba. Methods and Results: DNA extracted from Acanthamoeba castellanii taken from water and biofilms of cooling towers was analysed by the Riviere and Qvarnstrom assays. To quantify environmental Acanthamoeba, the calibration curves (DNA quantity vs cell number) were constructed with samples spiked with A. castellanii. The calibration curves for both quantitative PCR assays showed low variation (coefficient of variation of C_t≤ 5·7%) and high linearity (R² ≥ 0·99) over six orders of magnitudes with detection limit of three cells per water sample. DNA quantity determined by Qvarnstrom assay was equivalent between trophozoites and cysts (P = 0·49), whereas a significant difference was observed with Riviere assay (P < 0·0001). Riviere assay failed to detect Acanthamoeba in 21% (15/71) of the environmental samples which were positively detected by Qvarnstrom assay, while one sample (1·4%) was shown positive by Riviere assay but negative by Qvarnstrom assay. Moreover, Acanthamoeba counts by Qvarnstrom assay were greater than those by Riviere assay (P < 0·0001). Conclusions: Qvarnstrom assay performs better than Riviere assay for detection and quantification of Acanthamoeba in anthropogenic water and biofilms. Significance and Impact of the Study: Qvarnstrom assay may significantly contribute to a better knowledge about the distribution and abundance of Acanthamoeba in environments.     

Data‐independent acquisition‐based SWATH‐MS for quantitative proteomics: a tutorial

Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent and quantitatively accurate proteomics data from many samples. SWATH‐MS is a specific variant of data‐independent acquisition (DIA) methods and is emerging as a technology that combines deep proteome coverage capabilities with quantitative consistency and accuracy. In a SWATH‐MS measurement, all ionized peptides of a given sample that fall within a specified mass range are fragmented in a systematic and unbiased fashion using rather large precursor isolation windows. To analyse SWATH‐MS data, a strategy based on peptide‐centric scoring has been established, which typically requires prior knowledge about the chromatographic and mass spectrometric behaviour of peptides of interest in the form of spectral libraries and peptide query parameters. This tutorial provides guidelines on how to set up and plan a SWATH‐MS experiment, how to perform the mass spectrometric measurement and how to analyse SWATH‐MS data using peptide‐centric scoring. Furthermore, concepts on how to improve SWATH‐MS data acquisition, potential trade‐offs of parameter settings and alternative data analysis strategies are discussed.     

Long‐range quantitative PCR for determining inactivation of adenovirus 2 by ultraviolet light

Aims

An extra‐long‐range quantitative PCR (LR‐qPCR) method was developed for estimating genome damage to adenovirus 2 caused by UV irradiation. The objective was to use LR‐qPCR as a rapid method to determine adenovirus UV inactivation.

Methods

The LR‐qPCR consisted of two steps: a long‐range PCR (up to 10 kb fragment) and a real‐time, quantitative (q) PCR for quantifying the products of the first PCR. We evaluated LR‐qPCR with adenovirus irradiated with medium‐pressure (MP, polychromatic emission) and low‐pressure (LP, 254 nm) mercury vapour lamps and compared results with cell culture infectivity.

Results

Using LR‐qPCR, a fragment of 6 kb estimated DNA damage in a linear relationship to doses between 0 and 20 mJ cm^?2, and a 1‐kb fragment related linearly to doses between 20 and 100 mJ cm^?2. The LR‐qPCR results for the 6‐kb fragment were similar to infectivity assays results for adenovirus exposed to MP UV. For adenovirus irradiated with LP lamps, LR‐qPCR results for the shorter fragment size (1 kb) were similar to reduction in viral infectivity. No difference was observed between 10 and 6 kb LR‐qPCR results.

Conclusion

The LR‐qPCR can be used as a tool for estimating DNA damage caused by UV in adenovirus. The LR‐qPCR results were related to reduction in viral infectivity.

Significance and Impact of the Study

The use of LR‐qPCR to determine DNA damage and estimate inactivation of adenovirus 2 from UV disinfection allows for same‐day results compared with >7 days required for cell culture. This accelerates adenovirus inactivation results for the water industry where adenovirus is used as a representative virus for crediting UV systems. This PCR approach provides a framework that can be used for other viral viability assays using the inhibition of amplification of viral nucleic acid after pretreatments, such as propidium monoazide, and for cellular biology studies of DNA damage.