二重逆转录—聚合酶链反应及微孔板反向杂交法检？…

目的 为适应临床上需同时检测庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）感染情况，建立了二重逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）及微孔板反向杂交法。方法 根据ＨＣＶ与ＨＧＶ基因与ＨＧＶ特异探针微孔板反向杂交检测ＣＰＲ产物。结果 ＰＣＲ产物经测序，ＨＣＶ与Ｔａｋａｍｉｚａｗａ等及Ｃｈｏｏ等报道的核苷酸同源性分别为９３．１％￣９４．１％与９２．５％￣９３．７％，ＨＧＶ与Ｓｉｍｏｎｓ等、Ｌｉｎｎｅｎ等     

肝细胞癌组织中庚型肝炎病毒感染的研究

目的探讨庚型肝炎病毒（ＨＧＶ）在肝细胞癌（ＨＣＣ）组织中存在状态。方法采用抗ＨＧＶＮＳ５单克隆抗体,以免疫组织化学法检测５３例ＨＣＣ患者石蜡包埋的肝癌组织。结果ＨＣＣ组织中检出ＨＧ抗原（ＨＧＡｇ）２０例（３７７％）,在非乙非丙、乙型及丙型肝炎病毒组中,分别检出ＨＧＡｇ２例（２５％）、１６例（３９％）和２例。结论ＨＣＣ患者中ＨＧＶ感染较常见;ＨＧＶ感染似乎不是ＨＣＣ发生的高危因素。     

一种同时检测丙型与庚型肝炎病毒的逆转录-巢式聚合酶链反应方法

目的庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）同属黄病毒科，且传播途径相似，重叠感染率高，本研究旨在建立一种同时检测ＨＧＶ与ＨＣＶ感染的方法。方法根据ＨＣＶ与ＨＧＶ的基因序列分别选取５’－ＵＴＲ（ＨＣＶ）与ＮＳ３（ＨＧＶ）的两套引物，在同一管内进行逆转录－巢式聚合酶链反应，并初步应用于１５３例标本。结果该方法能同时检出ＨＧＶ与ＨＣＶ感染，扩增片段大小与设计相符。结论该方法简便特异，适用于临床检测     

逆转录─巢式PCR在一管中同时检测丙型与庚型肝炎病毒

逆转录┐巢式ＰＣＲ在一管中同时检测丙型与庚型肝炎病毒谭文杰夏宁邵丛郁庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）同属黄病毒科，且传播途径相似，重叠感染率高。本室根据对ＨＧＶ与ＨＣＶ基因序列的分析，分别选取５′ＵＴＲ（ＨＣＶ）与ＮＳ３（ＨＧＶ）区的两...     

一种同时检测丙型与庚型肝炎病毒的逆转录—巢式？…

目的 庚型肝炎病毒（ＨＧＶ）与丙型肝炎病毒（ＨＣＶ）同属黄病毒科，且传播途径相似，重叠感染率高，本研究旨在建立一种同时检测ＨＧＶ与ＨＣＶ感染的方法。方法 根据ＨＣＶ与ＨＧＶ的基因序列分别选取５‘－ＵＴＲ（ＨＣＶ）与ＮＳ３（ＨＧＶ）的两套引物，在同一管内进行逆转录－巢式聚合酶锭反应，并初步应用于１５３例标本。结果 该方法能同时检出ＨＧＶ与ＨＣＶ感洒，扩增片段大小与设计相符。结论 该方法简便特异，适用     

丙型肝炎病毒感染者中抗病毒IgM检测的意义

建立了抗ＨＣＶＩｇＭ间接ＥＬＩＳＡ方法，并用之检测ＨＣＶ不同感染人群。抗ＨＣＶＩｇＭ在不同感染人群中的检出率变化很大。急性输血后丙型肝炎患者检出率可高达９３．８％，而正常献血员中可低至０．６８％。比较抗ＨＣＶＩｇＭ和抗ＬｇＧ阳性中ＨＣＶＲＮＡ的检测结果发现，抗ＩｇＭ阳性者中，不同ＨＣＶ感染人群的ＨＣＶＲＮＡ检出率很高（９３．０％～１００％）；而ＩｇＧ阳性者中ＨＣＶＲＮＡ的检出率变化很大（３７．５％～９３．８％）。在４３例血液透析者中抗ＩｇＭ与ＨＣＶＲＮＡ检测的一致性为８３．７％；抗ＩｇＭ与抗ＩｇＧ检测的一致性为８８．４％，结果提示：（１）抗ＨＣＶＩｇＭ与ＨＣＶ活跃复制有关，（２）抗ＨＣＶＩｇＭ与抗ＨＣＶＩｇＧ检出不完全一致。因此，临床检测抗ＨＣＶＩｇＭ有其特殊意义。     

庚型肝炎病毒IgM抗体检测方法的建立及其临床意义

目的建立一种早期、快速诊断庚型肝炎的血清学检测方法。方法以辣根过氧化物酶标记庚型肝炎病毒（ＨＧＶ）多肽ＮＳ３,ＮＳ５区段抗原,建立了捕获酶联免疫吸附试验（ＥＬＩＳＡ）,用于检测血清中ＨＧＶＩｇＭ抗体。结果本法不受特异性ＩｇＧ的竞争和类风湿因子的干扰;与其它致肝炎的病毒（ＨＡＶ、ＨＢＶ、ＨＣＶ、ＨＥＶ、ＣＭＶ、ＥＢＶ）无交叉反应。检测４６例非甲、乙、丙、戊型肝炎患者血清,抗－ＨＧＶＩｇＭ阳性１４例,阳性率３０．４３％,其中,６例同时为ＨＧＶＲＮＡ阳性,阳性符合率为４２．８６％（６／１４）,检测１２例庚型肝炎病人双份血清,其中,急性期血ＨＧＶＩｇＭ抗体均为阳性。结论该法检测ＨＧＶＩｇＭ抗体特异性强,敏感性高,且简便快速,适用于临床对庚型肝炎新近感染的早期诊断,有推广应用价值。     

谷氨酰胺对缺氧复氧损伤人小肠上皮细胞谷胱甘肽的影响

目的：探讨谷氨酰胺（ＧＬＮ）对缺氧复氧损伤人小肠上皮细胞（ＩＥＣ）内还原型谷胱甘肽（ＧＳＨ）和丙二醛（ＭＤＡ）的影响。方法：采用原代培养人ＩＥＣ作为实验模型,用荧光法测定体外培养ＩＥＣ内ＧＳＨ和ＭＤＡ含量。结果：正常对照组ＧＳＨ含量最高,在对照组加入ＧＬＮ并不影响ＧＳＨ含量（Ｐ〉０．０５）。缺氧６０ｍｉｎ或９０ｍｉｎ复氧３０ｍｉｎ损伤ＩＥＣ细胞内ＧＳＨ含量均显著低于正常对照组（Ｐ〈０．０５）,预先     

肝细胞癌组织中转化生长因子α表达及其与乙型肝炎病毒感染的 …

目的 研究肝细胞（ＨＣＣ）中转化生长因子α（ＴＧＦα）的表达及其与ＨＢＶ感染的关系。方法 应用原位杂交及免疫组化ＳＰ法对５３例ＨＣＣ及１４例正常肝组织吕ＴＧＦαｍＲＮＡ、ＴＧＦα蛋白、ＨＢＶ ＤＮＡ进行检测。结果 ５３例ＨＣＣ组织中ＴＧＦαｍＲＮＡ、ＴＧＦα蛋白的阳性率分别为３０．２％（１６例）、７３．６％（３９例）,显著高于无表达的正常对照组（Ｐ〈０．０５）。癌旁不典型增生肝细胞组ＴＧＦαｍＲＮ     

软骨发育不全FGFR3基因突变的研究

目的了解中国人软骨发育不全患者成纤维细胞生长因子受体３（ｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒｒｅｃｅｐｔｏｒ３，ＦＧＦＲ３）基因变异情况。方法应用ＰＣＲ－ＳＳＣＰ和限制性内切酶酶切方法，分析辽宁地区７例软骨发育不全（ａｃｈｏｎｄｒｏｐｌａｓｉａ，ＡＣＨ）患者外周血ＤＮＡ标本中ＦＧＦＲ３基因第１０外显子区域。结果７例患者均检测到相同的Ｇ３８０Ｒ点突变。结论表明Ｇ３８０Ｒ为中国人ＡＣＨ患者常见突变。应用ＰＣＲ－ＳＳＣＰ和限制性内切酶酶切的方法检测ＦＧＦＲ３基因突变是产前诊断和早期诊断ＡＣＨ患者的简便、快速、可靠的手段     

Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2

The Sindbis virus mutant SB-RL, in contrast to its parent, Sindbis strain AR339 (SB), is attenuated in neonatal mice, has an increased rate of penetration in tissue culture cells, and is more sensitive to neutralization by E2-specific monoclonal antibodies (MCAbs) R6 and R13. These phenotypic differences are controlled by substitution of an arginine for serine at amino acid 114 of the E2 glycoprotein. To explore these relationships further, MCAb R6 and R13 neutralization escape mutants of both SB and SB-RL were isolated and characterized. All mutants bound both MCAb R6 and R13 significantly less effectively in ELISA, and were more resistant to complement-mediated neutralization than their respective parental strains. Single coding changes in the E2 glycoprotein gene of each 11 mutants were identified. SB/R6, SB/R13, and SB-RL/R13 mutants contained a mutation at either E2 codon 96 or 159. SB-RL/R6 mutants contained changes at E2 codon 62, 96, or 159. These coding changes included two intragenic suppressor mutations. Mutation of E2 codon 159 from lysine to glutamate or codon 62 from asparagine to aspartate suppressed the attenuated phenotype conferred by E2 arginine 114 in SB-RL. However, only the change at E2 codon 62 significantly suppressed the rapid penetration phenotype of SB-RL. Mutation in E2 codon 96 of SB, replacing tyrosine with histidine, reduced the virulence of SB for neonatal mice but had no effect on penetration of cultured cells. Therefore, mutation in E2 codons 62, 96, 114, or 159 affected both virulence in animals and the binding or biological activity of these E2c-specific MCAbs. These results suggest that an E2 antigenic site (E2c), defined by MCAbs R6 and R13, is conformational in nature and may constitute a surface domain on Sindbis virions important for virulence in neonatal mice.     

流式免疫微球捕获技术和荧光定量PCR检测BCR-ABL融合基因的方法学比较

目的:对流式微球捕获技术和荧光定量PCR技术在BCR-ABL融合基因中的检测进行比较和评价。方法:选取18例慢性粒细胞性白血病(chronic myeloid leukemia,CML)患者、6例急性髓系白血病(acute myeloblasticleukemia,AML)患者、9例缺铁性贫血(iron deficiency anemia,IDA)患者,分别用流式微球捕获技术和荧光定量PCR技术检测其骨髓BCR-ABL融合基因蛋白和mRNA。结果:6例AML和9例IDA患者均未检出阳性。18例CML患者荧光定量PCR检出16例P210和2例P190,流式微球捕获技术则均检出阳性。流式微球捕获技术的lgMFI(平均荧光强度)和荧光定量PCR法的lgCopies(拷贝数)的相关系数为0.708(P<0.01)。结论:流式免疫微球捕获技术对BCR-ABL各种断裂类型敏感性高,但不能区分断裂类型,适合作为初筛实验。     

A comparison of three methods for titration of poliovirus vaccines

Two methods for in vitro endpoint titration of poliovirus — the roller tube and the microtitration assay — were compared with each other and with the plaque assay, using secondary vervet monkey kidney cells and Vero cells as indicators. The roller tube method is the most reliable under difficult working conditions, but is otherwise cumbersome and expensive. The microtitre method is the most economical and the plaque assay the most sensitive. By suspending freshly trypsinized indicator cells with the virus dilutions before planting, it was possible to simplify the microtitre method considerably. The sensitivity of the plaque assay was improved for Vero cells by absorbing the virus onto freshly planted monolayers. The method was scaled down to a semi-micro level by using 24-well cell culture trays. The slower rate of plaque development under a low calcium overlay medium facilitated a more accurate plaque count.     

Evaluation of a quantitative plasma PCR plate assay for detecting cytomegalovirus infection in marrow transplant recipients.

A plasma PCR test, using a nonradioactive PCR plate assay, was evaluated for detection of human cytomegalovirus reactivation. This assay was compared to Southern blotting and found to perform well. As a noncompetitive method of quantitation, it was similar to a competitive method for detecting the number of genome copies per milliliter of plasma in marrow transplant recipients. This is a technically simplified assay with potential for adaptation to automation.     

Evaluation of a novel solid-phase immunoassay, Clearview Chlamydia, for the rapid detection of Chlamydia trachomatis.

Clearview Chlamydia (Unipath Limited, Bedford, United Kingdom) is a rapid immunoassay for the direct detection of Chlamydia trachomatis antigen. This assay was evaluated against the tissue culture method by using 376 paired endocervical specimens. The Clearview assay had a sensitivity of 93.5% and a specificity of 99% when it was compared with the tissue culture method. This assay does not require specialized equipment or trained personnel and yields results within 30 min from the time that a specimen is collected.     

Ultrarapid detection of blaKPC₁/₂-₁₂ from perirectal and nasal swabs by use of real-time PCR

The novel real-time PCR assay developed as described here was able to detect bla(KPC1/2-12) (bla(KPC-1/2) to bla(KPC-12)) from easily available clinical specimens in less than 2 h. The genotypic assay was highly sensitive (100%) and specific (98%). In some cases, it was able to detect bla(KPC) 48 h before positive detection by standard phenotypic assay on patients who were monitored daily. The high sensitivity and rapidity of the molecular method make it the method of choice for KPC surveillance and, thus, containment purposes.     

A new quantitative assay for the determination of complement activity

The suitability of the algal assay and the hemolysis method for the determination of complement activity was investigated with regard to reproducibility and quantification. It was shown that the day-to-day reproducibility is higher in the algal assay than in the hemolysis method in test tubes. The lysis of algal cells depends on serum concentrations. The extensive linear relationship between cytotoxic activity and serum concentration prevails at lysis rates between 20 and 80%. Therefore, it is often sufficient to measure the lysis rate at one serum concentration only. The algal assay enables the investigation of changes in complement activity, even if the changes are not expected to be drastic.     

Intra-laboratory validation of chronic bee paralysis virus quantitation using an accredited standardised real-time quantitative RT-PCR method

Chronic bee paralysis virus (CBPV) is responsible for chronic bee paralysis, an infectious and contagious disease in adult honey bees (Apis mellifera L.). A real-time RT-PCR assay to quantitate the CBPV load is now available. To propose this assay as a reference method, it was characterised further in an intra-laboratory study during which the reliability and the repeatability of results and the performance of the assay were confirmed. The qPCR assay alone and the whole quantitation method (from sample RNA extraction to analysis) were both assessed following the ISO/IEC 17025 standard and the recent XP U47-600 standard issued by the French Standards Institute. The performance of the qPCR assay and of the overall CBPV quantitation method were validated over a 6 log range from 10(2) to 10(8) with a detection limit of 50 and 100 CBPV RNA copies, respectively, and the protocol of the real-time RT-qPCR assay for CBPV quantitation was approved by the French Accreditation Committee.     

Increased sensitivity for detecting avian influenza-specific antibodies by a modified hemagglutination inhibition assay using horse erythrocytes

The hemagglutination inhibition (HI) assay is a widely used serological method to measure the levels of protective antibody responses against influenza viruses. However, the traditional HI assay which uses chicken erythrocytes is not sufficiently sensitive for detecting HI antibodies specific to avian influenza viruses. Previously, it was demonstrated that employing an assay using horse erythrocytes was able to increase the sensitivity of HI assay. The current report describes further optimization of this modified HI assay. It was shown that this method was able to increase detection of HI activities in rabbit sera immunized with H5 HA antigens, and proved that this increased sensitivity is useful in dissecting the strain specificity of HI antibody responses. In addition, the modified HI assay using horse erythrocytes increased the sensitivity of detecting HI antibodies specific for three major serotypes of avian influenza viruses, H5, H7 and H9, in people who may have asymptomatic infection with avian influenza viruses. Based on these results, the optimized use of horse erythrocytes should be standard practice for detecting HI activities against avian influenza viruses.     

测定淋巴细胞转化和鼠白细胞间素2活性的新方法——MTT比色分析法

细胞增殖通常用~3H-Tdr掺入法或活细胞计数法进行测定。由于前者使用放射性同位素易污染环境并且需要昂贵的仪器,而后者易受主观因素的影响误差较大,因此两者的应用都有一定局限性。最近Mosmann根据活的增殖细胞能代谢MTT[3-(4,5-di-methylthiazol-2-y1)2,5-diphenyl te-trazollum bromide]产生MTT甲 (for-