Polymerase Chain Reaction

聚合酶链反应
  • 文章类型: Journal Article
    这项研究评估了使用地方性肠冠状病毒聚合酶链反应(PCR)阴性测试结果作为检测具有类似临床疾病表现的动物健康威胁的替代方法。这项回顾性研究,在美国进行,在六个兽医诊断实验室测试的猪样本的PCR阴性检测结果。作为概念的证明,该数据库首次在1月1日之间搜索传染性胃肠炎病毒(TGEV)阴性提交,2010年4月29日,2013年,第一例猪流行性腹泻病毒(PEDV)病例被诊断出来。其次,TGEV和PEDV阴性提交用于检测2014年出现的猪delta冠状病毒(PDCoV)。最后,实施遇到的最佳检测算法,以前瞻性监测2023年肠道冠状病毒阴性提交。时间序列(每周TGEV阴性计数)和季节性自回归综合移动平均(SARIMA)用于控制异常值,趋势,和季节性。然后对SARIMA的拟合和残差进行异常检测算法(EARS,EWMA,CUSUM,法灵顿)来识别警报,定义为TGEV阴性比PEDV出现前的模型预测的更高的周。性能最好的检测算法具有最低的假警报(在基线期间检测到的警报数量)和最高的检测时间(在第一次警报和PEDV出现之间的周数)。表现最好的检测算法是CUSUM,EWMA,和法林顿灵活使用SARIMA拟合值,在PEDV和PDCoV出现前4至17周具有较低的误报率和已识别的警报。在2023年的肠测试结果中没有发现警报。在PEDV传播流行的情况下以及在PDCoV出现的同时传播流行的情况下,基于阴性的监测系统起作用。它证明了其作为诊断数据监测具有与所监测的地方性病原体相似的临床疾病的紧急病原体的附加工具的适用性。
    This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA\'s fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens.
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  • 文章类型: Journal Article
    背景:伤寒沙门氏菌是沙门氏菌的特异性菌株,负责引发伤寒;发展中国家的重大公共卫生问题。
    目的:本研究旨在鉴定胆囊中的细菌,在患者的胆囊切除术中,通过分离伤寒沙门氏菌并利用微观特征,生化和聚合酶链反应(PCR)测试。
    方法:从巴格达教学医院收集了120个标本,伊拉克。从10月开始进行横断面描述性研究,2021年至7月,2022年。在这项研究中,26例(54.2%)男性患者的沙门氏菌检测呈阳性,22例(45.8%)女性患者。患者的年龄从<30岁到>60岁不等。P值>0.05被认为是显著的,以证实患者的年龄与伤寒沙门氏菌效应之间的关系。
    结果:在这项研究采集的120个血液样本中,48(40%)通过PCR测试呈阳性,40(33.3%)通过使用Widal测试测试为阳性,35例(29.1%)活检培养阳性,血培养阳性35例(29.1%)。发现所有伤寒沙门氏菌分离株对亚胺培南敏感,头孢吡肟,和头孢曲松,但是对庆大霉素有抗药性,环丙沙星,阿米卡星,红霉素,和四环素(72%,29%,43%,100%,100%,分别)。
    结论:实时聚合酶链反应(RT-PCR)测试和Vitek2紧凑系统在伤寒沙门氏菌的检测中显示出很高的准确性。观察到多药耐药性,这应该是减少抗生素消费的信号。
    BACKGROUND: Salmonella typhi is a specific strain of the Salmonella bacterium, responsible for triggering typhoid fever; a significant public health concern in developing nations.
    OBJECTIVE: The current study aimed to identify the bacteria from the gallbladder, taken during cholecystectomies of patients, by isolating Salmonella typhi and by using microscopic characteristics, biochemical and polymerase chain reaction (PCR) tests.
    METHODS: A total of 120 specimens were collected from the Baghdad Teaching Hospital, Iraq. A cross-sectional descriptive study was carried out from October, 2021, to July, 2022. During that study, 26 (54.2%) male patient tested positive for Salmonella typhias well as 22 (45.8%) female patients. The age of the patients varied from < 30 to > 60 years. p-value > 0.05 was considered significant to confirm a relationship between age and Salmonella typhi effect for patients.
    RESULTS: Out of the 120 blood samples taken for this study, 48 (40%) tested positive by use of PCR test, 40 (33.3%) tested positive by use of the Widal test, 35 (29.1%) were positive for biopsy culture, and 35 (29.1%) were positive for blood culture. All Salmonella typhi isolates were found to be sensitive to the imipenem, cefepime, and ceftriaxone, but were resistant to gentamycin, ciprofloxacin, amikacin, erythromycin, and tetracycline (72%, 29%, 43%, 100%, 100%, respectively).
    CONCLUSIONS: The real time polymerase chain reaction (RT-PCR) tests and the Vitek 2 compact system showed a high level of accuracy in the detection of Salmonella typhi. Multidrug resistance was observed, which should be a signal to reduce antibiotic consumption.
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  • 文章类型: Journal Article
    使用耐酸杆菌(AFB)染色和结核分枝杆菌(Mtb)聚合酶链反应(PCR)的实验室算法通常用于消除分离预防措施。对52例培养证实为肺Mtb的患者的回顾性病例回顾显示,有4例痰AFB涂片阴性和MtbPCR阴性的受试者。所有患者都有明显的Mtb危险因素,并且干扰素γ释放试验呈阳性。PCR检测结果阴性并不排除Mtb诊断。
    Laboratory algorithms using Acid Fast Bacilli (AFB) staining and Mycobacterium tuberculosis (Mtb) polymerase chain reaction (PCR) are often used to remove isolation precautions. A retrospective case review of 52 patients with culture confirmed pulmonary Mtb revealed 4 subjects with negative sputum AFB smears and negative Mtb PCRs. All had significant risk factors for Mtb and had a positive interferon gamma release assay. A negative PCR test result does not exclude an Mtb diagnosis.
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  • 文章类型: Journal Article
    Enterocytozoon bieneusi is an obligate intracellular microsporidian parasite with a worldwide distribution. As a zoonotic pathogen, E. bieneusi can infect a wide range of wildlife hosts through the fecal-oral route. Although the feces of flying squirrels (Trogopterus xanthipes) are considered a traditional Chinese medicine (as \"faeces trogopterori\"), no literature is available on E. bieneusi infection in flying squirrels to date. In this study, a total of 340 fresh flying squirrel fecal specimens from two captive populations were collected in Pingdingshan city, China, to detect the prevalence of E. bieneusi and assess their zoonotic potential. By nested PCR amplification of the ITS gene, six specimens tested positive, with positive samples from each farm, with an overall low infection rate of 1.8%. The ITS sequences revealed three genotypes, including known genotype D and two novel genotypes, HNFS01 and HNFS02. Genotype HNFS01 was the most prevalent (4/6, 66.7%). Phylogenetic analysis showed that all genotypes clustered into zoonotic Group 1, with the novel genotypes clustering into different subgroups. To our knowledge, this is the first report of E. bieneusi infection in flying squirrels, suggesting that flying squirrels could act as a potential reservoir and zoonotic threat for E. bieneusi transmission to humans in China.
    UNASSIGNED: Occurrence et génotypage d’Enterocytozoon bieneusi chez les écureuils volants (Trogopterus xanthipes) de Chine.
    UNASSIGNED: Enterocytozoon bieneusi est un parasite microsporidien intracellulaire obligatoire présent dans le monde entier. En tant qu’agent pathogène zoonotique, E. bieneusi peut infecter un large éventail d’hôtes sauvages par la voie fécale-orale. Bien que les excréments d’écureuils volants (Trogopterus xanthipes) soient considérés comme un ingrédient de médecine traditionnelle chinoise (comme « faeces trogopterori »), aucune littérature n’est disponible à ce jour sur l’infection par E. bieneusi chez les écureuils volants. Dans cette étude, un total de 340 spécimens fécaux frais d’écureuils volants provenant de deux populations captives ont été collectés dans la ville de Pingdingshan, en Chine, pour détecter la prévalence d’E. bieneusi et évaluer leur potentiel zoonotique. Par amplification PCR nichée du gène ITS, six échantillons se sont révélés positifs, avec des échantillons positifs dans chaque ferme, et un taux d’infection global faible, à 1,8 %. Les séquences ITS ont révélé trois génotypes, dont le génotype D connu et deux nouveaux génotypes, HNFS01 et HNFS02. Le génotype HNFS01 était le plus répandu (4/6, 66,7 %). L’analyse phylogénétique a montré que tous les génotypes se regroupaient dans le groupe zoonotique 1, les nouveaux génotypes se regroupant en différents sous-groupes. À notre connaissance, il s’agit du premier rapport d’infection par E. bieneusi chez des écureuils volants, ce qui suggère que les écureuils volants pourraient agir comme un réservoir potentiel et une menace zoonotique pour la transmission d’E. bieneusi aux humains en Chine.
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  • 文章类型: Journal Article
    Sauroleishmaniaspp.包括四个利什曼原虫亚属之一,历史上被认为是爬行动物的非致病性原生动物。然而,一些菌株似乎对哺乳动物有短暂的感染,最近的发现已经在利什曼病流行地区的狗和人类中发现了这些寄生虫。在这里,234bp-hsp70片段的PCR-RFLP消化模式被评估为一种更简单,更便宜的工具,可以区分Sauroleishmania物种与其他利什曼原虫亚属。因此,用HaeIII消化234bp-hsp70片段产生了对所评估的四种Sauroleishmania菌株具有特异性的条带模式。这项技术可能有助于鉴定从沙蝇中分离出的利什曼原虫寄生虫,爬行动物,甚至是野外工作中的哺乳动物,作为使用费力和昂贵方法的替代方法。
    Sauroleishmania spp. comprises one of the four Leishmania subgenera, which has been historically considered a non-pathogenic protozoan of reptiles. However, some strains appear to be transiently infective to mammals, and recent findings have detected these parasites in dogs and humans in areas where leishmaniasis is endemic. Herein, the digestion pattern of PCR-RFLP of the 234 bp-hsp70 fragment was evaluated as a simpler and cheaper tool to distinguish the Sauroleishmania species from the other Leishmania subgenera. As a result, the digestion of the 234 bp-hsp70 fragments with HaeIII produced a banding pattern specific to the four Sauroleishmania strains assessed. This technique could contribute to the identification of Leishmania parasites isolated from sandflies, reptiles, or even mammals in fieldworks as an alternative to the use of laborious and expensive methodologies.
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  • 文章类型: Journal Article
    In patients with end-stage kidney disease, kidney transplantation is the kidney replacement therapy option that provides the most successful survival. However, immunosuppression agents administered after kidney transplantation can increase the risk of opportunistic infections. Microsporidia are obligate intracellular pathogens that can be fatal in immunosuppressed patients. The present study aimed to determine the prevalence of microsporidia in kidney transplantation recipients and the molecular characterization of the detected species.
    To evaluate the prevalence of renal microsporidiosis in kidney transplant recipients, the urine samples from a total of 325 patients were analyzed by real-time and nested polymerase chain reaction for Encephalitozoon spp. and Enterocytozoon bieneusi.
    Only one (0.4%) sample from the adult patient was positive for the Encephalitozoon species, while no positivity was found in pediatric patients. It was determined as Encephalitozoon intestinalis by ITS rRNA gene region sequence analysis. A microsporidia species obtained from humans in Türkiye has been characterized for the first time and registered in GenBank.
    Our epidemiological results show that the prevalence of renal microsporidiosis in kidney transplant recipients is very low. In addition, as a result of the phylogenetic analysis of the detected isolate, it was observed that it was 100% identical to the isolates reported from dogs in Kayseri, Türkiye. This situation provided essential data regarding the zoonotic transmission dynamics of microsporidia.
    Böbrek nakli, son dönem böbrek yetmezliği olan hastalarda en başarılı sağkalım sağlayan renal replasman tedavi seçeneğidir. Ancak böbrek nakli sonrasında uygulanan immün baskılayıcı ajanlar fırsatçı enfeksiyon riskini artırmaktadır. Microsporidialar, immün sistemi baskılanmış hastalarda ölümcül olabilen zorunlu hücre içi patojenlerdir. Bu çalışmada böbrek nakil hastalarında microsporidia prevalansının belirlenmesi ve tespit edilen türlerin moleküler karakterizasyonunun yapılması amaçlandı.
    Böbrek nakli hastalarında renal microsporidiosis prevalansını değerlendirmek için toplam 325 hastadan alınan idrar örnekleri Encephalitozoon spp. ve Enterocytozoon bieneusi açısından gerçek zamanlı ve nested polimeraz zincir reaksiyonu ile analiz edildi.
    Erişkin hastalardan sadece biri (%0,4) Encephalitozoon türleri yönünden pozitif belirlendi, çocuk hastalarda ise pozitiflik saptanmadı. ITS rRNA gen bölgesi sekans analizi sonucunda tespit edilen türün Encephalitozoon intestinalis olduğu görüldü. Bu çalışma ile Türkiye’de ilk kez insanlardan izole edilen bir microsporidia türü karakterize edilerek GenBank’a kaydedildi.
    Elde edilen epidemiyolojik sonuçlar, renal transplant hastalarında renal microsiporidiosis prevalansının çok düşük olduğunu göstermektedir. Ayrıca tespit edilen izolatın filogenetik analizi sonucunda Kayseri’de köpeklerden bildirilen izolatlarla %100 benzer olduğu görüldü. Bu çalışma microsporidiaların zoonotik bulaşma dinamikleri açısından önemli bir veri sağlamaktadır.
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  • 文章类型: Journal Article
    野生啮齿动物可以作为E.bieneusi的水库或载体,从而使寄生虫传播给家畜和人类。本研究旨在调查中国内蒙古自治区和辽宁省野生啮齿动物中E.bieneusi的流行情况。此外,为了评估基因型水平的人畜共患传播的可能性,对分离株进行了遗传分析.
    从中国两个省份共捕获了486只野生啮齿动物。进行聚合酶链反应(PCR)以扩增啮齿动物粪便DNA中的脊椎动物细胞色素b(cytb)基因,以检测其物种。通过rDNA的内部转录间隔区(ITS)区域的PCR扩增确定E.bieneusi的基因型。遗传特征和人畜共患潜力的检查需要应用相似性和系统发育分析。
    在四种确定的啮齿动物中,E.bieneusi的感染率为5.2%(n=89),黄鲸4.5%(n=96),小家鼠11.3%(n=106),褐家鼠为38.5%(n=195)。在486只啮齿动物中,平均感染率为17.4%。在确定的11种基因型中,已知9个:SHR1(在32个样品中检测到),D(30个样本),EbpA(9个样品),PigEbITS7(8个样品),HNR-IV(6个样品),IV型(5个样品),HNR-VII(2个样品),HNH7(1个样品),和HNPL-V(1个样品)。还发现了两种新的基因型,NMR-I和NMR-II,每个包含一个样本。通过系统发育分析将基因型分为第1组和第13组。
    根据初始报告,E.bieneusi在各自省和地区的野生啮齿动物中非常普遍,并且遗传多样性。这表明这些动物对于E.bieneusi的传播至关重要。携带人畜共患E.bieneusi的动物对当地居民构成重大危害。因此,有必要提高对这些啮齿动物带来的危险的认识,并减少其数量,以防止环境污染。
    UNASSIGNED: Wild rodents can serve as reservoirs or carriers of E. bieneusi, thereby enabling parasite transmission to domestic animals and humans. This study aimed to investigate the prevalence of E. bieneusi in wild rodents from the Inner Mongolian Autonomous Region and Liaoning Province of China. Moreover, to evaluate the potential for zoonotic transmission at the genotype level, a genetic analysis of the isolates was performed.
    UNASSIGNED: A total of 486 wild rodents were captured from two provinces in China. Polymerase chain reaction (PCR) was performed to amplify the vertebrate cytochrome b (cytb) gene in the fecal DNA of the rodents to detect their species. The genotype of E. bieneusi was determined via PCR amplification of the internal transcribed spacer (ITS) region of rDNA. The examination of genetic characteristics and zoonotic potential requires the application of similarity and phylogenetic analysis.
    UNASSIGNED: The infection rates of E. bieneusi in the four identified rodent species were 5.2% for Apodemus agrarius (n = 89), 4.5% for Cricetulus barabensis (n = 96), 11.3% for Mus musculus (n = 106), and 38.5% for Rattus norvegicus (n = 195). Infection was detected at an average rate of 17.4% among 486 rodents. Of the 11 identified genotypes, nine were known: SHR1 (detected in 32 samples), D (30 samples), EbpA (9 samples), PigEbITS7 (8 samples), HNR-IV (6 samples), Type IV (5 samples), HNR-VII (2 samples), HNH7 (1 sample), and HNPL-V (1 sample). Two novel genotypes were also discovered, NMR-I and NMR-II, each comprising one sample. The genotypes were classified into group 1 and group 13 via phylogenetic analysis.
    UNASSIGNED: Based on the initial report, E. bieneusi is highly prevalent and genetically diverse in wild rodents residing in the respective province and region. This indicates that these animals are crucial for the dissemination of E. bieneusi. Zoonotic E. bieneusi-carrying animals present a significant hazard to local inhabitants. Therefore, it is necessary to increase awareness regarding the dangers presented by these rodents and reduce their population to prevent environmental contamination.
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  • 文章类型: Journal Article
    从汇集的血浆制造的因子VIII和IX凝血因子浓缩物在1970年代和1980年代已被鉴定为血友病(PWHs)患者的有效病毒感染源。为了调查这一时期病毒的范围和多样性,我们分析了24种血液传播病毒的凝血因子浓缩物。核酸是从14种商业生产的凝血因子和10种无偿捐献者中提取的,以冻干形式保存(有效期:1974-1992年)。凝血因子通过商业和内部定量PCR检测血源性病毒甲型肝炎,B,C和E病毒(HAV,HBV,HCV,HEV),HIV-1/2型,细小病毒B19V和PARV4,以及人类pegivirus1和2型(HPgV-1,-2)。HCV和HPgV-1是最常见的检测病毒(14/24测试)主要在商业凝血因子,在1970年代末-1985年,病毒载量经常极高,HCV基因型范围也各不相同。引入病毒灭活后,检测频率急剧下降。HIV-1,HBV,和HAV的检出频率较低(分别为3/24、1/24和1/24);无HEV阳性。相反,在整个研究期间检测到B19V和PARV4,即使在引入干热处理后,与20世纪90年代初正在进行的有据可查的传输到PWHs是一致的。虽然在英国和其他地方,血友病治疗现在主要基于重组因子VIII/IX,对历史血浆来源的凝血因子的全面筛选表明,在整个1970年代至1990年代初,PWHs广泛暴露于血液传播病毒,以及影响凝血因子污染的流行病学和制造参数。
    Factor VIII and IX clotting factor concentrates manufactured from pooled plasma have been identified as potent sources of virus infection in persons with hemophilia (PWHs) in the 1970s and 1980s. To investigate the range and diversity of viruses over this period, we analysed 24 clotting factor concentrates for several blood-borne viruses. Nucleic acid was extracted from 14 commercially produced clotting factors and 10 from nonremunerated donors, preserved in lyophilized form (expiry dates: 1974-1992). Clotting factors were tested by commercial and in-house quantitative PCRs for blood-borne viruses hepatitis A, B, C and E viruses (HAV, HBV, HCV, HEV), HIV- types 1/2, parvoviruses B19V and PARV4, and human pegiviruses types 1 and 2 (HPgV-1,-2). HCV and HPgV-1 were the most frequently detected viruses (both 14/24 tested) primarily in commercial clotting factors, with frequently extremely high viral loads in the late 1970s-1985 and a diverse range of HCV genotypes. Detection frequencies sharply declined following introduction of virus inactivation. HIV-1, HBV, and HAV were less frequently detected (3/24, 1/24, and 1/24 respectively); none were positive for HEV. Contrastingly, B19V and PARV4 were detected throughout the study period, even after introduction of dry heat treatment, consistent with ongoing documented transmission to PWHs into the early 1990s. While hemophilia treatment is now largely based on recombinant factor VIII/IX in the UK and elsewhere, the comprehensive screen of historical plasma-derived clotting factors reveals extensive exposure of PWHs to blood-borne viruses throughout 1970s-early 1990s, and the epidemiological and manufacturing parameters that influenced clotting factor contamination.
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  • DOI:
    文章类型: Case Reports
    A 21-year-old retired polo Argentinian thoroughbred horse from a teaching herd was presented for a routine bronchoalveolar lavage demonstration, during which an incidental finding of a granulomatous mass on the dorsal aspect of the epiglottis was made. Rhinosporidium seeberi was suspected from a histological section obtained from an initial biopsy, and the mass was removed via laser surgery for cytology and PCR. Sequencing of the PCR amplicons confirmed the diagnosis of R. seeberi. A treatment protocol of nebulized voriconazole for 10 d postoperatively was used. Long-term follow-up required 2 more laser surgeries plus oral fluconazole to resolve the remaining fungal spores. However, 2.5 y later, there was no evidence of remaining fungal spores. Key clinical message: Horses from endemic regions can potentially be exposed to R. seeberi. Based on its travel history, this horse may have contracted the infection in South America, California, or Alberta. Treatments administered, including diode laser resection, voriconazole antifungal nebulization, and oral fluconazole administration, were successful but required repeated interventions.
    Suivi à long terme du Rhinosporidium seeberi laryngé diagnostiqué par PCR et traité par ablation au laser et nébulisation au voriconazole chez un cheval de polo thoroughbred pur-sang à la retraiteUn cheval thoroughbred argentin de polo retraité de 21 ans, issu d’un troupeau d’enseignement, a été présenté pour une démonstration de lavage broncho-alvéolaire de routine, au cours de laquelle une découverte fortuite d’une masse granulomateuse sur la face dorsale de l’épiglotte a été faite. Rhinosporidium seeberi a été suspecté à partir d’une coupe histologique obtenue à partir d’une biopsie initiale, et la masse a été retirée par chirurgie au laser pour cytologie et PCR. Le séquençage des amplicons PCR a confirmé le diagnostic de R. seeberi. Un protocole de traitement au voriconazole nébulisé pendant 10 jours après l’opération a été utilisé. Le suivi à long terme a nécessité 2 autres interventions chirurgicales au laser et du fluconazole oral pour éliminer les spores fongiques restantes. Cependant, 2,5 ans plus tard, il n’y avait aucune trace de spores fongiques restantes.Message clinique clé:Les chevaux des régions endémiques peuvent potentiellement être exposés à R. seeberi. D’après ses antécédents de voyage, ce cheval pourrait avoir contracté l’infection en Amérique du Sud, en Californie ou en Alberta. Les traitements administrés, notamment la résection au laser à diode, la nébulisation antifongique au voriconazole et l’administration orale de fluconazole, ont été efficaces mais ont nécessité des interventions répétées.(Traduit par Dr Serge Messier).
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  • 文章类型: Journal Article
    在过去的几年里,已经开发了几种基因克隆方法。选择克隆策略取决于各种因素,其中,简单性和可负担性一直被考虑。本研究的目的,一方面,是通过跳过体外组装反应来简化基因克隆,另一方面,通过消除相对昂贵的材料来降低成本。我们研究了一个克隆系统,使用带有两个质粒的大肠杆菌,pLP-AmpR和pScissors-CmR。pLP-AmpR包含由两个基因(λint和λgam)组成的着陆区(LP),该基因允许使用位点特异性重组替换转化的线性DNA。在更换过程之后,例如,来自pScissors-CmR(CRISPR/Cas9)载体的可诱导表达SpCas9和特异性sgRNA导致非重组pLP-AmpR质粒的去除。通过直接转化PCR产物来探索LP的功能。评价pScissors-CmR质粒对三种载体的固化作用,包括pBR322,p15A,pSC101用PCR产物替换LP并快速根除含有pSC101来源的载体是成功的。在基因置换和质粒固化过程后确认重组菌落。结果使我们乐观地认为,这种策略可能是一种简单而廉价的克隆方法。关键点:•通过用着陆垫替换靶基因进行体内克隆。•通过适应关键载体,非重组质粒的快速根除是可能的。•该策略不依赖于体外组装反应和昂贵的材料。
    Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.
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