变性高效液相色谱检测沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌

应用多重PCR 反应(multiplex PCR,mPCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的快速检测方法.以编码沙门氏菌的fimY基因、编码空肠弯曲菌的gyrA基因和编码肠出血性大肠杆菌O157:H7的rfbE基因为靶基因,选择3对引物,建立并优化了快速鉴别沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的多重PCR体系,扩增产物分别为284、159和499 bp,并验证了该多重PCR具有特异性.沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7标准菌株稀释成不同梯度,做灵敏度检测.试验结果表明该方法有很好的特异性,且灵敏度高,检测限可达到:沙门氏菌1.5 CFU/ml、空肠弯曲菌15 CFU/ml、肠出血性大肠杆菌O157:H7 15 CFU/ml.在随机采集的226份冷冻鸡肉类样品中,检出了7份样品为沙门氏菌阳性、10份为空肠弯曲菌阳性、1份为肠出血性大肠杆菌O157:H7阳性.研究建立的多重PCR-DHPLC方法可特异、灵敏地实现对沙门氏菌、空肠弯曲菌和肠出血性大肠杆菌O157:H7的快速检测.     

肉类中大肠杆菌O157:H7多重PCR检测方法的建立

以编码大肠杆菌O157抗原的rfbE基因、 编码H7抗原的fliC基因以及编码毒力因子的eaeA基因为靶基因, 选择3对引物, 建立并优化了检测大肠杆菌O157:H7的多重PCR体系, 扩增产物分别为291 bp、625 bp、368 bp, 采用30株细菌验证了该多重PCR具有特异性。PCR检测的灵敏度在DNA水平上达到91.35 pg; 在存在干扰菌鼠伤寒沙门氏菌(Salmonella?typhimurium)的情况下, 当起始污染量为1.4 CFU/mL时, 37 ℃培养6 h 即可检出。在30份肉类样品中, 有3份检出了大肠杆菌O157:H7。本研究建立的多重PCR方法可特异、灵敏地实现对大肠杆菌O157:H7的检测。     

基于环介导恒温扩增技术的大肠杆菌O157:H7快速检测试剂盒的评价

【目的】评价基于环介导恒温扩增技术(LAMP)的大肠杆菌O157:H7(Escherichia coli O157:H7)快速检测试剂盒的实效性。【方法】测定快速检测试剂盒的特异性、灵敏度、重复性、保质期以及运输稳定性,并与传统方法对比检测实际样品。【结果】大肠杆菌O157:H7标准菌株样品均检测为阳性,非大肠杆菌O157:H7标准菌株样品均检测为阴性,未发现有交叉反应;试剂盒最低检验限为29 CFU;该试剂盒的特异性、灵敏度及准确度与传统方法相比具有较高的一致性;试剂盒对高菌量目标菌和阴性菌样品的检测重复率均为100%,对低菌量目标菌样品的批间检测重复率为94%。试剂盒可在4°C保存9个月以上,并且可进行变温储存72 h以上。【结论】该试剂盒特异性好,灵敏度高,重复性好,储存方便,检测结果稳定、可靠,适用于对食品中大肠杆菌O157:H7的检测需求。     

多种食源性致病菌检测的多重PCR 方法的研究

目的:利用多重PCR技术,建立可以同时检测多种食源性致病菌的多重PCR方法。方法:分别选择沙门氏菌invA基因,志贺氏菌的ipaH基因,单核细胞增生李斯特氏菌的hlyA基因,大肠杆菌O157:H7的eaeA基因,副溶血弧菌的toxR基因,设计多重PCR引物,建立多重PCR检测体系,并对该体系进行特异性和灵敏度实验。结果:通过对19株菌株进行实验,所有的目标菌株均为阳性,而其余菌株为阴性。对多重PCR体系的灵敏度进行考察,沙门菌的灵敏度为5000 CFU/mL;志贺氏菌的灵敏度为5500CFU/mL;单核细胞增生李斯特氏菌的灵敏度为5200 CFU/mL;O157:H7的灵敏度为5000CFU/mL;副溶血弧菌的灵敏度为6300CFU/mL。结论:建立的多重PCR体系能实现多种致病菌同时检测。     

改良环介导等温扩增技术快速检测肉类中的大肠杆菌O157: H7

针对大肠杆菌O157:H7(Escherichia coli O157:H7,E.coli O157:H7)传统检测方法检测周期长的问题,建立了肉类中的E.coli O157:H7的改良环介导等温扩增(LAMP)快速检测方法。以E.coli O157:H7的O157特异性抗原rfbE基因、鞭毛H7特异性抗原fliC基因序列作为靶序列,分别设计2套增加了环引物的改良LAMP引物序列,单管同时检测,通过肉眼观察白色沉淀,判断检测结果。采用36株细菌验证了该改良LAMP引物的特异性。热裂解法提取的DNA经改良LAMP体系扩增20 min,检测E.coli O157:H7的灵敏度为1.4 CFU/mL,人工污染肉中的E.coli O157:H7检出限为1.8 CFU/g。137份实样中,检测出1份E.coli O157:H7假阳性,与行业标准SNT0973-2000符合率达到99.3%。     

肉类中大肠杆菌O157:H7多重PCR检测方法的建立

以编码大肠杆菌 O157 抗原的 rfbE 基因、编码 H7 抗原的 fliC 基因以及编码毒力因子的eaeA 基因为靶基因,选择3对引物,建立并优化了检测大肠杆菌 O157:H7 的多重 PCR 体系,扩增产物分别为291 bp、625 bp,368 bp,采用30株细菌验证了该多重 PCR 具有特异性.PCR 检测的灵敏度在 DNA 水平上达到91.35 Pg;在存在干扰菌鼠伤寒沙门氏(Salmonella typhimurium)的情况下,当起始污染量为1.4 CFU/mL时,37℃培养6 h即可检出.在30份肉类样品中,有3份检出了大肠杆菌 O157:H7.本研究建立的多重 PCR 方法可特异、灵敏地实现对大肠杆菌 O157:H7 的检测.     

基于免疫纳米颗粒强化的压电免疫传感器检测大肠杆菌O157︰H7

摘要：【目的】结合纳米技术建立检测大肠杆菌（Escherichia coli）O157︰H7高灵敏检测技术。【方法】采用化学共沉淀法制备出核心粒径约为10 nm的免疫纳米磁颗粒,柠檬酸钠还原法制备粒径约为20 nm的免疫胶体金。压电免疫传感器通过金黄色葡萄球菌蛋白A（Protein A from Staphylococcus aureus SPA）法将抗体固定于石英晶振上,两种免疫纳米颗粒借助不同的抗体连接于传感器上对检测频率信号进行放大。【结果】SPA在石英晶振上的最佳固定浓度和时间为1.2 mg/mL和40 min,抗体的最佳固定浓度和时间为1.0 mg/mL和60 min。压电免疫传感器通过两种免疫纳米颗粒的放大作用,使其对大肠杆菌O157︰H7的检测限从104 cfu/mL提高到101 cfu/mL。【结论】免疫纳米颗粒强化对压电免疫传感器的检测频率信号具有很好的放大效应,可以明显提高其检测灵敏度。     

噬菌体裂解酶Lysin1902原核表达及其与ε-聚赖氨酸联用效果评价北大核心

【目的】研究裂解酶Lysin1902与ε-聚赖氨酸(ε-PL)对大肠杆菌O157:H7的协同抗菌作用。【方法】通过Mega构建进化树、使用在线工具等分析大肠杆菌噬菌体裂解酶Lysin1902氨基酸序列组成和结构等;原核表达并纯化Lysin1902;通过平板裂解实验检测Lysin1902对大肠杆菌O157:H7灭活菌株的裂解活性;用96孔板法检测Lysin1902或ε-PL的活菌裂解能力;棋盘法验证Lysin1902和ε-PL联用效果。【结果】体外成功表达并纯化了Lysin1902。Lysin1902对大肠杆菌O157:H7灭活菌株具有裂解活性,但不能有效裂解活的大肠杆菌。噬菌体裂解酶与ε-PL联用结果表明,加入Lysin1902后,ε-PL能够完全控制大肠杆菌O157:H7增殖的使用浓度由0.7mg/mL降低到0.1 mg/mL。【结论】体外原核表达并纯化Lysin1902,其单独使用对活的大肠杆菌O157:H7无裂解活性,但与ε-PL联用可显著提高ε-PL对大肠杆菌O157:H7的裂解能力。     

大肠杆菌O157显色培养基检测效果的评价

【目的】评价显色培养基对大肠杆菌O157:H7(Escherichia coli O157:H7)的检测效果。【方法】本实验室研制的大肠杆菌O157显色培养基(HKM),与国外梅理埃、科玛嘉及国内厂家的同类产品及传统培养基CT-SMAC作比较,对相关菌株以及污染样品和实际样品进行对比测试。【结果】实验室研制的HKM大肠杆菌O157显色培养基与科玛嘉同类产品在特异性、灵敏度及检测效果方面均无明显差异,均优于梅里埃、国内厂家产品及CT-SMAC。【结论】HKM大肠杆菌O157显色培养基具有高检出率及高特异性的特点,具有较好的应用价值和前景。     

短双歧杆菌对鼠伤寒沙门氏菌的抑制

【背景】鼠伤寒沙门氏菌是主要的肠道病原菌之一,利用益生菌治疗肠道病原菌感染已成为一种新型、绿色的微生态疗法。【目的】研究筛选出的短双歧杆菌无细胞发酵上清液(Cell-free supernatant,CFS)对鼠伤寒沙门氏菌的体外抑制作用及机制。【方法】采用微量稀释法测定短双歧杆菌YH68 CFS对鼠伤寒沙门氏菌的最小抑菌浓度(Minimum inhibitory concentration,MIC)和亚抑制浓度(Sub-inhibitory concentrations,SIC),并从鼠伤寒沙门氏菌的细胞形态、细胞膜通透性、膜完整性以及毒力基因表达的变化探讨YH68 CFS对鼠伤寒沙门氏菌的抑菌机理,同时检测YH68 CFS对鼠伤寒沙门氏菌粘附和侵袭肠上皮细胞HT29的影响。【结果】YH68 CFS (3×109 CFU/mL)对鼠伤寒沙门氏菌具有较好的抑制效果,抑菌圈直径为22.27±0.44 mm,最小抑菌浓度为250μL/mL,对鼠伤寒沙门氏菌的抑制机制是通过增加其细胞膜通透性破坏其完整性,形成难以修复的孔洞,最终达到抑菌的目的;亚抑制浓度为62.5μL/mL时YH68 CFS并不能影响鼠伤寒沙门氏菌的生长,但仍然能通过下调毒力基因表达的方式抑制其对肠上皮细胞的粘附和入侵。【结论】短双歧杆菌YH68对鼠伤寒沙门氏菌具有良好的抑菌作用,可作为治疗沙门氏菌感染的潜在益生菌。     

Recent advances in the study of Kaposi's sarcoma-associated herpesvirus replication and pathogenesis

It has now been over twenty years since a novel herpesviral genome was identified in Kaposi's sarcoma biopsies. Since then, the cumulative research effort by molecular biologists, virologists, clinicians, and epidemiologists alike has led to the extensive characterization of this tumor virus, Kaposi's sarcoma-associated herpesvirus(KSHV; also known as human herpesvirus 8(HHV-8)), and its associated diseases. Here we review the current knowledge of KSHV biology and pathogenesis, with a particular emphasis on new and exciting advances in the field of epigenetics. We also discuss the development and practicality of various cell culture and animal model systems to study KSHV replication and pathogenesis.     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

The structure, reproduction and taxonomy of Vidalia and Osmundaria (Rhodophyta, Rhodomelaceae)

Comprises species occurring mostly in subtidal habitats in tropical, subtropical and warm-temperate areas of the world. An analysis of the type species, V. spiralis (Sonder) Lamouroux ex J. Agardh, a species from Australia, establishes basic characters for distinguishing species in the genus. These characters are (1) branching patterns of thalli, (2) flat blades that may be spiralled on their axis, (3) width of the blade, (4) primary or secondary derivation of sterile and fertile branchlets and (5) position of sterile and fertile branchlets on the thalli. Application of the latter two characters provides an important basic method for separation of species into three major groups. Osmundaria , a genus known only in southern Australia, was studied in relation to Vidalia , and its separation from the Vidalia assemblage is not accepted. Species of Vidalia therefore are transferred to the older genus name, Osmundaria. Two new species, Osmundaria papenfussii and Osmundaria oliveae are described from Natal. Confusion in the usage of the epithet, Vidalia fimbriala Brown ex Turner has been clarified, and Vidalia gregaria Falkenberg, described as an epiphyte on Osmundaria pro/ifera Lamouroux, is revealed to be young branches of the host, Osmundaria prolifera.     

New or rare chromosome counts in Artemisia L. (Asteraceae, Anthemideae) and related genera from Kazakhstan

Fifteen chromosome counts of six Artemisia taxa and one species of each of the genera Brachanthemum, Hippolytia, Kaschgaria, Lepidolopsis and Turaniphytum are reported from Kazakhstan. Three of them are new reports, two are not consistent with previous counts and the remainder are confirmations of very scarce (one to four) earlier records. All the populations studied have the same basic chromosome number, x = 9, with ploidy levels ranging from 2x to 6x. Some correlations between ploidy level, morphological characters and distribution are noted.