Modern Probe-Assisted Methods for the Specific Detection of Bacteria

This review intends to present an overview of methods currently under development for the specific and sensitive detection of pathogenic bacteria that exist in a variety of human environments. Bacteria continue to be a major health threat in general, and much effort is being deployed to counteract this problem. In a first instance, current and efficient techniques in use for the detection of bacteria are described. In a second instance, this review serves to compare the more conventional techniques to emerging technologies for the direct (non-labelled) detection of bacteria (referred to as “biosensors”). These approaches are mainly optical, piezoelectric, and electro-chemical in nature. They are cost-effective, quite sensitive, and potentially portable for rapid on-site/real-time detection, and rapid prevention. These devices are comprised of specific chemical/ biochemical probes immobilized onto physical transducers. This work also presents comparisons between the efficiencies (assay time and sensitivity) of various techniques being employed.

Detection and Identification of Six Foodborne Bacteria by Two-tube Multiplex Real Time PCR and Melting Curve Analysis

Objective This study is aimed to develop a two-tube melting curve-based multiplex real time PCR assay （MCMRT-PCR） for the simultaneous detection of six common foodborne pathogenic bacteria （diarrhoeagenic Escherichia coli, Salmonella, and 5higella in tube 1, Staphylococcus aureus, Vibrio parahaemolyticus, and Listeria monocytogenes in tube 2）. Methods A two-tube MCMRT-PCR assay was performed on 7900HT Fast Real-Time PCR System {Applied Biosystems, USA）. Amplification by PCR was optimized to obtain high efficiency. The sensitivity and specificity of assays were investigated. Results The detection limit of optimized MCMRT-PCR assay was 3.9x102 CFU/mLfor S. aureus, 4.4x102 CFU/mL for L. monocytogenes, 3.0x102 CFU/mL for Salmonella, 2.5x102 CFU/mL for Shigella, 2.1x102 CFU/mL for V. parahaemolyticus, and 1.2x102 CFU/mL for E. coll. The feasibility of MCMRT-PCR was further evaluated using artificially contaminated milk, the sensitivity was at the level of 10s CFU/mL. Conclusion A two-tube MCMRT-PCR assay using six primer sets was developed for detection of multiple pathogens. Our findings demonstrates that the proposed two-tube assay is reliable, useful and rapid for simultaneous detection of six foodborne pathogenic bacteria with an intended application in provincial Centers for Diseases Control and Prevention （CDC）.

Scanning electron microscopy coupled with energydispersive X-ray spectrometry for quick detection of sulfuroxidizing bacteria in environmental water samples

Detection of sulfur-oxidizing bacteria has largely been dependent on targeted gene sequencing technology or traditional cell cultivation, which usually takes from days to months to carry out. This clearly does not meet the requirements of analysis for time-sensitive samples and/or complicated environmental samples. Since energy-dispersive X-ray spectrometry(EDS) can be used to simultaneously detect multiple elements in a sample, including sulfur, with minimal sample treatment, this technology was applied to detect sulfur-oxidizing bacteria using their high sulfur content within the cell. This article describes the application of scanning electron microscopy imaging coupled with EDS mapping for quick detection of sulfur oxidizers in contaminated environmental water samples, with minimal sample handling. Scanning electron microscopy imaging revealed the existence of dense granules within the bacterial cells, while EDS identified large amounts of sulfur within them. EDS mapping localized the sulfur to these granules. Subsequent 16S rRNA gene sequencing showed that the bacteria detected in our samples belonged to the genus Chromatium, which are sulfur oxidizers. Thus, EDS mapping made it possible to identify sulfur oxidizers in environmental samples based on localized sulfur within their cells, within a short time(within 24 h of sampling). This technique has wide ranging applications for detection of sulfur bacteria in environmental water samples.

Green Fluorescent Protein Recombinant Nisin as a Probe for Detection of Gram-Positive Bacteria

A great amount of foodborne pathogens were Gram-positive (G+) bacteria, a threat to public health. In this study, considering the binding ability of nisin towards G+ bacteria and the stable fluorescent ability of EGFP protein, a fluorescent nisin–EGFP protein probe was constructed by a gene engineering method. Nisin and EGFP were used as the receptor and fluorophore, respectively, to detect G+ bacteria. The nisin and egfp gene were amplified separately according to the sequence published in GenBank using unique primers. The two genes were cloned into a pET-28b(+) vector resulting in a pET-28b(+)–nisin–egfp vector. The vector was transferred into Escherichia coli (E. coli) BL21 (DE3) for expression. The expressed protein was extracted, purified by a Ni–NTA column, and then tested by the SDS-PAGE method to confirm its molecular weight. Listeria monocytogenes (L. monocytogenes), Staphylococcus aureus (S. aureus), and Micrococcus luteus (M. luteus) were used as the representations of G+ bacteria. E. coli O157, representing the gram-negative (G−) bacteria, was used as a negative control. The binding specificity of the recombinant protein was performed on two types of bacteria and then detected through fluorescent microscopy. The results indicated that the nisin–EGFP probe could detect G+ bacteria at 108CFU/mL. © 2017, Tianjin University and Springer-Verlag Berlin Heidelberg.

Rapid detection of Shigella and Salmonella in rhesus monkeys by loop-mediated isothermal amplification assay

Objective To establish a loop-mediated isothermal amplification( LAMP) method for detecting diarrhea pathogens( Shigella and Salmonella) in rhesus monkeys and evaluate the application of the LAMP method for detecting bacterial diseases in nonhuman primate laboratory animals. Materials and Methods A total of 205 fecal samples of rhesus monkeys were detected in this LAMP assay. The specificity and sensitivity of LAMP for Shigella and Salmonella were analyzed,and real-time polymerase chain reaction( REAL-TIME PCR) assay was employed as control. Results The LAMP method established here needed only 45 min to complete the reaction at 63℃. Its detection limit was 10 pg / μL and with a high specificity. The positive rate of Shigella and Salmonella was 1. 5% and 6. 3%,respectively. Conclusions Here we have established a fast and simple Shigella and Salmonella LAMP detection method that has strong specificity and high sensitivity and is suitable for rapid detection of bacterial disease in macaques. The development of this rapid detection kit is underway,and it will be helpful to the diarrhea detection.

Fluorescent aptasensor for detection of live foodborne pathogens based on multicolor perovskite-quantum-dot-encoded DNA probes and dual-stirring-bar-assisted signal amplification

In this study,a fluorescent(FL)aptasensor was developed for on-site detection of live Salmonella typhimurium(S.T.)and Vibrio parahaemolyticus(V.P.).Complementary DNA(cDNA)of aptamer(Apt)-functionalized multicolor polyhedral oligomeric silsesquioxane-perovskite quantum dots(cDNA-POSSPQDs)were used as encoded probes and combined with dual-stirring-bar-assisted signal amplification for pathogen quantification.In this system,bar 1 was labeled with the S.T.and V.P.Apts,and then bar 2 was functionalized with cDNA-POSS-PQDs.When S.T.and V.P.were introduced,pathogen-Apt complexes would form and be released into the supernatant from bar 1.Under agitation,the two complexes reached bar 2 and subsequently reacted with cDNA-POSS-PQDs,which were immobilized on MXene.Then,the encoded probes would be detached from bar 2 to generate FL signals in the supernatant.Notably,the pathogens can resume their free state and initiate next cycle.They swim between the two bars,and the FL signals can be gradually enhanced to maximum after several cycles.The FL signals from released encoded probes can be used to detect the analytes.In particular,live pathogens can be distinguished from dead ones by using an assay.The detection limits and linear range for S.T.and V.P.were 30 and 10 CFU/mL and 10^(2) -10^(6) CFU/mL,respectively.Therefore,this assay has broad application potential for simultaneous on-site detection of various live pathogenic bacteria in water.

Microwave Detection, Disruption, and Inactivation of Microorganisms

This paper reviews three complex interactions between microwave energy and microorganisms (bacteria, fungi, and viruses). The first interaction comprises the detection of viruses within human blood using a 50-Ohm transmission-line vector net-analyzer (typically 0 to 10 dBm @ 2 to 8.5 GHz) where the blood is placed within a test chamber that acts as a non-50-Ohm discontinuity. The second interaction employs 1 to 6.5 W @ 8 to 26 GHz for microwave feed-horn illumination to inactivate microorganisms at an applied power density of 10 to 100 mW-2. The third interaction is within multi-mode microwave ovens, where microorganism cell membrane disruption occurs at a few 100 s of W @ 2.45 GHz and microorganism inactivation between 300 to 1800 W @ 2.45 GHz. Within the first microwave interaction, blood relaxation processes are examined. Whereas in the latter two microwave interactions, the following disruption, and inactivation mechanisms are examined: chemical cellular lysis and, microwave resonant absorption causing cell wall rupture, and thermodynamic analysis in terms of process energy budget and suspension energy density. In addition, oven-specific parameters are discussed.

Community Structure of Endophytic Bacteria in Chestnut, Castanea mollissima

[ Objective] The paper was to detect the species of endophytic bacteria in Chestnut （Castanea mollissima）. [ Method ] The 16S rDNA-V4 region of endophytic bacteria in chestnut （sample name BL） was sequenced by Illumina MiSeq high-throughput sequencing technology. The number of sequences and op- erational taxonomic units （OTUs） for each sample was sorted and calculated using Qiime and Mothur software. The abundance and α-diversity of species were analyzed. [ Result] The number of effective sequences and OTUs were 21 167/191. The rarefaction curves showed that adequate sampling was achieved, and the number of OTUs was close to saturation. The endophytic bacteria of chestnut belonged to 10 genera, including Koribacter （3.48%） , Solibacter （2.59%） , Bra- d^hizobium （ 3. 57% ）, Rhodoplanes （ 7. 41% ）, Methylobacterium （ 4. 20% ）, Agrobacterium （ 19. 73% ）, Kaistobacter （ 2. 05% ）, Sphingomonas （18.66%）, Acidovorax （35.98%）, and Methylibium （2.32%）. The dominant species were Acidovorax, Agrobacterium and Sphingomonas. [ Conclusion] Illumina high-throughput sequencing technology provided more accurate and scientific data resources for the study of endophytic bacteria in chestnut.

Effects of Physical Parameters on Bacterial Cell Adsorption onto Pre-Imprinted Sol-Gel Films

Organically modified silica (ORMOSILS) thin films produced by sol-gel method were imprinted with two bacterial strains as whole cells in order to develop an easy, fast and specific probe to detect and specifically identify these micro-organisms when present in water samples. An important feature of the imprinting process was the molecular finger-prints left by these microorganisms alongside morphology, into imprinted film cavities. The films also showed high selectivity toward the imprinted template and were able to discriminate between two very close bacterial species (E. coli and S. typhimurium). In addition, several central physical parameters of the experimental water solution were examined (i.e., pH, ionic strength and the organic load exemplified by NaCl and TOC concentration, respectively). The method sensitivity to different bacterial concentrations was studied by confocal microscopy (CLSM) and quartz crystal microbalance (QCM) tools. Results showed that increased bacterial concentrations favor rapid adsorption onto imprinted sol-gel films with high affinity, while low pH, increased organic load and high ionic concentrations (i.e., seawater) interfere with bacteria re-adsorption, reducing detection capability. Under average drinking water chemical composition the method proved to be highly efficient.

Comparative quantification of human intestinal bacteria based on cPCR and LDR/LCR

AIM: To establish a multiple detection method based on comparative polymerase chain reaction (cPCR) and ligase detection reaction (LDR)/ligase chain reaction (LCR) to quantify the intestinal bacterial components. METHODS: Comparative quantification of 16S rDNAs from different intestinal bacterial components was used to quantify multiple intestinal bacteria. The 16S rDNAs of different bacteria were amplified simultaneously by cPCR. The LDR/LCR was examined to actualize the genotyping and quantification. Two beneficial (Bifidobacterium , Lactobacillus ) and three conditionally pathogenic bacteria (Enterococcus , Enterobacterium and Eubacterium ) were used in this detection. With cloned standard bacterial 16S rDNAs, standard curves were prepared to validate the quantitative relations between the ratio of original concentrations of two templates and the ratio ofthe fluorescence signals of their final ligation products. The internal controls were added to monitor the whole detection flow. The quantity ratio between two bacteria was tested. RESULTS: cPCR and LDR revealed obvious linear correlations with standard DNAs, but cPCR and LCR did not. In the sample test, the distributions of the quantity ratio between each two bacterial species were obtained. There were significant differences among these distributions in the total samples. But these distributions of quantity ratio of each two bacteria remained stable among groups divided by age or sex. CONCLUSION: The detection method in this study can be used to conduct multiple intestinal bacteria genotyping and quantification, and to monitor the human intestinal health status as well.

MALDI-TOF质谱技术检测豆类作物病原细菌

本文通过基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术分析豆类作物上7种主要病原细菌,建立了MALDI-TOF MS分析方法。结果表明:构建的MALDI-TOF质谱检测数据库可以快速、准确地区分和鉴定该作物上的主要病原菌。该分析方法具有属特异性和致病变种特异性,可用于豆类作物主要病原细菌的检测。

OFDM系统中基于因子图的信道估计算法

为提高正交频分复用(OFDM)系统的信道估计精度,根据频谱资源的无线信道特性,提出基于因子图的OFDM系统信道估计算法,包括二维联合信道估计算法和2个级联的一维信道估计算法。将时变频率选择性衰落信道建模为一阶自回归模型,使信道参数之间的交互信息近似为高斯分布,利用和积算法实现OFDM系统的联合信道估计和符号检测。仿真结果表明,该信道估计算法能够以较低的计算复杂度逼近最优的估计性能。

基于国产质谱Autof ms1000快速检测耐β-内酰胺类抗菌药物肠杆菌的研究评价

目的建立一种基于基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)快速检测不同肠杆菌耐药性的方法,对比MALDI-TOF MS检测结果与常规药敏检测结果,评估MALDI-TOF MS在检测细菌耐药性方面的可行性。方法收集10株肠杆菌科菌株进行预实验(8株耐药,2株敏感质控),用β-内酰胺类抗菌药物(氨苄西林、亚胺培南、美罗培南、厄他培南、头孢噻肟、头孢他啶)孵育耐药细菌一定时间后,耐药菌会引起抗菌药物的水解,从而造成+18的质量偏移,通过MALDI-TOF MS检测药物水解情况,得到细菌耐药性与敏感性分类。同时用微量肉汤稀释法进行药敏实验,对比两种方法的药敏结果。采用预实验中的最佳实验条件,选择120株肠杆菌科菌株进行批量验证实验,对两种结果进行分析统计。结果抗菌药物与耐药菌孵育4 h内,氨苄西林水解在相对分子质量为350、372、394处峰消失,亚胺培南在相对分子质量为300处峰消失、美罗培南在相对分子质量为384、406处峰消失,厄他培南在相对分子质量为475、498、520处峰消失,头孢噻肟在相对分子质量为456、478处峰消失,头孢他啶在相对分子质量为468处峰消失,敏感菌株特征峰仍然存在,由此可判断菌株的耐药性。批量验证结果与微量肉汤稀释法结果一致,且可在4 h内得到药敏结果。结论国产质谱Autof ms1000可用于快速检测β-内酰胺类细菌耐药情况,结果准确可靠,与常规药敏实验方法符合率高,并且缩短了检测时间,可提前指导临床早期合理使用β-内酰胺类抗菌药物。

Integrated Application Technology of Feces Processing in Duck Farm at Scale Based on Configuration Technology

[Objective] The aim was to explore technology on processing feces in duck farms at scale, providing guiding method for effective control of environmental issues in breeding farms. [Methed] Bio-safety disposal and recovering processing of excrements in a duck farm were researched based on technology of configuration, detection, digestion, EM and poultry breeding. [Result] The integrated application technology is quite simple and the cost is not high. During breeding period, excellent organic fertilizers and high-protein forages could be obtained without any antibiotics. Furthermore, secondary wastes and pollution would not occur. In addition, ammonia was lower in excrements processed with earthworm and air pollution was reduced; the produced humus provided organic fertilizers and improved barren soils. [Conclusion] The research provides references of multi-technology integration for related industries.

MALDI-TOF技术在重症肺炎痰液样本病原菌检测和鉴定中的应用

目的:观察基质辅助激光解吸电离飞行时间(Matrix-assisted laser desorption ionization-time of flight,MALDI-TOF)技术在重症肺炎痰液样本病原菌检测和鉴定中的应用效果。方法:选取我院2021年7月至2021年12月收治的214例重症肺炎患者为研究对象,分别采用MALDI-TOF技术、VITEK-2 Compact全自动微生物鉴定系统(VITEK 2 Compact automatic microbial analyze,VITEK-2)和基因测序法对患者痰液样本进行病原菌检测和鉴定,以基因测序法为金标准,比较两种检测方法对病原菌的鉴定结果,对比其他两种检测技术对病原菌的鉴定率以及鉴定结果差异。结果:214例重症肺炎痰液样本中,包括金黄葡萄球菌46例、表皮葡萄球菌33例、头部葡萄球菌7例、孔氏葡萄球菌6例、粪肠球菌4例、铜绿假单胞杆菌38例、肺炎克雷伯杆菌14例、不动杆菌12例、大肠埃希菌37例、阴沟肠杆菌7例、嗜麦芽杆菌3例、其他类型病菌7例。MALDI-TOF技术对重症肺炎痰液样本病原菌鉴定准确率明显高于VITEK-2技术(P<0.05)。两组检测技术漏诊率比较差异无统计学意义(P>0.05),MALDI-TOF技术对重症肺炎痰液样本病原菌误诊率明显低于VITEK-2技术(P<0.05)。结论:MALDI-TOF技术在重症肺炎痰液样本病原菌检测和鉴定中,能快速且准确鉴定出病原菌信息,为临床诊治提供可靠依据。

Study on Spoilage Microorganism of Cabbage During Storage

[Objectives]This study was conducted to investigate the spoilage microorganisms in the storage process of Chinese flowering cabbage.[Methods]Pathogenic bacteria were separated and purified from rotted Chinese flowering cabbage during storage.The gradient dilution culture method and streaking purification method were applied to selectively cultivate spoilage microorganisms for separation and observation.The isolated strains were identified through the ITS and sequence analysis of 16 S rDNA combined with the morphological characteristics and physiological and biochemical properties of the microbes.On the basis of morphology,combined with gene sequence analysis,the isolated pathogenic bacteria A1,A2,A3,and A4 were identified by PCR using the bacterial universal primer 16 S rDNA sequences,and B1 was amplified using the fungal universal primer ITS sequence.The gene sequences obtained by sequencing were subjected to homologous sequence alignment in the NCBI gene library to determine the biological classification of the spoilage bacteria.[Results]The results showed that the four bacteria numbered A1,A2,A3,and A4 were Klebsiella,Acinetobacter baylyi,Staphylococcus epidermidis,and Pseudomonas,respectively.The saprophytic fungus labeled B1 was Streptomyces albus.Re-contacting it to Chinese flowering cabbage caused the cabbage to rot,so it was the main saprophytic fungus that caused the cabbage to rot after picking.Therefore,the main spoilage microorganisms during storage of Chinese flowering cabbage were Klebsiella,A.baylyi,S.epidermidis,Pseudomonas,and S.albus.[Conclusions]This study provides a certain scientific basis and theoretical basis for the storage and preservation of Chinese flowering cabbage.

基于BCUSUM的多参数变点估计

文章基于递归残差的逆序特征和隔离检测研究了回归模型多参数变点的检测方法。首先,构建带有变点的回归模型,考虑到多元正向CUSUM检验能防止协变量均值与偏移量正交时损失功效,但其变点检测效果并不理想的情况,引入修正的检验统计量BCUSUM。其次,结合快速高效的隔离检测技术,提出MCPDP算法用于估计变点数目及位置。最后,模拟结果表明,所提出的方法能较好地控制检验水平,有更高的功效;评价结果显示,MCPDP算法在变点估计性能方面表现较优;实例分析表明,交通流变点符合实际交通情况,验证了该方法的有效性,且所构建的模型可以作为交通参数确定性经验关系的一种修正。

Detecting Strength and Location of Jump Discontinuities in Numerical Data

In [1] and some following publications, Tadmor and Gelb took up a well known property of conjugate Fourier series in 1-d, namely the property to detect jump discontinuities in given spectral data. In fact, this property of conjugate series is known for quite a long time. The research in papers around the year 1910 shows that there were also other means of detecting jumps observed and analysed. We review the classical results as well as the results of Gelb and Tadmor and demonstrate their discrete case using different estimates in all detail. It is worth noting that the techniques presented are not global but local techniques. Edges are a local phenomenon and can only be found appropriately by local means. Furthermore, applying a different approach in the proof of the main estimate leads to weaker preconditions in the discrete case. Finally an outlook to a two-dimensional approach based on the work of Móricz, in which jumps in the mixed second derivative of a 2-d function are detected, is made.

基于改进CUSUM算法的路由器异常流量检测

针对核心路由器端口的输入、输出流量的变化,用改进的CUSUM(cumulativesum)算法对其统计特性进行实时监控,检测网络流量异常.基于路由器多端口的特点,提出了矩阵式的多统计量CUSUM算法(M-CUSUM),并提出了可调的参数设定体系,以提高准确性.M-CUSUM算法通过对输入、输出端口流量的绝对差与和之比进行统计,实时地监控其均值的偏移情况.通过对该算法在计算机中的模拟实现,验证了该算法对DOS/DDOS攻击具有较高的检测速度和精度,且系统开销小,已成功运行在软件路由器之上.

基于工业控制模型的非参数CUSUM入侵检测方法

为解决日趋严重的工业控制系统(industrial control system,ICS)信息安全问题,提出一种针对工业控制网络的非参数累积和(cumulative sum,CUSUM)入侵检测方法.利用ICS输入决定输出的特性,建立ICS的数学模型预测系统的输出,一旦控制系统的传感器遭受攻击,实际输出信号将发生改变.在每个时刻,计算工业控制模型的预测输出与传感器测量信号的差值,形成基于时间的统计序列,采用非参数CUSUM算法,实现在线检测入侵并报警.仿真检测实验证明,该方法具有良好的实时性和低误报率.选择适当的非参数CUSUM算法参数τ和β,该入侵检测方法不但能在攻击对控制系统造成实质伤害前检测出攻击,还对监测ICS中的误操作有一定帮助.