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.     

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.     

多重PCR-DHPLC快速检测食品中产志贺毒素大肠杆菌

应用多重PCR(motiplex PCR)结合变性高效液相色谱技术(denaturing high-performanceliquid chromatography,DHPLC)建立了快速检测食品中产志贺毒素大肠杆菌O111和O157的方法.以基因wzxO111、rfbEO157为靶基因,建立多重PCR-DHPLC方法,进行特异性和灵敏度测试,同时进行RT-PCR检测比较灵敏度.该方法具有良好特异性,可以一次PCR扩增同时检测O111、O157;灵敏度达到25 CFU/mL.129份牛肉样品中检出1例O111,3例O157阳性;74份鸡肉样品中检测出O111、O157阳性各1例,67份蔬菜样品中未检测到O111、O157.本文建立O111、O157多重PCR-DHPLC检测方法,操作简便,特异性强,适用于产志贺毒素大肠杆菌筛选检测.     

实时荧光定量PCR技术鉴别非O157∶H7大肠杆菌产志贺毒素菌株(STEC)的研究

本文运用实时荧光定量PCR的技术对菌株进行stx1基因、stx2基因、eaeA毒力基因检测;并对stx阳性、eaeA阳性的菌株进行O抗原基因rfbE(O157)、wzx(O26)、wbdI(O111)、ihp1(O145)、wzx(O103)检测。探究了实验室保存的94株非O157:H7大肠杆菌是否存在产志贺毒素菌株(STEC)存在;结果表明94株大肠杆菌中检出3株含有stx基因、12株含有eaeA基因;对stx和eaeA阳性菌株O抗原基因试验,检出2株含有wzx(O26)基;这2株大肠杆菌血清凝集试验结果为阳性。研究结果显示,实时荧光定量PCR技术具有特异性强,灵敏度高等特点,可用于产志贺毒素菌株(STEC)前期筛查。     

畜禽肉沙门氏菌和大肠杆菌O157多重PCR检测研究

沙门氏菌和大肠杆菌O157都是目前世界公认引起食源性疾病的重要致病菌.本研究针对致病茵传统检测方法耗时长、过程繁琐的缺点,建立了同时检测畜禽肉及其制品中沙门氏菌和大肠杆菌O157的多重PCR分子检测方法.结果表明:分别针对沙门氏茵侵袭基因invA、大肠杆菌O157抗原基因rfbE建立的多重PCR方法可简便、快速、灵敏地实现对沙门氏菌和大肠杆菌O157的同时检测,整个过程在9h～10h内完成,人工污染猪肉检测限分别达到2.4×102cfu/mL(沙门氏菌)和2.2×102 cfu/mL(大肠杆菌O157);为食源性致病菌的检测提供了理想手段,有良好的应用前景.     

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%。     

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。     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

肠出血性大肠杆菌O157∶H7前噬菌体CP-933Y缺失突变株的构建

目的:利用Red重组系统敲除肠出血性大肠杆菌O157∶H7前噬菌体片段CP-933Y,进而构建CP-933Y缺失突变株。方法:以肠出血性大肠杆菌O157∶H7菌株为模板,加入酶切位点PCR扩增前噬菌体CP-933Y上、下游各600 bp的同源臂序列;酶切后分别连接到p UC19-kan质粒的卡那霉素(包含FRT位点)抗性基因两侧,构建中间是卡那霉素抗性基因标记含有目的基因上、下游同源序列的线性片段;导入含有p KD46质粒的O157∶H7菌株中,利用Red编码的同源重组酶使该片段与目的基因上、下游发生同源重组,卡那霉素抗性基因置换菌株中CP-933Y前噬菌体片段,最后导入p CP20质粒去除卡那霉素抗性标记基因。结果:经PCR及测序验证,O157∶H7菌株中前噬菌体片段CP-933Y被敲除,敲除株与野生株具有相似的生长曲线。结论:构建了大肠杆菌O157∶H7前噬菌体CP-933Y缺失株,为进一步研究前噬菌体CP-933Y的功能奠定了基础。     

大肠杆菌O157:H7核酸探针检测方法的建立

目的：应用核酸探针方法快速检测大肠杆菌O157：H7。方法：通过使用吖啶酯标记的特异DNA探针方法检测大肠杆菌O157：H7,对此种方法的特异性、敏感性、准确性进行研究,比较该方法与传统国标法的检测结果。结果：核酸探针方法检测大肠杆菌O157：H7特异性以及敏感性强,检出大肠杆菌O157：H7菌液浓度最低限约为106cfu/ml,检测大肠杆菌O157：H7的结果与国标法相一致;对O157：H7鉴定时间仅需30min,简便快捷。结论：核酸探针方法可用于大肠杆菌O157：H7的快速检测。     

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.