实时荧光RT-PCR与细胞培养法在流感病毒检测中的应用

目的探讨实时荧光逆转录聚合酶链式反应(rRT-PCR)在流感病毒检测中的应用,并比较与常规细胞培养方法的差异,以确定两种检测方法各自更适合的应用领域。方法收集烟台市两所国家级哨点医院就诊的流感样病例(ILI)咽拭子标本,应用实时荧光RT-PCR检测流感病毒核酸,阳性标本用狗肾传代细胞(MDCK)培养、分离流感病毒,比较两种检测方法的灵敏度及相关性差异。结果在617份标本中,实时荧光RT-PCR检测阳性152份,阳性率为24.64%;MDCK细胞培养法分离流感病毒79株,阳性率为12.80%。两者的阳性率差异有统计学意义(χ2=28.38,P<0.01)。结论实时荧光RT-PCR和细胞培养方法检测流感病毒具有一致的特异性。实时荧光RT-PCR可作为一种快速有效的流感病毒核酸检测法,适用于公共卫生应急疫情的实验室快速诊断;细胞培养法作为流感病原学监测的基础,用于核实暴发疫情的实验室诊断,对病毒进行抗原性和基因特性分析。     

3种方法对发热伴血小板减少综合征检测结果比较

目的 采用实时荧光PCR法、酶联免疫吸附法及细胞培养分离病毒株的方法,对2013年临床疑似发热伴血小板减少综合征患者急性期血进行检测比较,寻求快速、准确的鉴定新布尼亚病毒感染的方法。方法 对180例临床疑似病例的急性期血液首先采用实时荧光PCR法检测,筛选出新布尼亚病毒核酸阳性的样本,同时将阳性样本采用酶联免疫吸附的方法检测抗体及采用VERO-E6细胞培养的方法分离病毒株。结果 180份样本经实时荧光PCR法检测,阳性率为42.78%(77/180 Ct值≤35曲线形态与阳性对照一致呈S型的),ELISA方法检测抗体阳性率为44.16%(34/77),将核酸检测阳性的77份样本全部感染VERO-E6细胞获得病毒株32株,阳性率为38.55%(32/77)。结论 实时荧光PCR法更适合于新布尼亚病毒感染者早期实验室快速诊断,发病在1w左右的病例不适合用ELISA的方法做确证实验。细胞培养分离病毒虽然是金标准,但耗时、费力,对早期诊断意义不大,但适于研究或疫苗研发。     

实时荧光定量PCR技术检测肺炎衣原体的诊断价值

目的探讨实时荧光定量PCR技术检测肺炎衣原体的诊断价值。方法对呼吸道感染的186例患者的痰液分别采用实时荧光定量PCR技术和基因测序检测,以评价实时荧光定量PCR技术检测肺炎衣原体感染的准确性。结果实时荧光定量PCR检测肺炎衣原体的灵敏度为100.0%,特异度为93.1%,阳性预期值为80.8%,阴性预期值为100.0%,准确率为94.6%。结论实时荧光定量PCR技术检测肺炎衣原体具有较高的可靠性,适用于临床快速诊断肺炎衣原体感染的相关疾病。     

定量PCR检测HCMV-DNA结合细胞培养分离病毒法诊断小儿HCMV感染

目的探讨实时荧光定量PCR检测HCMV-DNA结合细胞培养分离病毒,对诊断小儿HCMV感染的价值。方法使用实时荧光定量PCR法检测尿液HCMV-DNA与细胞培养分离尿液HCMV二种方法检测。结果（1）该实时荧光定量PCR检测HCMV-DNA体系,与相关的三种病毒（I型单纯疱疹病毒、腺病毒和呼吸道合胞病毒）无交叉反应,20例正常幼儿皆为阴性。（2）40例临床患儿实时荧光定量PCR检测尿液HCMV-DNA,阳性10例。其中,唇腭裂组阳性6例（6/11）,肺炎组阳性4例（4/29）,两组检出结果比较,差异有显著性（χ2=5.06,P〈0.05）,唇腭裂组阳性检出高于肺炎组。（3）40例临床患儿细胞培养法HCMV分离检测,阳性8例,其中,唇腭裂组阳性4例（4/11）,肺炎组阳性为4例（4/29）,两组检出结果比较,差异无显著性（χ2=1.21,P〉0.05）。（4）40例临床患儿,实时荧光定量PCR检测尿液HCMV-DNA与细胞培养分离病毒结果符合度比较,差异无显著性（χ2=3.33,P〉0.05）。二种方法阳性检出比较,差异无显著性（χ2=0.38,P〉0.05）。结论实时荧光定量PCR检测尿液HCMV-DNA,与细胞培养法分离病毒联合应用可提高HCMV感染诊断阳性率。     

实时荧光PCR技术用于RHc检测的研究

目的:探讨实时荧光PCR方法用于中国人群RHc基因检测的可行性。方法:收集144例大连地区RhD阳性健康献血者血样，使用血清学分型方法检测样本RhCcEe血型表型。使用检测RHCE基因第2外显子178C>A和307C>T多态性位点的TaqMan探针实时荧光PCR方法对血液标本进行RHc基因分型，并将基因分型结果与血清学分型结果相比较。结果:经血清学检测，144例样本中有60例为CCee、48例为CcEe、17例为ccEE、11例为Ccee、7例为ccEe、1例为ccee;经TaqMan探针实时荧光PCR方法检测，发现RHc基因阳性为84例、阴性为60例，基因分型结果与血清学分型结果一致，该方法的灵敏度、特异度和准确度均为100%。结论:TaqMan探针实时荧光PCR方法适用于检测中国人群RHc基因，可用于临床实验室对Rhc血型进行基因检测。     

实时荧光PCR检测儿童肺结核粪便中Mtb-DNA的效果评价

目的 应用实时荧光PCR快速检测肺结核患儿粪便中Mtb-DNA,并初步评估其临床效果。 方法 收集肺结核住院儿童粪便标本76份,应用实时荧光PCR检测Mtb-DNA,检测结果与20例其他呼吸系统疾病患儿的粪便实时荧光PCR检测Mtb-DNA、76例粪便抗酸杆菌涂片检查以及41例患儿痰标本的涂片和(或)培养、实时荧光PCR检测结果进行比较。 结果 粪便实时荧光PCR检测敏感度达到23.68%(18/76),特异度为100.00％(20/20),肺结核患儿粪便PCR阳性率［23.68%(18/76)］明显高于涂片阳性率［6.58%(5/76)］,41例患儿中痰涂片和(或)培养阳性例数(15例)和痰PCR检测阳性例数(18例)高于粪便PCR检测阳性例数(11例)。 结论 实时荧光PCR检测儿童粪便Mtb-DNA,是一种特异度高、比较敏感、非侵入性且相对安全的儿童肺结核快速诊断方法。     

基于GroEL基因的实时荧光定量PCR检测恙虫病东方体方法的建立及评价

目的 建立一种敏感、特异的实时荧光定量PCR方法,用于检测临床标本中的恙虫病东方体。方法 根据恙虫病东方体GroEL蛋白基因序列设计引物和探针,建立实时荧光定量PCR检测方法。并从灵敏度、特异度、重复性及临床标本的检测能力等方面对本方法进行综合评价。结果 建立的实时荧光定量PCR方法具有良好的特异性,可检测出恙虫病东方体Gilliam、Karp、Kato和Kawasaki株。建立的标准曲线循环阈值(Ct)与模板拷贝数呈现良好的线性关系(r=0.99),灵敏度分析显示样品最低检出浓度为21.8拷贝/μL。批内及批间重复性实验变异系数均小于1.5%,显示本方法具有良好的重复性。对本实验室保存的82份临床标本进行检测,并与常规巢式PCR方法比较,本方法检测出26份阳性标本中的25份,检出率为96.15%。结论 本研究建立的实时荧光定量PCR方法具有较高的敏感性、特异性和稳定性,可用于临床病人及暴发疫情的实验室快速检测。     

实时荧光定量PCR法分析结核分枝杆菌对异烟肼耐药的分析

目的利用实时荧光定量PCR技术快速检测异烟肼耐药结核分枝杆菌。方法收集到医院就诊的结核病疑似患者痰液样本,提取痰液样本的总DNA,利用实时荧光定量PCR(Real-time PCR)技术对结核分枝杆菌感染进行快速筛查,并与传统药敏试验进行比较,对两者的灵敏度、特异性、一致性进行比较分析。结果检测346例结核病人临床分离培养样本,药敏试验检出257例异烟肼敏感标本,101例异烟肼耐药标本;实时荧光定量PCR法共检测出异烟肼敏感和耐药标本225例98例,灵敏度为86.64%,特异性为93.92%,一致率为93.12%。结论跟传统药物敏感性实验相比,实时荧光定量PCR法检测速度快速、特异性强、灵敏度较高,可用于结核分枝杆菌耐异烟肼突变的快速检测,适于耐多药结核病的快速筛查。     

TaqMan MGB探针实时荧光定量PCR快速检测布鲁氏菌

目的利用新一代TaqMan MGB探针技术,建立特异敏感的实时荧光定量PCR方法,用于布鲁氏菌的快速检测。方法针对布鲁氏菌基因组中16SrRNA序列设计特异性引物和探针,建立一套基于TaqMan MGB探针技术的实时荧光定量PCR方法,验证方法的特异性、敏感性和稳定性,对2008-2010年期间采集的773份动物样本中的布鲁氏菌进行检测,并与普通PCR方法进行比较分析。结果建立的TaqMan MGB探针实时荧光定量PCR方法对布鲁氏菌的检测具有高度的特异性,对小肠结肠耶尔森菌、假结核耶尔森菌、肠炎沙门氏菌、大肠埃希菌、铜绿假单胞菌、空肠弯曲菌、泰泽氏菌均无交叉反应。生成的标准曲线的相关系数为0.999,斜率为-3.301,TaqMan MGB探针实时荧光定量PCR效率为100.872%。TaqMan MGB探针实时荧光定量PCR能够准确地从布鲁氏菌阳性样本中检测到布鲁氏菌DNA,最低能够检测到的布鲁氏菌数量为9.3拷贝,比常规PCR方法的灵敏度高100倍。对773份动物样本进行检测,结果TaqMan MGB探针实时荧光定量PCR能检出53份布鲁氏菌阳性样本,而常规PCR只检出37份阳性。结果显示,TaqMan MGB探针实时荧光定量PCR方法比常规PCR方法更敏感,能够直接从动物样本中检出布鲁氏菌DNA,检测时间仅为2h。结论本研究首次建立了直接用于检测动物样本中布鲁氏菌的TaqMan MGB探针实时荧光定量PCR方法,该技术适用于传染性病原体的快速检测与鉴定。     

实时荧光定量PCR在手足口病肠道病毒快速检测中的应用

目的建立快速检测手足口病肠道病毒的实时荧光定量PCR法,并作出应用分析。方法采用卫生部《手足口病预防控制指南》(2008年版)推荐的RT-PCR法,对丽水市2008年4～5月间发生的172例临床诊断手足口病患者的324份标本进行人肠道病毒、柯萨奇病毒A组16型和肠道病毒EV71型特异性核酸的检测,同时采用实时荧光定量PCR法进行平行检测,比较两者的实验结果,分析实时荧光定量PCR方法的特异性和灵敏度。结果实时荧光定量PCR检测324份标本,肠道病毒通用核酸阳性70份,柯萨奇A16核酸阳性22份,肠道EV71病毒核酸阳性15份,与RT-PCR结果一致,符合率100%。结论实时荧光定量PCR法具有特异性强、灵敏度高等优点,是一种理想的手足口病肠道病毒的检测方法。     

胰岛素瘤的解剖分布特点分析

目的胰岛素瘤是最常见的胰腺神经内分泌肿瘤，因其临床表现多样，导致诊断困难。影像学诊断尤其是超声内镜(EUS)在胰岛素瘤的诊断中起着重要作用，拥有较高的敏感性和特异性。本研究拟通过明确胰岛素瘤的解剖分布特点，以期有助于提高影像学的诊断准确率和降低漏诊率，尤其是在教育和培训实践中对于EUS的学习者更具有指导价值。 方法回顾性分析解放军总医院第一医学中心病案资料数据库1993年1月至2019年11月经外科手术、病理确诊为胰岛素瘤的患者的临床资料，检索方法采取搜索术后病理诊断为"胰岛素瘤"的病例，通过查阅病例的方法，提取出胰岛素瘤的大小和解剖分布等数据，进一步分析其特点。 结果共检索到确诊为胰岛素瘤的患者116例，其中，男45例、女71例，年龄13～76岁，平均年龄(44.4±14.85)岁。胰岛素瘤单发110例(94.8%)、多发6例(5.2%)。位置分布：头颈部46例(39.7%)，单发45例、多发1例；体尾部68例(58.6%)，单发65例、多发3例；全胰腺多发2例(1.7%)。病变大小特点：最大径0.4～3.4 cm，平均大小(1.53±0.58)cm。≤1 cm 29例、>1 cm而≤1.5 cm41例、>1.5 cm而≤2.0 cm28例，≤3 cm 15例，>3 cm 3例。年龄与肿瘤的大小相关，≤44岁患者肿瘤平均大小为(1.36±0.51)cm、>44岁患者肿瘤平均大小为(1.70±0.60)cm，P<0.05。头颈部的肿瘤大于体尾部的肿瘤，头颈部肿瘤平均大小(1.66±0.63)cm，体尾部(1.42±0.52)cm，P<0.05。 结论胰岛素瘤在胰腺体尾部较头颈部更好发；绝大多数单发，但可以全胰腺多发；多数小于1.5 cm，肿瘤的大小与患者年龄和肿瘤的解剖分布相关。     

Pathogenesis of carcinoma of the papilla of Vater

Most adenomas and carcinomas of the small intestine and extrahepatic bile ducts arise in the region of the papilla of Vater. In familial adenomatous polyposis (FAP) it is the main location for carcinomas after proctocolectomy. In many cases symptoms due to stenosis lead to diagnosis at an early tumor stage. In about 80%, curative intended resection is possible. Operability is the most relevant prognostic factor. Most ampullary carcinomas resp. carcinomas of the papilla of Vater develop from adenomatous or flat dysplastic precursor lesions. They can be sited in the ampulloduodenal part of the papilla of Vater, which is lined by intestinal mucosa. They also can develop in deeper parts of the ampulla, which are lined by pancreaticobiliary duct mucosa. Intestinal-type adenocarcinoma and pancreaticobiliary-type adenocarcinoma represent the main histological types of ampullary carcinoma. Furthermore, there exist unusual types and undifferentiated carcinomas. Many carcinomas of intestinal type express the immunohistochemical marker profile of intestinal mucosa (keratin 7?, keratin 20+, MUC2+). Carcinomas of pancreaticobiliary type usually show the immunohistochemical profile of pancreaticobiliary duct mucosa (keratin 7+, keratin 20?, MUC2?). Even poorly differentiated carcinomas, as well as unusual histological types, may conserve the marker profile of the mucosa they developed from. These findings underline the concept of histogenetically different carcinomas of the papilla of Vater which develop either from intestinal- or from pancreaticobiliary-type mucosa of the papilla of Vater. Molecular alterations in ampullary carcinomas are similar to those of colorectal as well as pancreatic carcinomas, although they appear at different frequencies. In future studies, molecular alterations in ampullary carcinomas should be correlated closely with the different histologic tumor types. Consequently, the histologic classification should reflect the histogenesis of ampullary tumors from the two different types of papillary mucosa.     

Demonstration of the mechanism of action of biguanides

Summary Palmitic acid oxidation in rat diaphragm homogenate is depressed by biguanide concentrations that are still incapable of inhibiting oxidative phosphorylation. Glucose oxidation is not directly effected by the same biguanide concentrations: however, the inhibitory effect of palmitic acid on glucose oxidation is partly removed by biguanides. Inhibition of fatty acid oxidation, which accounts for most of the metabolic effects caused by these drugs, can be regarded as the fundamental mechanism of action of biguanides. There is some evidence suggesting that these drugs might interact with carnitine, thus preventing long-chain fatty acids from being transported across the mitochondrial membrane to the site of oxidation. Traduzione a cura degli AA.     

Lack of correlation of degree of villous atrophy with severity of clinical presentation of coeliac disease

BACKGROUND AND AIM: Both the clinical presentation and the degree of mucosal damage in coeliac disease vary greatly. In view of conflicting information as to whether the mode of presentation correlates with the degree of villous atrophy, we reviewed a large cohort of patients with coeliac disease. PATIENTS AND METHODS: We correlated mode of presentation (classical, diarrhoea predominant or atypical/silent) with histology of duodenal biopsies and examined their trends over time. RESULTS: The cohort consisted of 499 adults, mean age 44.1 years, 68% females. The majority had silent coeliac disease (56%) and total villous atrophy (65%). There was no correlation of mode of presentation with the degree of villous atrophy (p=0.25). Sixty-eight percent of females and 58% of males had a severe villous atrophy (p=0.052). There was a significant trend over time for a greater proportion of patients presenting as atypical/silent coeliac disease and having partial villous atrophy, though the majority still had total villous atrophy. CONCLUSIONS: Among our patients the degree of villous atrophy in duodenal biopsies did not correlate with the mode of presentation, indicating that factors other than the degree of villous atrophy must account for diarrhoea in coeliac disease.     

血吸虫童虫生长发育及其机制的研究进展

血吸虫童虫是宿主免疫系统攻击的重要靶标,包括皮肤型、肺型和肝门型童虫。宿主分子对童虫生长发育具有重要作用。童虫生长发育机制包括免疫调节、信号转导、性别发育及凋亡等。肌动蛋白、组织蛋白酶、烯醇化酶和葡萄糖基转移酶等分子为血吸虫童虫生长发育的重要分子。本文对血吸虫童虫生长发育及其机制的研究进展做一综述。     

124例耐多药结核分枝杆菌基因突变特征分析

目的对临床分离的耐多药结核分枝杆菌相关基因的突变特征进行分析。方法对124例耐多药结核分枝杆菌以及50株敏感株的耐药相关基因(包括异烟肼inh A、kat G、oxyR-ahp C间隔区以及利福平rpo B)进行序列测定,分析其基因突变情况。结果异烟肼耐药inh A基因突变率为14.5%;kat G基因突变率为70.2%(87/124),主要位于315位;oxyR-ahp C间隔区突变率为15.3%;inh A、kat G两种基因同时突变率75.0%,三种基因同时突变率为89.5%。利福平rpo B基因突变的检出率高达95.2%,突变主要发生在531、526、516位点。结论我省耐多药菌异烟肼耐药相关基因最常见突变为kat G 315、inh A C-T(-15)、axyR-ahp C间隔区(-10)C-T,利福平为rpo B531、526、516。结合MDR-TB耐药相关基因的特征分析,可以建立一种快速、准确、特异的适合于我省的检测结核菌耐多药性的新方法。     

氯硝柳胺悬浮剂的毒性评价

目的评价氯硝柳胺悬浮剂的毒性，为现场大规模应用灭螺提供依据。方法按照中华人民共和国国家标准GB 15670-1995《农药登记毒理学试验方法》和鱼类毒性试验方法进行。结果经口、经皮肤的LDso雌、雄性大鼠均>5 000 mg/kg，经呼吸道的LCso雌、雄性大鼠均>5 000mg/m3，该药经口、经皮肤、经呼吸道毒性均属微毒类药物；兔眼用药后，观察期内无不良反应，对眼无刺激性；皮肤用药后对皮肤无刺激性。与氯硝柳胺原药、氯硝柳胺乙醇胺盐原药和氯硝柳胺乙醇胺盐可湿性粉剂相比，氯硝柳胺悬浮剂对鱼急性毒性最低。结论氯硝柳胺悬浮剂属微毒类药物，对鱼的毒性低于其乙醇胺盐可湿性粉剂，适合于现场应用。     

Assessment of quality of life of parents of children with juvenile chronic arthritis

The aim of the study was to assess the quality of life (QOL) and the psychological status of parents of children with juvenile chronic arthritis (JCA). The QOL, anxiety and depression of the parents of 28 children with JCA were evaluated and compared to those of the parents of 28 healthy children. Mothers of JCA children and mothers of healthy children reported similar QOL. The reported anxiety and depression levels were similar for mothers and fathers in both groups. The parents of children with pauciarticular-type JCA reported lower QOL and higher levels of anxiety and depression than the parents of children with other types, namely polyarticular and systemic JCA. These findings may be explained by the fact that the pauciarticular patients had shorter disease duration and were less frequently seen in the outpatient clinic. The QOL of mothers of children with JCA was found to be slightly impaired in the group of children with pauciarticular JCA. Future larger studies are needed to confirm these results, as the number of subjects in the three groups was rather low. Received: 26 September 2001 / Accepted: 8 February 2002     

Numerical Simulation of the Effect of Rate of Change of Glucose on Measurement Error of Continuous Glucose Monitors

Background

A 5-day in-patient study designed to assess the accuracy of the FreeStyle Navigator® Continuous Glucose Monitoring System revealed that the level of accuracy of the continuous sensor measurements was dependent on the rate of glucose change. When the absolute rate of change was less than 1 mg•dl⁻¹•min⁻¹ (75% of the time), the median absolute relative difference (ARD) was 8.5%, with 85% of all points falling within the A zone of the Clarke error grid. When the absolute rate of change was greater than 2 mg•dl⁻¹•min⁻¹ (8% of the time), the median ARD was 17.5%, with 59% of all points falling within the Clarke A zone.

Method

Numerical simulations were performed to investigate effects of the rate of change of glucose on sensor measurement error. This approach enabled physiologically relevant distributions of glucose values to be reordered to explore the effect of different glucose rate-of-change distributions on apparent sensor accuracy.

Results

The physiological lag between blood and interstitial fluid glucose levels is sufficient to account for the observed difference in sensor accuracy between periods of stable glucose and periods of rapidly changing glucose.

Conclusions

The role of physiological lag on the apparent decrease in sensor accuracy at high glucose rates of change has implications for clinical study design, regulatory review of continuous glucose sensors, and development of performance standards for this new technology. This work demonstrates the difficulty in comparing accuracy measures between different clinical studies and highlights the need for studies to include both relevant glucose distributions and relevant glucose rate-of-change distributions.     

Study of constancy of hydrogen-consuming flora of human colon

The constancy of the hydrogen consuming flora of the human colon was studied in 15 healthy subjects via two measurements obtained 18 to 36 months apart. Hydrogen disappearance rate and the major products of H₂-consuming bacteria, methane and sulfide, were measured during incubation of fecal homogenates with excess hydrogen and sulfate. In 11/15, the hydrogen consumption rate and the predominant hydrogen-consuming pathway (methanogenesis, sulfate reduction, or neither) remained constant. However, major shifts in these pathways were observed in four subjects, with two losing and two gaining the ability to produce methane. Methanogenesis was associated with the highest hydrogen consumption rate. This study demonstrates that clinically unrecognizable, major alterations of the colonic flora occur in healthy subjects. Understanding of the factors responsible for these alterations might allow for therapeutic manipulation of the colonic flora.Supported in part by the Department of Veterans Affairs and NIDDKD RO1 DK 13309-25.