Development of Quantitative Real-time Polymerase Chain Reaction for the Detection of Vibrio vulnificus Based on Hemolysin (vvhA) Coding System

Objective To establish a TaqMan real-time fluorescent quantitative PCR to detect Vibrio vulnificus based on the hemolysin gene （vvhA） coding cytolysin. Methods Primers and probes in the conserved region of the vvhA gene sequence were designed for the TaqMan real-time PCR to detect 100 bp amplicon from V. vulnificus DNA. Recombinant plasmid pMD19-vvhA100 was constructed and used as a positive control during the detection. Minimal amplification cycles （Ct value） and fluorescence intensity enhancement （ARn value） were used as observing indexes to optimize the reaction conditions of TaqMan real-time PCR. The TaqMan assay for the detection of Vbirio vulnificus was evaluated in pure culture, mice tissue which artificially contaminated Vibrio vulnificus and clinical samples. Results The established TaqMan real-time PCR showed positive results only for Vibrio vulnificus DNA and pMD19-vvhA100. The standard curve was plotted and the minimum level of the vvhA target from the recombinant plasmid DNA was 103 copies with a Ct value of 37.94±0.19, as the equivalent of 0.01 ng purified genomic DNA of Vibrio vulnificus. The results detected by TaqMan PCR were positive for the 16 clinical samples and all the specimens of peripheral blood and subcutaneous tissue of mice which were infected with Vibrio vulnificus. Conclusion TaqMan real-time PCR is a rapid, effective, and quantitative tool to detect Vibro vulnificus, and can be used in clinical laboratory diagnosis of septicemia and wound infection caused by Vibrio vulnificus.

海洋细菌Aeromonas hydrophila和Vibrio vulnificus对单萜柠檬烯的生物转化产物分析

研究了南海海洋细菌嗜水性气单胞菌Aerom onas hydrophila(5-213)和创伤弧菌Vibrio vulnificus(13-203)在培养基中添加单萜(D)-柠檬烯后提取产物的组成变化情况。经GC-MS分析发现该两细菌对(D)-柠檬烯有显著的生物催化转化作用,能对底物进行不同程度的氧化、还原、水解、酯化、开环等反应,并产生丰富的系列结构不同的其它萜类化合物:包括单萜、倍半萜、二萜、以及三萜等。通过单萜微生物转化和生物合成必将获得新的活性萜类产品,也为丰富的天然单萜资源的开发与利用提供新的途径和方法。

Sensitivities of Vibrio vulnificus to Chinese Herbs and Effect of Medicinal Bath on Vibrio vulnificus

Vibrio vulnificus can cause rotten-skin disease in marine fish aquaculture, resulting in a large number of deaths. The occurrence of rotten-skin disease is becoming severer and severer, leading to serious economic losses. In this study, the sensitivities of V. vulnificus to the 18 kinds of herbs, such as stone calamus and Chinese rhubarb, were determined using the agar diffusion method （susceptibility paper）. The results showed that Chinese gall, dark plum, sea buckthorn and eucalyptus leaves have bacteriostatic and bactericidal effects, while sappan wood, Chi- nese rhubarb, pomegranate bark, mentha and clove have strong bacteriostatic ef- fects. The Chinese medicinal herb bath showed good control effect on V. vulnificus.

Dynamic Expressions of Liver Tissue Apoptosis-related Genes of Vibrio Vulnificus Sepsis Rats and the Effects of Antibacterial Agents

Dynamic changes in mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax and apoptosis-inhibiting gene Bcl-2 of vibrio vulnificus sepsis rats were detected and the effects of antibacterial agents were examined. The rat model with Vibrio vulnificus sepsis （VV group） was established and some of the Vibrio vulnificus sepsis rats were treated with antibacterial agents （AA group）. The mRNA expressions of Fas, Bax and Bcl-2 were measured by reverse transcription polymerase chain reaction （RT-PCR）. As compared with normal control group （NC group）, the ex- pressions of Fas and Bax mRNA in liver tissue at all different time points in VV group were increased significantly （P〈0.05）, and the highest levels of Fas and Bax mRNA expressions were 6 and 12 h after the infection, respectively. At the same time, the expression of Bcl-2 mRNA in liver tissue at all different time points in VV group were decreased significantly （P〈0.05）, and the lowest level of Bcl-2 mRNA expression appeared 2 h after the infection. The mRNA expressions of Bcl-2 in liver tissue 9 and 12 h after the infection in AA group were increased significantly （P〈0.05） compared with NC group, while the expressions of Fas and Bax mRNA were not significantly different from those of NC group. Compared with VV group, the expression of Fas mRNA in AA group was decreased （P〈0.05） and Bax rnRNA was decreased significantly 12 and 16 h after the infection （P〈0.05）, while the expressions of Bcl-2 mRNA were increased significantly 9, 12 and 16 h after the infection （P〈0.05）. It is concluded that the mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax were increased remarkably in vibrio vulnificus sepsis rats, whereas the expression of apoptosis-inhibiting gene Bcl-2 mRNA was decreased obviously in sepsis rats in early stage. The treatment with cefoperazone sodium and levofloxacin lactate could inhibit the expression of Fas mRNA and Bax mRNA and enhance the expression ofBcl-2 rnRNA at the same time.

Resveratrol Prevents Vibrio vulnificus-Induced Sepsis by Attenuating Necroptosis

Objective This study investigated how the natural phytophenol and potent SIRT1 activator resveratrol(RSV)regulate necroptosis during Vibrio vulnificus(V.vulnificus)-induced sepsis and the potential mechanism.Methods The effect of RSV on V.vulnificus cytolysin(VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays.Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction,western blot,and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V.vulnificus-induced sepsis mouse model.Results RSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells.RSV also inhibited the inflammatory response,had a protective effect on histopathological changes,and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages,lung,spleen,and liver tissues of V.vulnificus-induced septic mice in vivo.Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V.vulnificusinduced septic mice.RSV also improved the survival of V.vulnificus-induced septic mice.Conclusion Our findings collectively demonstrate that RSV prevented V.vulnificus-induced sepsis by attenuating necroptosis,highlighting its potency in the clinical management of V.vulnificus-induced sepsis.

Vibrio vulnificus Disease Prevalence

Vibrio vulnificus is a deadly disease that has been increasing in prevalence. In this study, we review both primary and secondary data to discuss the factors that are contributing to the increase of vibrio disease, causing a 41% increase between 1996 and 2005. It has also been shown that public health campaigns to limit vibrio infections have focused on raw seafood consumption. However, an estimated 42% of infections are now being caused by wound infection, rather than through contaminated seafood. This shows the disparities in addressing vibrio contamination through contaminated seawater and open wounds. This is particularly stressing as global warming is causing an increase in risk of contaminated seawater. Reasons for this increase are discussed, and also possible solutions are presented for public health interventions to help mitigate this rise in vibrio infections.

<i>Vibrio vulnificus</i>septicemia and necrotizing fasciitis in the patients with liver cirrhosis and diabetes mellitus

Vibrio vulnificus (V. vulnificus) infection is a rare disease in Japan but the leading cause of death related to raw seafood consumption. We hereby reported a successfully treated case of V. vulnificus septicemia, severe necrotizing fasciitis, disseminated intravascular coagulation and multiple organ failure after raw perch consumption with underlying alcoholic liver cirrhosis and diabetes mellitus. It is the first report of a case of V. vulnificusinfection caused by eating raw perch, whereas V. vulnificus infection should be suspected in all of middle-aged to elderly men with underlying immunosuppressive diseases, who have recent consumption of raw seafood or contact with seawater, especially in the summer. The levels of HbA1c and glycoalbumin were not high in the present case, however, obvious hyperglycemia was found even after the infection had completely healed. On reviewing 166 case of V. vulnificus infection in Japan including ours, the complication of diabetes mellitus, one of immunocompromised condition, was found only in 11%, although it had been reported that individuals strongly suspected of having diabetes were 17.2% among the Japanese male population aged from 40 to 74 years. Because diabetes mellitus might be underdiagnosed in the previous reports, intensive examinations are considered to be necessary in order to correctly diagnose diabetes mellitus in patients with severe V. vulnificus infection.

A case report of Vibrio vulnificus infection in the lower limbs by sea fish stabbing and literature review

Objective: To summarize and analyze the clinical treatment experience of Vibrio vulnificus sepsis and improve the success rate of treatment. Methods: The clinical data of a patient with vibrio vulnificus sepsis admitted to the Department of Critical Care Medicine, the First Affiliated Hospital of Hainan Medical College in October 2019 were analyzed. Summarize the experience of treatment and compare with other literatures to analyze the most reasonable clinical treatment of vibrio vulnificus sepsis. Results: The patient was acutely ill, the disease progressed rapidly, fever, soft tissue swelling and pain, necrosis, ulceration and pus, systemic multiple organ failure, family members finally gave up treatment, microbiology culture prompted: Vibrio parahaemolyticus. Conclusions: Early intervention, blood purification, debridement, removal of infection sources and inflammatory mediators can improve the survival rate of patients with Vibrio vulnificus infection. Due to the high mortality rate, clinicians should pay attention to it.

创伤弧菌溶血素基因vvhA质粒定性标准样品的研制及其在水产品检测中的应用

针对我国缺乏适用于创伤弧菌检测的质粒标准样品的现状,本研究开展了创伤弧菌鉴定即用型定性质粒标准样品的研制和应用工作。首先构建了创伤弧菌毒力基因vvhA的重组质粒,经测序验证后制备成质粒标准样品冻干粉,然后对其进行PCR定性检测及紫外分光光度计法定量分析。均匀性检验结果表明,样品间无显著差异,均匀性良好,符合预期目标;短期稳定性检验结果表明,样品能在4℃、37℃条件下稳定保存14天;长期稳定性检验结果表明,样品能在-20℃条件下稳定保存至少12个月。研究结果表明,创伤弧菌质粒定性标准样品的均匀性和稳定性均符合国家定性标准样品的要求,为创伤弧菌的快速、高通量的定性鉴定分析提供了可靠的参考物质。将标准样品应用于鱼类、贝类等10份海鲜类食品样品的检测中,经传统培养法验证,检测结果准确无误。本研究所研制的创伤弧菌质粒定性标准样品具有较好的商业应用潜力,为其在食品检测领域的推广应用奠定了重要基础。

3种水产病原弧菌(Vibrio)的16S—23S rDNA间区(IGSs)的克隆、测序与分析

根据细菌16S rDNA3端和23S rDNA5端的高度保守区设计引物,PCR扩增了5株溶藻弧菌、2株河流弧菌和2株创伤弧菌的16S—23S rDNA间区,克隆到pGEM-T载体上,测序;采用BLAST和DNAstar软件对16S—23S rDNA间区序列及其内的tRNA基因进行比较分析研究。结果表明,3种弧菌共测出的16S—23S rDNA间区有33条,类型有6种:IGSGLAV、IGSGLV、IGSIA、IGSG、IGSA和IGS0,其中IGSIA和IGSG最为常见,9株弧菌均包含有此两类IGS;在3种弧菌中,大部分IGS序列tRNA基因两端的非编码区具有较高的种内同源性,可以成为设计种特异性引物和探针的靶区。16S—23S rDNA间区结构的差异为建立一类新的弧菌检测方法奠定了基础。溶藻弧菌的16S—23S rDNA间区序列为首次报道。

PGE_2免疫干预对大鼠Vv攻击后TNF-α、IL-10以及SOD和NO的影响

目的:探讨前列腺素E2(Prostagland in E2,PGE2)免疫干预对大鼠创伤弧菌(V ibrio vu ln ificus,Vv)攻击后TNF-α、IL-10以及SOD和NO的影响。方法:正常大鼠和肝病大鼠各27只,分别为Vv攻击对照、Vv攻击后氧氟沙星药物治疗和Vv攻击PGE2氧氟沙星联合保护组(n=9)。结果:无论是正常还是肝病大鼠,PGE2免疫干预组IL-10均比相对应组高,有显著意义(P<0.01),而TNF-α又均比相对应组低(P<0.01);PGE2免疫干预组SOD均较相对应组显著升高,而NO明显降低,有显著意义(P<0.01)。结论:PGE2免疫干预对大鼠Vv攻击后具有免疫保护作用。

抗重组蛋白rVVC多克隆抗体的制备及其保护作用的鉴定

目的:纯化rVVC免疫家兔,制备并纯化多克隆抗体。观察复性rVVC、纯化多克隆抗体、rVVC和纯化多克隆抗体混合作用肝癌细胞SMMC7721后,肝癌细胞SMMC7721形态学和活性变化,评估抗重组rVVC蛋白多克隆抗体对肝癌细胞SMMC772的保护作用。方法:纯化重组蛋白rVVC免疫日本大耳兔获得多克隆抗体,多克隆抗体经过硫酸铵盐析、Sephadex G25除盐和DEAE-Sephadex G100层析纯化并进行Westerm Blot鉴定和抗体效价分析。复性rVVC、纯化多克隆抗体、rVVC和纯化多克隆抗体混合物作用肝癌细胞SMMC7721后,显微镜观察并以MTT法确定抗重组rVVC蛋白多克隆抗体对肝癌细胞SMMC7721的保护作用。结果:兔抗重组rVVC多克隆抗体经盐析法、分子筛和阴离子交换层析法纯化后,得到较纯的IgG分子。免疫印迹结果显示,抗重组rVVC蛋白多克隆抗体与纯化抗原呈现特异性反应条带。显微镜观察和MTT结果表明,纯化多克隆抗体能够阻断重组rVVC活性蛋白对肝癌细胞SMMC772的损伤。结论:成功制备了具有保护和阻断作用的兔抗重组rVVC多克隆抗体,为以后rVVC促炎症、促应激作用机制和信号传导机制研究提供一个重要的阻断剂。

创伤弧菌vvhA基因的克隆、原核表达系统的构建及鉴定

目的克隆创伤弧菌(Vibrio vulnificus,Vv)溶细胞素基因(vvhA),构建原核表达系统并鉴定其表达产物的免疫性。方法采用PCR技术从Vv GTC333和WZ01株DNA中扩增全长vvhA基因,T-A克隆后测定其核苷酸序列。采用pET32a质粒构建vvhA基因原核表达载体,在E.coliBL21(DE3)宿主菌中用不同浓度的IPTG诱导目的重组蛋白rVvhA表达,采用Ni-NTA亲和层析法提纯rVvhA,SDS-PAGE检测表达和提纯效果。采用兔抗Vv全菌抗体的Western Blot和兔抗rVvhA血清的免疫扩散试验鉴定其免疫反应性和免疫原性。结果所克隆的vvhA基因核苷酸序列与GeneBank公布的同源性分别为96.09%和98.26%。在0.5 mmol/L IPTG诱导下,rVvhA产量可占细菌总蛋白的18%。提纯的rVvhA经SDS-PAGE后仅显示单一的蛋白条带。重组蛋白rVvhA能与兔抗Vv全菌抗体发生特异性结合,免疫家兔可获得高效价抗体。结论该研究成功地构建了创伤弧菌vvhA基因高效原核表达系统,所表达的rVvhA具有良好的免疫原性和免疫反应性,可作为Vv免疫检测试剂盒及疫苗的抗原。

The cytochrome P4501A1(CYP1A1)inhibitor bergamottin enhances host tolerance to multidrug-resistant Vibrio vulnificus infection

Purpose:Vibrio vulnificus (V. Vulnificus) infection is characterized by rapid onset, aggressive progression, and challenging treatment. Bacterial resistance poses a significant challenge for clinical anti-infection treatment and is thus the subject of research. Enhancing host infection tolerance represents a novel infection prevention strategy to improve patient survival. Our team initially identified cytochrome P4501A1 (CYP1A1) as an important target owing to its negative modulation of the body''s infection tolerance. This study explored the superior effects of the CYP1A1 inhibitor bergamottin compared to antibiotic combination therapy on the survival of mice infected with multidrug-resistantV. Vulnificus and the protection of their vital organs.Methods:An increasing concentration gradient method was used to induce multidrug-resistantV. Vulnificus development. We established a lethal infection model in C57BL/6J male mice and evaluated the effect of bergamottin on mouse survival. A mild infection model was established in C57BL/6J male mice, and the serum levels of creatinine, urea nitrogen, aspartate aminotransferase, and alanine aminotransferase were determined using enzyme-linked immunosorbent assay to evaluate the effect of bergamottin on liver and kidney function. The morphological changes induced in the presence of bergamottin in mouse organs were evaluated by hematoxylin and eosin staining of liver and kidney tissues. The bacterial growth curve and organ load determination were used to evaluate whether bergamottin has a direct antibacterial effect on multidrug-resistantV. Vulnificus. Quantification of inflammatory factors in serum by enzyme-linked immunosorbent assay and the expression levels of inflammatory factors in liver and kidney tissues by real-time quantitative polymerase chain reaction were performed to evaluate the effect of bergamottin on inflammatory factor levels. Western blot analysis of IκBα, phosphorylated IκBα, p65, and phosphorylated p65 protein expression in liver and kidney tissues and in human hepatocellular carcinomas-2 and human kidney-2 cell lines was used to evaluate the effect of bergamottin on the nuclear factor kappa-B signaling pathway. One-way ANOVA and Kaplan-Meier analysis were used for statistical analysis.Results:In mice infected with multidrug-resistantV. Vulnificus, bergamottin prolonged survival (p = 0.014), reduced the serum creatinine (p = 0.002), urea nitrogen (p = 0.030), aspartate aminotransferase (p = 0.029), and alanine aminotransferase (p = 0.003) levels, and protected the cellular morphology of liver and kidney tissues. Bergamottin inhibited interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α expression in serum (IL-1β:p = 0.010, IL-6:p = 0.029, TNF-α:p = 0.025) and inhibited the protein expression of the inflammatory factors IL-1β, IL-6, TNF-α in liver (IL-1β:p = 0.010, IL-6:p = 0.011, TNF-α:p = 0.037) and kidney (IL-1β:p = 0.016, IL-6:p = 0.011, TNF-α:p = 0.008) tissues. Bergamottin did not affect the proliferation of multidrug-resistantV. Vulnificus or the bacterial load in the mouse peritoneal lavage fluid (p = 0.225), liver (p = 0.186), or kidney (p = 0.637).Conclusion:Bergamottin enhances the tolerance of mice to multidrug-resistantV. Vulnificus infection. This study can serve as a reference and guide the development of novel clinical treatment strategies forV. Vulnificus.

Extracellular matrix(ECM)pathway involved in skin immune response of Cynoglossus semilaevis upon Vibrio vulnificus infection

Half-smooth tongue sole(Cynoglossus semilaevis)is regarded as a significant commercial marine fish species in China,and frequent outbreaks of vibriosis has led to substantial economic losses.In this study,we investigated the molecular mechanisms underlying the involvement of the extracellular matrix(ECM)signaling pathway in the skin immune response of C.semilaevis infected with Vibrio vulnificus.The results showed that most differentially expressed proteins(DEPs)were identified through iTRAQ proteome analysis,and ten ECM-related proteins were screened at 12 and 36 h post-infection(hpi).Notably,several DEPs associated with ECM including HSPG,FN,COCH,LAMB1,LAMC1,TSPN,COL6A1,AMBP,HX and ITGA6 were tested for their expression at the transcriptional level during V.vulnificus infection using qRT-PCR assay.The analysis of protein-protein interaction(PPI)showed that the integrin ITGA6 exhibited obvious interactions among ECM-related DEPs.Furthermore,the spatio-temporal expression of the Csitga6 gene was highest in the skin,gill,muscle,and spleen,but lower in the liver,kidney and intestine.To our knowledge,this is the first report on the involvement of ECM pathway in the skin immune response to V.vulnificus infection,and provides a reference for further study of the ECM mechanism in the mucosal immune response of marine fish.

A culture-free method for detection of Vibrio vulnif icus from coastal seawater based on loop-mediated isothermal amplification targeting vcgC gene

Vibrio vulnificus is an opportunistic pathogen widely distributed in estuarine and coastal seawaters. In this study, a culture-free method was developed to rapid detection of Vo vulnificus in all seasons, based on loop-mediated isothermal amplification （LAMP） targeting virulent-correlated gene （vcg）. The new assay method allows differentiation between the virulent and non-virulent strain of V. vulnificus accurately. This method also allows effective detection of the pathogeny in winter when the bacterium lives in the viable but non-culturable （VBNC） state. A total of 30 costal seawater samples collected in all seasons were used for the evaluation of this method. The results show that the method is sensitive, accurate and convenient.

青蛤(Cyclina sinensis)AP-1基因的克隆及在鳗弧菌(Vibrio anguillarum)侵染下的表达分析

为了探究青蛤(Cyclina sinensis)转录因子(transcription factor gene,AP-1)在病原微生物侵染下的免疫防御应答过程中的潜在作用,本研究采用转录组测序筛选、设计特异性引物并成功克隆得到青蛤AP-1基因的c DNA序列(Gen Bank注册号:KX840340),利用生物信息学在线软件进行了生物信息学分析,结果显示:AP-1基因全长1914bp,结构域全长195bp,编码65个氨基酸,开放阅读框全长825bp,编码274个氨基酸,存在一个保守的b ZIP功能域;是重要的转录因子。运用实时荧光定量PCR(q PCR)的方法分析了Ap-1基因在青蛤5个组织中表达过程变化,结果表明:Ap-1基因在血淋巴、外套膜、肝脏、闭壳肌、腮等组织中广泛表达;在血淋巴中表达量最高,在闭壳肌中次之,然后为鳃和外套膜,在肝脏中表达量最低,约为血淋巴的1/7。另外,我们还分析了鳗弧菌(Vibrio anguillarum)侵染青蛤血淋巴后的变化情况,结果显示:在鳗弧菌侵染后,与对照组相比,青蛤血淋巴中的AP-1表达量明显升高,在24h时Ap-1基因表达量最大,与对照组差异极显著(P<0.01)。说明Ap-1基因在青蛤的免疫防御反应中具有重要作用,同时也为进一步研究AP-1在病原微生物侵染青蛤过程中的功能和作用机制奠定了基础。

创伤弧菌VV106的分离鉴定及其生物学特性分析

为研究斜带石斑鱼致病菌VV106的生物学特性,对其进行菌株鉴定、理化因素对生长的影响和药敏性分析.结果表明:VV106为创伤弧菌,具有典型的海洋细菌特征,最适生长温度为30℃、氯化钠浓度为3%、pH值为8.创伤弧菌VV106对硫酸新霉素、氨苄青霉素、磺胺嘧啶、磺胺间甲氧嘧啶和多西环素耐药,对甲砜霉素、氟苯尼考、硫酸卡那霉素、恩诺沙星、盐酸土霉素、磺胺二甲氧嘧啶和交沙霉素敏感.在1/2 MIC氟苯尼考、恩诺沙星和交沙霉素条件下分别诱导获得了Flo20、Enr20和Jos20,它们不仅具有稳定可遗传的耐药性,而且交叉耐药比率分别为75%、50%和62.5%.本研究可为防治创伤弧菌引起的疾病、指导精准用药、提高水产养殖经济效益奠定基础.

实时荧光定量聚合酶链式反应快速检测4种食源性致病弧菌

目的建立和优化4种食源性致病弧菌的实时荧光定量聚合酶链式反应(quantitative real-time polymerase chain reaction,qPCR)快速检测方法。方法基于副溶血性弧菌的toxR基因(GenBank ID:MH047287.1)、霍乱弧菌的ompW基因(GenBank ID:MF100046.1)、拟态弧菌的toxR基因(GenBank ID:EF693743)和创伤弧菌的vvhA基因(GenBank ID:KC821520.1)设计常规PCR引物和qPCR特异性引物和探针。分别建立副溶血性弧菌、霍乱弧菌、拟态弧菌和创伤弧菌4种食源性致病弧菌的单重qPCR检测体系,根据扩增曲线和Ct值对探针浓度进行优化,设置探针浓度梯度。结果4种弧菌的探针最适浓度均为0.1µmol/L。对设计的4种弧菌的引物分别进行常规PCR的扩增,其最低检出限均为1×10^(5) copies/µL,4种弧菌分别进行单重qPCR的最低检出限均为1×10^(1) copies/µL,扩增效率均接近100%。qPCR的灵敏度均高于常规PCR。特异性实验结果表明,4种目的弧菌DNA扩增出特异性曲线,其他的非目的基因未被扩增出扩增曲线。结论该方法准确度和灵敏度高,前处理简单快速,适用于4种食源性致病弧菌的荧光定量PCR快速检测。

创伤弧菌核酸检测试剂国家参考品的建立

目的建立创伤弧菌核酸检测试剂国家参考品并制定质量标准,用于该类试剂的质量评价。方法收集培养多种创伤弧菌及其他弧菌病原体,经菌落鉴定、16s rRNA测序分析及实时荧光定量PCR试剂检测后筛选出18份样本,稀释、分装后组成创伤弧菌核酸检测试剂国家参考品。邀请不同的实验室对参考品进行协作标定,并对参考品进行均匀性和稳定性考察。结果建立的国家参考品,包括8份阳性参考品P1~P8、10份阴性参考品N1~N10、1份重复性参考品R和1份检测限参考品L;L使用菌落计数法测定浓度为1×10^(8)CFU/mL。4家实验室参与了国家参考品的协作标定,并根据结果制定质量标准为阳性符合率8/8,阴性符合率10/10,检出限要求至少为1×10^(3)CFU/mL及以上阳性,重复性要求检测10次均为阳性,且Ct值或定量值变异系数不大于5.0%。国家参考品均匀性检测的Ct值变异系数均不高于5%,符合要求;在2~8℃冷藏条件、室温放置7天,均未影响参考品稳定性。结论成功建立了创伤弧菌核酸检测试剂国家参考品,可用于企业试剂研发的质量控制及评价。