生物传感器应用于食源性致病菌检测研究进展

生物传感器技术是一种由生物、化学、物理、医学、电子技术等多种学科互相渗透形成起来的高新微量分析技术,具有选择性好、灵敏度高、分析速度快、成本低、能在复杂的体系中进行在线连续监测的特点.本文根据生物传感器的分子识别元件将生物传感器分为DNA传感器、免疫传感器、细胞传感器三大类,简要介绍各种生物传感器的原理及其在检测食源性致病菌方面的应用情况,并对未来生物传感器应用于实际检测进行了展望.     

A universal fluorescent aptasensor based on AccuBlue dye for the detection of pathogenic bacteria

We report a universal fluorescent aptasensor based on the AccuBlue dye, which is impermeant to cell membranes, for the detection of pathogenic bacteria. The sensor consists of AccuBlue, an aptamer strand, and its complementary strand (cDNA) that partially hybridizes to the aptamer strand. We have fabricated two models by changing the sequence of the reaction between the elements in the system. One is the “signal on” model in which the aptamer is first bound to the target, followed by the addition of cDNA and AccuBlue, at which time the cDNA hybridizes with the free unreacted aptamer and forms a double-stranded DNA (dsDNA) duplex. Such hybridization causes AccuBlue to insert into the dsDNA and exhibit significantly increased fluorescence intensity because of the specific intercalation of the AccuBlue into dsDNA rather than single-stranded DNA (ssDNA). The other model, “signal off,” involves hybridization of the aptamer with cDNA first, resulting in high fluorescence intensity on the addition of AccuBlue. When the target is added, the aptamer binds the target, causing the cDNA to detach from the dsDNA duplex and resulting in low fluorescence as a result of the liberation of AccuBlue. Because this design is based purely on DNA hybridization, and AccuBlue is impermeant to cell membranes, it could potentially be adapted to a wide variety of analytes.     

Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria

The realization of rapid, sensitive, and specific methods to detect foodborne pathogenic bacteria is central to implementing effective practice to ensure food safety and security. As a principle of transduction, the impedance technique has been applied in the field of microbiology as a means to detect and/or quantify foodborne pathogenic bacteria. The integration of impedance with biological recognition technology for detection of bacteria has led to the development of impedance biosensors that are finding wide-spread use in the recent years. This paper reviews the progress and applications of impedance microbiology for foodborne pathogenic bacteria detection, particularly the new aspects that have been added to this subject in the past few years, including the use of interdigitated microelectrodes, the development of chip-based impedance microbiology, and the use of equivalent circuits for analysis of the impedance systems. This paper also reviews the significant developments of impedance biosensors for bacteria detection in the past 5 years, focusing on microfabricated microelectrodes-based and microfluidic-based Faradaic electrochemical impedance biosensors, non-Faradaic impedance biosensors, and the integration of impedance biosensors with other techniques such as dielectrophoresis and electropermeabilization.     

Method for colorimetric detection of double-stranded nucleic acid using leuco triphenylmethane dyes

Because loop-mediated isothermal amplification (LAMP) can amplify substantial amounts of DNA under isothermal conditions, its applications for simple genetic testing have attracted considerable attention. A positive LAMP reaction is indicated by the turbidity caused by by-products or by the color change after adding a metallochromic indicator to the reaction solution, but these methods have certain limitations. Leuco crystal violet (LCV), a colorless dye obtained after sodium sulfite treatment of crystal violet (CV), was used as a new colorimetric method for detecting LAMP. LCV is reconverted into CV through contact with double-stranded DNA (dsDNA). Therefore, the positive reaction of LAMP is indicated by color change from colorless to violet. The assay is sensitive enough to detect LAMP products, with a detection limit of 7.1 ng/μl for dsDNA. It is also highly selective to dsDNA, and interference with single-stranded DNA and deoxynucleotide triphosphates (dNTPs) is not observed. LCV facilitates direct colorimetric detection of the main product rather than a by-product of the LAMP reaction; therefore, this method can be used under various reaction conditions such as those with added pyrophosphatase in solution. This colorimetric LAMP detection method using LCV is useful for point-of-care genetic testing given its simplicity.     

西藏拉萨地区常见致病菌及其耐药性分析

目的研究2009年至2011年西藏拉萨地区主要医院的常见致病菌及其耐药性情况。方法采集拉萨市临床医院细菌感染性疾病临床标本1 200份进行致病菌的分离。采用法国梅里埃-ATB菌种鉴定仪对分离的菌株鉴定到种,参照2010年CLSI推荐方法进行耐药性分析。结果对拉萨地区主要临床医院的感染者标本分离鉴定出332株临床致病菌,其中细菌304株(91.57%),真菌28株(8.43%)。病原细菌分布主要为革兰阳性球菌97株,占29.22%;革兰阴性杆菌200株,占60.24%;其他菌7株,占2.11%。凝固酶阴性葡萄球菌对苯唑西林﹑头孢曲松和环丙沙星的耐药率分别为72%﹑40%和44%。大肠埃希菌对氨苄西林﹑哌拉西林和头孢唑林耐药率分别为83%﹑53%和43%。克雷伯菌属对氨苄西林﹑头孢唑林耐药率分别为86%和58%。铜绿假单胞菌和不动杆菌对亚胺培南的耐药率分别高达20%和19%。结论拉萨地区的细菌感染及其耐药菌株分布较为广泛,该地区应加强常规临床致病菌的耐药性监测以指导临床医师合理使用抗菌药物。     

Activities of different types of Thai honey on pathogenic bacteria causing skin diseases,tyrosinase enzyme and generating free radicals

Background

Honey is a natural product obtained from the nectar that is collected from flowers by bees. It has several properties, including those of being food and supplementary diet, and it can be used in cosmetic products. Honey imparts pharmaceutical properties since it has antibacterial and antioxidant activities. The antibacterial and antioxidant activities of Thai honey were investigated in this study.

Results

The honey from longan flower (source No. 1) gave the highest activity on MRSA when compared to the other types of honey, with a minimum inhibitory concentration of 12.5% (v/v) and minimum bactericidal concentration of 25% (v/v).Moreover, it was found that MRSA isolate 49 and S. aureus were completely inhibited by the 50% (v/v) longan honey (source No. 1) at 8 and 20 hours of treatment, respectively. Furthermore, it was observed that the honey from coffee pollen (source No. 4) showed the highest phenolic and flavonoid compounds by 734.76 mg gallic/kg of honey and 178.31 mg quercetin/kg of honey, respectively. The antioxidant activity of the honey obtained from coffee pollen was also found to be the highest, when investigated using FRAP and DPPH assay, with 1781.77 mg FeSO₄•7H₂O/kg of honey and 86.20 mg gallic/kg of honey, respectively. Additionally, inhibition of tyrosinase enzyme was found that honey from coffee flower showed highest inhibition by 63.46%.

Conclusions

Honey demonstrates tremendous potential as a useful source that provides anti-free radicals, anti-tyrosinase and anti-bacterial activity against pathogenic bacteria causing skin diseases.     

The synthesis of novel chromogenic enzyme substrates for detection of bacterial glycosidases and their applications in diagnostic microbiology

The preparation and evaluation of chromogenic substrates for detecting bacterial glycosidase enzymes is reported. These substrates are monoglycoside derivatives of the metal chelators catechol, 2,3-dihydroxynaphthalene (DHN) and 6,7-dibromo-2,3-dihydroxynaphthalene (6,7-dibromo-DHN). When hydrolysed by appropriate bacterial enzymes these substrates produced coloured chelates in the presence of ammonium iron(III) citrate, thus enabling bacterial detection. A β-d-riboside of DHN and a β-d-glucuronide derivative of 6,7-dibromo-DHN were particularly effective for the detection of S. aureus and E. coli respectively.     

Age influences resistance of Caenorhabditis elegans to killing by pathogenic bacteria

Caenorhabditis elegans has previously been proposed as an alternative host for models of infectious disease caused by human pathogens. When exposed to some human pathogenic bacteria, the life span of nematodes is significantly reduced. We have shown that mutations in the age-1, and/or age-2 genes of C. elegans, that normally enhance life expectancy, can also increase resistance to killing by the bacterial pathogens Pseudomonas aeruginosa, Salmonella enterica var. Typhimurium, Burkholderia cepacia or Yersinia pseudotuberculosis. We also found that the rate at which wild-type C. elegans was killed by the bacterial pathogens tested increased as nematodes aged. In the case of P. aeruginosa infection, the difference in life span of wild type and age-1 mutants of C. elegans was not due to differences in the level of bacterial colonisation of the gut.     

临床标本优势菌种分布及耐药分析

目的了解临床标本中微生物分离率及其耐药性,为微生物检验教学改革提供临床依据。方法对近一年来临床标本的细菌分离情况及耐药性进行回顾性统计和分析,使用SPSS 13.0软件,采用计数资料进行统计描述分析。结果从2 989份标本中分离出644株病原菌,其中G-杆菌占67.7%,G+球菌占23.29%,真菌58株占9.01%。药敏结果显示大多细菌呈现泛耐药性。结论临床感染以G-杆菌为主,G-杆菌又以NFGNB常见,而G+球菌中肠球菌占主要地位。且这些细菌对常用抗菌药物耐药严重。     

重症监护病房的病原调查及耐药性监测研究

目的调查浙江中医药大学附属第一医院重症监护病房（ICU）临床分离株的病原分布及细菌耐药状况,并与非ICU相比较,观察二者的区别,为临床用药提供有效的参考价值。方法收集该院2010年1月至2011年6月临床送检的各类标本,采用VITEK-2 compact全自动微生物鉴定仪,用GPI、GNI、ANC、YST鉴定卡、AST—GN13、AST—GP67药敏卡进行菌株的鉴定和药敏,根据美国临床实验室标准化协会（CLSI2010）制定的指导原则,判断细菌的耐药率。结果共计分离到2341株细菌,其中ICU有505株占21．6％,非ICU有1836株占78．4％。在ICU分离到的细菌中,革兰阳性菌占23．2％（117／505）;非发酵菌占47．3％（239／505）。在非ICU中,革兰阳性菌占34．4％（632／1836）;非发酵菌20．2％（371／1836）。ICU前3位细菌分别为鲍曼不动杆菌、肺炎克雷伯杆菌、洋葱伯克霍尔德菌。非ICU前3位依次为大肠埃希菌、金黄色葡萄球菌、铜绿假单胞菌。非发酵菌中,铜绿假单胞菌和鲍曼不动杆菌对哌拉西林／他唑巴坦、头孢他啶、头孢吡肟、亚胺培南、美洛培南的耐药率,ICU和非ICU差异有统计学意义（P〈0．05）。亚胺培南对ICU铜绿假单胞中的MIC50是非ICU的8倍,MIC。值相当。ICU与非ICU分离的葡萄球菌属细菌对头孢唑啉、环丙沙星、左旋氧氟沙星的耐药率差异有统计学意义（P〈0．05）。ICU和非ICU葡萄球菌对利奈唑胺、万古霉素、替考拉宁全部敏感。结论ICU患者分离的细菌以革兰阴性菌为主,其中又以非发酵菌占大多数。非ICU患者分离的革兰阳性菌比例明显要比ICU高。在主要的致病菌中,ICU的耐药率明显高于非ICU。     

泌尿系感染病原菌分布及耐药性分析

目的了解深圳市人民医院泌尿系感染病原菌的分布及耐药性,为临床医师合理使用抗菌药物提供科学依据。方法对655株泌尿系统感染病原菌进行常规鉴定,用k-B法或ATB-FUNGUS 3对其进行药敏试验。结果病原菌构成比前5位分别为大肠埃希菌（37.9%）、假丝酵母（18.0%）、肠球菌（13.1%）、肺炎克雷伯菌（6.6%）、铜绿假单胞菌（6.3%）。病原菌对各种抗菌药物产生了不同程度的耐药,肺炎克雷伯菌、铜绿假单胞菌对亚胺培南和美罗培南的耐药率为14.3%～26.8%。结论深圳市人民医院泌尿系感染病原菌主要以大肠埃希菌、假丝酵母和肠球菌等为主,病原菌对抗菌药物已产生了一定的耐药性,应加强监测与控制。     

The merits of bipartite transition-state mimics for inhibition of uracil DNA glycosylase

The glycosidic bond hydrolysis reaction of the enzyme uracil DNA glycosylase (UDG) occurs by a two-step mechanism involving complete bond breakage to the uracil anion leaving group in the first step, formation of a discrete glycosyl cation-uracil anion intermediate, followed by water attack in a second transition-state leading to the enzyme-bound products of uracil and abasic DNA. We have synthesized and determined the binding affinities of unimolecular mimics of the substrate and first transition-state (TS1) in which the uracil base is covalently attached to the sugar, and in addition, bimolecular mimics of the second addition transition state (TS2) in which the base and sugar are detached. We find that the bipartite mimics of TS2 are superior to the TS1 mimics. These results indicate that bipartite TS2 inhibitors could be useful for inhibition of glycosylases that proceed by stepwise reaction mechanisms.     

G-quadruplex-generating polymerase chain reaction for visual colorimetric detection of amplicons

We have developed a self-reporting polymerase chain reaction (PCR) system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNPs). Amplification is performed using target-specific primers modified with a 5′-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point, G-quadruplexes are forced to fold from PCR-generated duplex DNA and then are used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles.     

不同病区血培养病原菌分布及耐药性分析

目的 了解各病区血培养分离病原菌的分布特点及其耐药性,指导临床医生合理有效地使用抗生素.方法 对宁波市泌尿肾病医院·鄞州第二医院2008年8月至2011年7月临床送检的8409例血液标本经Bact/A-lert3D全自动血培养仪培养,分离所得菌用美国德灵公司Walkaway 40系统进行鉴定和药敏.对检测结果进行统计学分析.结果 共检出病原菌853株,其中革兰阴性杆菌422株,革兰阳性球菌396株,真菌35株.血浆凝固酶阴性葡萄球菌占首位,其次分别为大肠埃希菌及肺炎克雷伯菌.检出菌数量居前三位的病区是重症监护病房、消化内科及肾内科.各病区的病原菌分布不尽相同.丁胺卡那、亚胺培南对大肠埃希菌、肺炎克雷伯菌显示出较高的敏感性,未发现耐利奈唑胺及万古霉素菌株.结论 该院血流感染病原菌以革兰阴性杆菌为主,各主要病区病原菌分布不尽相同,临床医生应跟据药敏结果合理有效地使用抗生素.     

2002年至2007年太原地区儿童细菌性腹泻病原菌分布及耐药分析

目的了解太原地区近6年儿童细菌性腹泻病原菌分布及耐药情况。方法对临床诊断细菌性腹泻病,便培养已分离到病原菌1080例作回顾性分析,分析其病原菌的分布及耐药情况。结果埃希菌属486株（45％）,居于首位,前5位的病原菌依次为埃希菌属、肠球菌属、酵母样真菌、志贺菌属、假单胞菌属。各年均以大肠埃希菌为主要检出菌,志贺菌逐年减少。年龄分布中,婴儿的构成比最高（44．4％）。埃希菌属、志贺菌属、假单胞菌属、沙门菌属、气单胞菌属此5种杆菌对13种抗生素的平均耐药率依次为舒普深、痢特灵、头孢他啶、庆大霉素、环丙沙星、头孢哌酮、头孢曲松、丁胺卡那、头孢噻肟、诺氟沙星、头孢呋辛、哌拉西林、头孢唑啉。从埃希菌属近6年的耐药性变迁资料可以看出,对13种抗生素的耐药率均有不同程度上升。结论传统的致病菌志贺菌属、沙门菌属较少,而肠球菌属、假单胞菌属、枸橼酸杆菌属、克雷伯杆菌属、肠杆菌属、酵母样真菌等条件致病菌肠炎占有相当比例。各种致病菌的耐药性增加,第三代头孢除头孢他啶的耐药率较低外,其余都较高。提示应严格掌握抗生素用药指证,合理选用抗生素。     

684例胆道感染患者胆汁病原菌分布及耐药性分析

目的研究胆道感染患者胆汁病原菌的分布及其耐药性,为临床合理使用抗菌药物提供依据。方法对684例胆道感染患者胆汁进行培养,应用Micro Scan Walk Away40细菌鉴定及药敏分析系统对分离菌进行鉴定及药敏测定,用WHONET 5.6软件进行数据分析。结果从684例患者胆汁中分离出315株病原菌,革兰阴性杆菌占65.1%,革兰阳性球菌占34.3%,真菌占0.6%。革兰阴性杆菌对亚胺培南、美罗培南、头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、左氧氟沙星、头孢吡肟、庆大霉素耐药率较低。肠球菌属、葡萄球菌属对喹诺酮类耐药率较低,未发现耐万古霉素、利奈唑胺的革兰阳性球菌。结论胆道感染仍以革兰阴性杆菌为主,病原菌分布广泛,耐药情况较严重,治疗使用抗生素需要进行病原菌检测及耐药性分析。     

开颅术后颅内感染的病原菌分布特征及耐药性分析

目的 分析行开颅手术患者术后颅内感染的病原菌分布及耐药性。方法 回顾性分析2013年6月—2017年6月在我院神经外科行开颅手术的328例患者临床资料,统计术后颅内感染发生率,并对颅内感染患者的脑脊液进行病原学分析。结果 开颅术后颅内感染的发生率为9.15%（30/328）;30例颅内感染脑脊液标本中共分离出47株病原菌,其中革兰阳性菌占65.96%（31/47）,革兰阴性菌占34.04%（16/47）;革兰阳性菌对青霉素、红霉素完全耐药,对万古霉素敏感;对苯唑西林的耐药率达90%以上,对磺胺甲恶唑/甲氨苄啶的耐药率为70%~85%,对利福平的耐药率为50%~65%,对哌拉西林/他唑巴坦的耐药率均在50%以下。革兰阴性菌对环丙沙星、氨曲南完全耐药,对亚胺培南敏感;对阿米卡星、头孢他啶的耐药率达80%以上,对头孢吡肟的耐药率为60%~75%,对头孢曲松、妥布霉素及庆大霉素的耐药率均在60%以下;其中肺炎克雷伯菌对美罗培南敏感,而鲍曼不动杆菌对美罗培南仍有耐药性,耐药率为16.67%。结论 开颅术后颅内感染的病原菌以革兰阳性菌为主,并具有多重耐药特点,临床上应加强颅内感染的病原菌监测,同时结合药敏分析结果,合理使用抗菌药物。     

石鲽细菌性败血感染症及其病原细菌研究

2001—2002年,分别对两起养殖的石鲽(Stone flounder,Kareius bicoloratusL.)病害进行了检验,均表现为败血症感染特征。经以20尾病(死)石鲽(每起病例各10尾)做病变组织中细菌检查、细菌分离与鉴定、人工感染试验等的病原学检验,表明两起被检病例均为同种的杀鲑气单胞菌(Aeromonas salmonicida)感染;另在其中1起病例所检10尾石鲽的2尾中,同时检出了呈继发感染的不动杆菌(Acinetobacter)。通过对所分离后做纯培养的60株杀鲑气单胞菌6、株不动杆菌分别进行较系统的表观分类学指征(Phenotypic information)及代表菌株16S rRNA基因序列测定与系统发育学分析,表明其中的杀鲑气单胞菌为一个新亚种,不动杆菌为琼氏不动杆菌(Acinetobacter junii)的一个新形态型;又择代表菌株送中国典型培养物保藏中心(CCTCC)进行了表观性状、DNA中C Cmol%的测定等的复核鉴定,并将其中的杀鲑气单胞菌新亚种定名为杀鲑气单胞菌杀鲽亚种(Aeromonas salmonicidasubsp.flounderacidasubsp.no...     

A simple colorimetric DNA detection by target-induced hybridization chain reaction for isothermal signal amplification

A novel DNA detection method is presented based on a gold nanoparticle (AuNP) colorimetric assay and hybridization chain reaction (HCR). In this method, target DNA hybridized with probe DNA modified on AuNP, and triggered HCR. The resulting HCR products with a large number of negative charges significantly enhanced the stability of AuNPs, inhibiting aggregation of AuNPs at an elevated salt concentration. The approach was highly sensitive and selective. Using this enzyme-free and isothermal signal amplification method, we were able to detect target DNA at concentrations as low as 0.5 nM with the naked eye. Our method also has great potential for detecting other analytes, such as metal ions, proteins, and small molecules, if the target analytes could make HCR products attach to AuNPs.     

Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections

A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.