五重荧光定量RT-PCR法检测甲型流感病毒

目的建立一种五重荧光定量RT-PCR检测并鉴别流感病毒A(Flu A)及血凝素H3、H5、H7基因亚型的方法。方法以人细胞核糖核酸酶P(RNase P)基因为内参照,用Primer Express 3.0软件设计PCR特异性引物和探针。构建质粒标准品用于分析该方法的灵敏度和重复性,利用不同来源的呼吸道病毒样本进行特异性分析。结果该法检测Flu A、H3、H5、H7以及RNase P质粒标准品的灵敏度均达102copies/m L,特异性达100%,各对引物和探针仅检测出相应的病毒,未有交叉反应,变异系数(CV)≤1.99%。结论建立的五重荧光定量RT-PCR法灵敏度、特异性高,重复性好,可用于检测出多种甲型流感亚型,对甲型流感病毒不同亚型早期诊断具有一定的价值。     

四重荧光定量RT-PCR检测人感染新型甲型禽流感H7N9病毒

将H7N9禽流感病毒HA和NA基因设为内部参考,并分别设计特异性探针和引物,分析不同来源呼吸道病毒的特异性,并对四重荧光定量RT-PCR检测方法的重复性和灵敏度进行评定。结果甲型H7N9禽流感病毒标本和RNase P检测灵敏度均为102copies/m L,特异性为99.7%,变异系数均低于2.20%,阳性符合率和阴性符合率均为100%。利用四重荧光定量RT-PCR检测H7N9病毒具有较高的灵敏度和特异性,且检测快速准确,对H7N9病毒感染者早期诊断有重要意义。     

五重荧光定量RT-PCR法检测呼吸道病毒

目的 建立一种五重荧光定量RT-PCR检测并鉴别流感病毒A( Flu A)、流感病毒B( Flu B)、呼吸道合胞病毒(RSV)以及新甲型H1N1流感病毒(nH1N1)的方法.方法 以人细胞RNA酶P基因为内参,用Primer Express 3.0设计PCR引物和探针.用一系列不同滴度的病毒培养物和不同来源的呼吸道...     

双重荧光定量RT-PCR快速检测甲型和甲型H1N1流感病毒

目的建立双重荧光RT-PCR快速检测方法,用于甲型和甲型H1N1流感病毒的同时检测和鉴别诊断。方法针对甲型流感病毒M基因和甲型H1N1流感病毒NA基因的保守区序列分别设计特异性引物和Taqman探针,建立优化双重荧光RT-PCR反应体系,评价所建双重RT-PCR反应体系的特异性、敏感性和稳定性,并应用于疑似流感或甲型H1N1流感含漱液标本检测。结果该方法对甲型、甲型H1N1流感病毒检测具有高度特异性,检出限分别为0.01 TCID50和0.1 TCID50,具有较好的稳定性。可从疑似流感或甲型H1N1流感患者含漱液中直接检测到流感病毒核酸。结论本研究建立的双重荧光定量RT-PCR可以同时准确检测甲型和甲型H1N1流感病毒,灵敏度高,稳定性好,是一种快速检测流感病毒的新方法。     

多重荧光定量RT-PCR检测高致病性H5N6禽流感病毒

目的建立一种快速检测流感病毒A型(FluA)及高致病性禽流感(HPAI)H5N6病毒感染的荧光定量RT-PCR法。方法以人细胞核糖核酸酶P(RP)基因作为临床样本的内参基因,用Primer Express 3.0软件设计PCR特异性引物和探针,建立一种检测FluA及HPAI-H5N6病毒的一步法多重荧光定量RT-PCR方法,评估其灵敏度、特异性,并与上海之江公司提供的试剂及测序法进行比较。结果建立的多重荧光定量RT-PCR法检测FluA、H5、N6及RP质粒标准品的灵敏度均达102copies/m L。各对引物和相应探针仅检测出相应的病毒,在这些病毒和常见病原分析中未发现存在交叉反应,特异性达100%。用该法检测135例临床标本,结果显示FluA为23例,未发现HPAI-H5N6病毒的感染者;与上海之江公司提供的试剂的检测结果及基因测序结果一致。结论建立的一步法多重荧光定量RT-PCR法可用于FluA及HPAI-H5N6病毒感染患者的早期诊断。     

江西省首起聚集性人感染H7N9禽流感实验室诊断与分析

目的通过real-time RT-PCR对江西省首起聚集性人感染H7N9禽流感病例进行实验室诊断与分析,进一步提高实验室对人感染禽流感的检测能力。方法采集人感染H7N9禽流感病例咽拭子并提取RNA,分别采用流感甲/乙型通用引物、季节性流感特异性引物(H1N1、H3N2、H1N1pdm)、禽流感特异性引物(H5、H7、N9、H9、H10)进行real-time RT-PCR检测。结果患者A甲型流感、禽流感H7、N9核酸扩增结果均为阳性;患者B(患者A的母亲)甲型流感、禽流感H7核酸扩增结果均为阳性。结论经江西省疾控中心流感实验室复核,患者A与患者B均感染H7N9禽流感病毒,为江西省首起人感染H7N9禽流感病毒聚集性病例。     

甲型H1N1流感病毒RNA检测方法的建立及临床应用

目的建立检测甲型H1N1流感病毒RNA的实时荧光定量逆转录聚合酶链反应(RT-PCR)方法,并探讨其临床应用价值。方法根据甲型H1N1流感病毒HA基因和NA基因的核苷酸序列和GenBank数据库中的核苷酸序列设计检测特异性引物和TaqMan探针,优化反应体系和反应条件,建立检测甲型H1N1流感病毒RNA的实时荧光定量RT-PCR方法,并对方法的敏感性、特异性和重复性进行评价。采用该方法检测21份鼻咽拭子样本的甲型H1N1流感病毒RNA,并与鸡胚培养法进行比较。结果采用实时荧光定量RT-PCR检测甲型H1N1流感病毒RNA结果为阳性,H1~H16流感病毒、季节性流感病毒、猪流感病毒RNA检测结果均为阴性。实时荧光定量RT-PCR的检测敏感性为10~3拷贝/μL RNA分子,标准曲线r~2=0.998。阳性RNA标准品重复检测的循环阈值(Ct)批内、批间变异系数(CV)均10%,阴性标准品[焦碳酸二乙酯(DEPC)水]重复检测的Ct值均0,重复性良好。21份鼻咽拭子样本中有2份样本甲型H1N1流感病毒检测结果为阳性,其他样本均为阴性,与鸡胚培养法的符合率为100%。结论建立的检测甲型H1N1流感病毒RNA的实时荧光定量RT-PCR方法操作简单、耗时短、特异性较强、敏感性较高,可在临床广泛使用。     

禽流感病毒H7N9型 实时荧光定量RT-PCR检测方法的研究

目的 建立一种快速准确诊断禽流感病毒H7N9型的实时荧光定量RT-PCR检测方法。方法 参照国家生物技术信息中心(NCBI)公布的H7N9基因HA序列,设计特异性引物及TaqMan探针; 构建阳性重组质粒,验证其最低检测拷贝数、重复性及特异度。结果 试验构建的阳性重组质粒,线性关系在6×10² copies/μl～6×10⁸ copies/μl范围内检测结果良好; 用该方法测得最低拷贝数为600 copies/μl,特异度和重复性(CV<1%)良好,无交叉反应; 用该荧光定量RT-PCR检测方法检测8份阳性样品和8份阴性样品,准确率为100%(16/16)。结论 实验建立的实时荧光定量RT-PCR检测方法可用于禽流感病毒H7N9型的快速检测。     

一步法实时荧光RT-PCR在快速检测甲型流感病毒中的应用

目的建立一种能快速检测甲型流感病毒的一步法实时荧光PCR方法,对样本进行准确检测。方法以甲型流感病毒特异M基因的保守区为靶区域设计特异引物和TaqMan荧光探针,建立一步法实时荧光RT-PCR方法,并对乙型流感病毒、呼吸道合胞病毒Long株、冠状病毒OC43及229E、副流感病毒1、2、3型以及季节性流感病毒H1、H3进行检测,验证该方法的特异性和灵敏度,并对临床样本进行检测应用。结果所建立的一步法实时荧光RT-PCR可对甲型流感病毒进行特异扩增,操作方便快速,对H1和H3亚型的最低检出限可达0.01TCID50/ml;对384例临床咽拭子样本的检测准确度达99.5%。结论本研究建立的一步法实时荧光RT-PCR方法可实现对甲型流感病毒快速准确的检测,有助于对临床流感病例的快速诊断。     

双重荧光定量RT-PCR法检测柯萨奇病毒A2和A5型

目的建立同时检测柯萨奇病毒A2和A5型(CVA2、CVA5)的双重实时荧光定量RT-PCR法。方法用Primer Express3.0软件设计高度特异性引物和荧光标记探针。通过优化反应体系,建立标准曲线,并评价双重荧光定量RT-PCR法的特异性、灵敏度和重复性。用建立的方法检测367例疑似手足口病患儿的粪便标本,并对阳性结果测序验证。结果荧光定量RT-PCR结果表明本实验所选的引物和探针可特异性检测并区分出CVA2、CVA5亚型,与其他肠道病毒的阳性核酸未发生交叉反应。该方法最低检测限达到102copies/m L,批内变异系数(CV)均1.49%。367份临床标本中CVA2型阳性23份,阳性率6.3%;CVA5阳性11份,阳性率3%,检测阳性结果与测序结果一致。结论成功建立了在单管中同时检测并鉴别CVA2、CVA5型的双重实时荧光定量RT-PCR检测方法。该方法具有较好的灵敏度、特异性、重复性,可用于CVA2、CVA5型引起的暴发疫情的快速筛查和手足口病的流行病学的研究。     

A Km mutant of arylsulfatase A

Semi-micro techniques are presented for the genotyping of galactokinase and galactose-1-phosphate uridyltransferase. Radioactive assays are used to quantitate the levels of enzyme activity, while agarose gel electrophoresis is used to type the variants of galactose-1-phosphate uridyltransferase. All three methods can be performed on 1 ml whole blood, making the procedures ideal for use in confirmation of galactosemia or galactokinase deficiency in newborn screening programs.     

Hepatitis A: A preventable threat

Hepatitis A is a major public health problem, particularly in the pediatric population. Although hepatitis A infection does not cause chronic liver disease, it is associated with significant morbidity. The virus is transmitted primarily by person-to-person contact via the fecal-oral route. The infection can be inapparent, subclinical, anicteric, or icteric. In general, the severity of the disease is inversely correlated with the age of the child. Occasionally, fulminant hepatitis, which is associated with a high mortality rate, may result. The diagnosis of acute hepatitis A is most commonly made through the detection of immunoglobulin M (IgM) anti-hepatitis A antibody. Treatment is generally supportive. General preventive measures include improved standards of hygiene and sanitation. Universal childhood vaccination is the most effective method for eradicating hepatitis A and preventing its transmission.     

RHD845A/1227A基因型个体家系调查分析

本研究对1例RHD845A/1227A基因型个体进行家系调查分析,并分析他们的遗传特性。通过盐水法和间接抗人球蛋白试验（IAT）检测RH（D）抗原,PCR—SSP法检测RHD1227A和RHD845A等位基因以及RHD合子型,基因测序方式分析RIHD基因编码区序列。研究结果表明：血清学检测发现1例RH（D）抗原盐水法检测阴性、IAT法检测阳性样本,经RHD基因编码序列分析发现,RHD第845位与第1227位均出现G／A碱基杂合现象,推测该个体基因型可能为RHD845A／1227A。家系调查显示,先证者父亲为RHD阴性,母亲为RHD阳性。PCR-SSP检测结果显示,父亲携带RHDl227A等位基因,基因型为RHD845A／RHD-;母亲携带RHD845A等位基因,基因型为RHD845A／RHD＋,证明先证者分别从父亲和母亲遗传RHD1227A和RHD845A等位基因,基因型为RHD845A／1227A。结论：经过家系调查分析发现了1例罕见的RHD845A／1227A基因型个体。根据家系调查证明,RHD845A和RHD1227A等位基因分别由遗传获得,而非由个体基因变异形成。     

Potential Therapeutic Applications of Adenosine A2A Receptor Ligands and Opportunities for A2A Receptor Imaging

Adenosine A_2A receptors (A_2ARs) are highly expressed in the human striatum, and at lower densities in the cerebral cortex, the hippocampus, and cells of the immune system. Antagonists of these receptors are potentially useful for the treatment of motor fluctuations, epilepsy, postischemic brain damage, or cognitive impairment, and for the control of an immune checkpoint during immunotherapy of cancer. A_2AR agonists may suppress transplant rejection and graft‐versus‐host disease; be used to treat inflammatory disorders such as asthma, inflammatory bowel disease, and rheumatoid arthritis; be locally applied to promote wound healing and be employed in a strategy for transient opening of the blood–brain barrier (BBB) so that therapeutic drugs and monoclonal antibodies can enter the brain. Increasing A_2AR signaling in adipose tissue is also a potential strategy to combat obesity. Several radioligands for positron emission tomography (PET) imaging of A_2ARs have been developed in recent years. This review article presents a critical overview of the potential therapeutic applications of A_2AR ligands, the use of A_2AR imaging in drug development, and opportunities and limitations of PET imaging in future research.     

A PHYSIOLOGICO-PATHOLOGICAL STUDY OF A CASE OF HEART-BLOCK OCCURRING IN A DOG AS A RESULT OF NATURAL CAUSES

What is believed to be the first known case of heart-block arising in a dog as a result of an ingenerate pathological lesion is here reported. The auriculo-ventricular dissociation was of that degree known as relatively complete block and became apparent on section of the right vagus nerve. Stimulation of the peripheral end of the cut vagus failed to inhibit the ventricles, although complete inhibition of the auricles occurred. The same results were obtained during the ventricular acceleration produced by strophanthin, so that the failure of the vagus to inhibit the ventricles is not due to the latter''s infrequent action, but more probably to a normal lack of direct chronotropic influence upon the ventricular muscle. These findings are similar to those obtained by Erlanger in experimental heart-block. To small repeated doses of strophanthin injected intravenously the heart reacted as follows: (a) irregular slowing of the auricles and conversion of the relatively complete into an absolutely complete a-v block; (b) a rise in the irritability of the cardiac muscle manifested by a rapidly progressing auricular and ventricular frequency, the ventricular frequency surpassing ultimately the auricular frequency; (c) complete arrest of the auricles, the ventricles continuing at their high rate; (d) sudden fibrillation of the ventricles and shortly afterwards arrest in diastole. There were found post mortem myxomatous-like thickenings at the free edge of the septal tricuspid leaflet and at the attached margin of the posterior aortic leaflet and along part of the right anterior aortic leaflet. There was also a grayish patch on the right side of the auricular septum above the auriculo-ventricular junction. The thickenings at the edge of the valves consisted of dense, circumscribed masses of what appeared to be new connective tissue. The same tissue was found pressing against the bundle along the greater part of the latter''s course. There was considerable fatty infiltration of the auricular musculature immediately above the bundle and, to a slight extent, of the bundle itself. The fibers of communication between the auricular muscle and the node of Tawara were relatively few as compared with those of the normal heart. A review of the pathology of heart-block is appended, showing the present status of the question, concerning the relationship existing between disease of the a-v conducting system and the various grades of heart-block.