用逆转录套式PCR检测临床标本中的腮腺炎病毒RNA

流行性腮腺炎是儿童常见呼吸道传染病,并发症多为睾丸炎、无菌脑炎等。个别患者甚至因严重的脑炎并发症而死亡［１］。故有必要对患者进行早期病原诊断,以利于早期治疗。腮腺炎病毒ＲＮＡ中小疏水蛋白（ＳＨ）基因作为高变区在不同毒株的分子特性鉴别中比其它基因片段更...     

一株腮腺炎野毒分离传代前后其SH基因cDNA的测序比较

为证实鸡胚分离传代是否引起腮腺炎病毒基因组高变区小疏水蛋白（ＳＨ）基因的变异,用来源于上海的腮腺炎野毒Ｗｓｈ３株在鸡胚尿囊腔分离前和传代５次后测定ＳＨ基因及其旁侧区３８０个核苷酸的ｃＤＮＡ序列,两者结果相同,未见该基因的突变。该基因核苷酸测序可用于腮腺炎病毒的分子流行病学和毒株基因鉴别研究。     

腮腺炎病毒分离株(SP株)SH基因及其旁侧序列的初步分析

目的 比较腮腺炎病毒分离株SP株与其他野毒株的序列差异,以确定其遗传特征。方法 将2005年在云南省石屏县收集到的腮腺炎患儿唾液标本,于Vero细胞培养7 d后观察病变并分离收获病毒,用血细胞吸附试验验证。同时提取病毒RNA,采用反转录-套式聚合酶链反应(RT-nPCR)法从病毒RNA中扩增出SH基因及其旁侧序列,测序并以VectorNT16．0软件分析。结果 SH基因旁侧区序列分析表明:SP株与国内已知野毒株具有明显的亲缘关系,在SH基因旁侧区范围内的序列一致性为93％～96％,差异均＜8％。氨基酸序列分析发现:该毒株与其他国内野毒株在此区域中的氨基酸序列一致性为93．3％～98．25％,与Wlz2株的遗传距离最近。结论 SP株属于F基因亚型。     

Characterization of a family B DNA polymerase from Thermococcus barophilus Ch5 and its application for long and accurate PCR

The family B DNA polymerase gene from the euryarchaeon Thermococcus barophilus Ch5 (Tba5) contains an open reading frame of 6198 base pairs that encodes 2065 amino acid residues. The gene is split by three inteins that must be spliced out to form the mature DNA polymerase. A Tba5 DNA polymerase gene without inteins (genetically intein-spliced) was expressed under the control of the pET-28b(+)T7lac promoter in E. coli Rosetta 2(DE3)pLysS cells. The molecular mass of the purified Tba5 DNA polymerase was about 90 kDa consistent with the 90,470 Da molecular mass calculated based on the 776 amino acid sequence. The optimal pH for Tba5 DNA polymerase activity was 7.5 and the optimal temperature was 70–75 °C. The enzyme possessed 3′ → 5′ exonuclease activity and was activated by magnesium ions. PCR amplification using Tba5 DNA polymerase enables high-yield for 1- to 6-kb target DNA products, while 8- to 10-kb target DNA products were amplified at low or inefficient levels. To simultaneously improve product yield and amplification fidelity, Tba5 plus DNA polymerase mixtures were constituted with various amounts of Tba5 DNA polymerase mixed with Taq DNA polymerase. The Tba5 plus DNA polymerase mixtures robustly amplified up to 25-kb λ DNA fragments. In addition, the PCR error rate of Tba5 plus3 and Tba5 plus4 mixtures were much lower than those of wild-type Tba5 DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, and Pfu plus DNA polymerase.     

PCR检测淋球菌的探讨

多聚酶链反应（ＰＣＲ）具有灵敏度高,特异性强,快速高效的特点,在病原微生物检测领域有广阔的前景。本文对４０８例疑似淋病患者的泌尿生殖道分泌物作了ＰＣＲ检测淋球菌的探索,结果１７０例阳性,占４１．７％,而培养阳性（２１．６％）,ＰＣＲ法显著高于培养法（Ｐ＜０．０１）。     

应用PCR技术检测病人血液标本中斑点热群立克次体DNA

本文以聚合酶链反应（PCR）,采用二次扩增法直接检测蜱传斑点热病人血液标本中斑点热群立克次体DNA,结果从7份标本中检出阳性1份,进一步的限制性片段多态性分析证明和斑点热群黑龙江立克次体基因型相同。实验证明：黑龙江立克次体对人具有致病性,同时也证明PCR技术快速、简单、敏感,用于斑点热群立克次体的早期诊断是可行的,并可作出分型鉴定。     

The Sequence of the Hartmannella vermiformis Small Subunit rRNA Coding Region

ABSTRACT The Hartmannella vermiformis small-subunit rRNA coding region was amplified, and the amplified DNA was cloned and sequenced. The coding region is 1,840 nucleotides long, and is typical of eukaryotic rRNA genes in both size and composition. Different clones contained different nucleotides at three positions.     

Detection of Tobacco mosaic virus and Tomato mosaic virus in pepper and tomato by multiplex RT-PCR

Aims: To develop a highly sensitive and rapid protocol for simultaneous detection and differentiation of Tobacco mosaic virus (TMV) and Tomato mosaic virus (ToMV) in pepper and tomato. In this study, we use the multiplex PCR technique to detect dual infection of these two viruses. Methods and Results: A multiplex RT–PCR method consisting of one‐tube reaction with two primer pairs targeted to replicase genes was developed to simultaneously detect TMV and ToMV in seed samples of pepper and tomato. Specific primers were designed from conserved regions of each of the virus genomes, and their specificity was confirmed by sequencing PCR products. RT–PCR detected up to 10^?6 dilution of total RNA extracted from infected leaves. Multiplex RT–PCR revealed the presence of both TMV and ToMV in three of 18 seed samples of tomato and one of 18 seed samples of pepper. Conclusions: The multiplex PCR assay was a cost effective, quick diagnostic technique, which was helpful in differentiating TMV and ToMV accurately. Significance and Impact of the Study: The multiplex PCR assay described in this study is a valuable tool for plant pathology and basic research studies. This method may facilitate better recognition and distinction of TMV and ToMV in both pepper and tomato.     

247例人乳头瘤病毒的PCR检测结果分析

２４７例生殖系分泌物标本用ＰＣＲ检测人乳头瘤病毒ＨＰＶ－ＤＮＡ。结果显示：阳性感染率达８７．６％,在与临床疾病密切的亚型中,６、１１型感染率（６０％）,高于１６、１８型感染率（５１．６％）,但统计学处理没有显著性差异。女性发病率明显高于男性。易感人群组以２０～４０岁最高（占感染人数的８４．４％）,并且有年轻化的趋势（１０～２０岁年龄组占８．４％）。     

用聚合酶链反应（PCR）检测支气管肺泡灌洗（BAL）液中的嗜肺军团杆菌

本文研究了由嗜肺军团杆菌的巨噬细胞感染性增效子（ｍｉｐ）基因设计的一对引物,用ＰＣＲ扩增嗜肺军团杆菌３、５、７、８血清型的４个标准菌株的特异ＤＮＡ序列,研究了用该引物扩增ＢＡＬ液中嗜肺军团菌特异ＤＮＡ序列的方法、灵敏度及特异性。结果表明：用上述引物扩增嗜肺军团菌４个标准菌株的ＤＮＡ,均可得到２０７ｂｐ的特异扩增产物,ＢＡＬ液中的军团菌经离心及裂解液裂解后,可直接进行ＤＮＡ扩增,当ＢＡＬ与液中军团菌量为２×１０３ＣＦＵ／ｍｌ时,即可检测出特异扩增带（电泳法）,除军团菌外,其它受试细菌均无此特异性扩增,用本法对４２例临床非典型肺炎患者的ＢＡＬ液进行嗜肺军团菌的检测,在４２份嗜肺军团菌培养均为阴性的ＢＡＬ液中,其中一例ＰＣＲ检测军团菌为阳性。本研究提示：用ＰＣＲ检测ＢＡＬ液中的军团菌是可行的,并有快速、灵敏、特异之忧点。     

2018-2019年中国流行性腮腺炎流行特征和病毒基因特征分析

阐明中国(未包括香港、澳门特别行政区和台湾地区,下同)2018-2019年流行性腮腺炎(流腮)流行特征和病毒基因特征。对2018-2019年中国流腮流行病学和病毒学监测数据进行描述流行病学和分子流行病学分析。2018-2019年中国流腮年报告发病率分别为18.65/10万和21.48/10万,15岁以下儿童和青少年是我国流腮的高发人群,分别占总病例数的85.30%和82.56%。流腮的流行具有明显的季节性特征。全国各省、自治区、直辖市份均有流腮病例报告,西部和中部地区发病率高于东部地区。2018-2019年共获得160条腮腺炎病毒(Mumps virus,MuV)SH基因序列,其中150条(93.75%)序列鉴定为F基因型MuV,在我国11个省份检测到;10条(6.25%)序列为G基因型MuV,2019年在广东、湖北和新疆3个省份检测到。和我国既往流行MuV代表株相比,2018-2019年流行的F基因型MuV代表株序列在基因亲缘性关系树上相对集中。现阶段我国流腮的流行病学特征未发生明显改变,仍呈现病毒自然流行模式;F基因型作为优势流行基因型,在我国大部分地区持续流行,但毒株的遗传多态性有所降低,这可能和我国实施1剂次腮腺炎疫苗常规免疫策略有关。G基因型MuV主要在我国局部地区流行,但流行范围在逐渐扩大。建议进一步加强两剂次腮腺炎疫苗接种工作,降低我国腮腺炎易感人群。同时持续性开展MuV流行学和病毒学监测工作,为鉴别病毒的来源,确定病毒传播途径和评估腮腺炎疫苗免疫策略奠定重要的基础。     

Detection of Various Variant Verotoxin Genes in Escherichia coli by Polymerase Chain Reaction

We constructed common primers for the polymerase chain reaction to detect the genes for various Verotoxins reported, that is, VT1 (or SLT-I), VT2 (or SLT-II), VT2vha, VT2vhb, SLT-IIv (or VT2vp1, VTe) and SLT-IIva (or VT2vp2). A total of 80 Verocytotoxin-producing Escherichia coli strains isolated from humans, domestic animals and meats gave a positive result by PCR with the designed common primers. Digestion by restriction endonucleases BglII and EcoT14I of the amplicon of the VT2vp2 gene gave specific bands of the expected sizes, but not of the amplicons of other VT genes, suggesting a possible method for identification of the VT2vp2 gene. Application of the PCR with the designed primers in diagnostic and epidemiological studies on VTEC infection is also discussed.     

用聚合酶链式反应（PCR）检测马铃薯纺锤块茎类病毒

用DNA合成仪合成两个马铃薯纺锤块茎类病毒(Potato spindle tuber viroid, PSTVd)特异性引物,从感病的马铃薯块茎组织的核酸抽提液中,用反转录酶合成PSTVd eDNA,然后用PCR法进行扩增,扩增产物用电泳检测,建立了用PCR法检测PSTVd的新方法。结果表明,该方法特异性强,灵敏度可达0.15pg,比现有其它检测方法高,而且样品用量少。     

透明颤菌血红蛋白的DNA改组研究

为提高透明颤菌血红蛋白在限氧条件下促进宿主细胞生长的能力,首先通过易错PCR向透明颤菌血红蛋白基因中引入突变,再结合DNA改组对其进行改造。将改组基因置于透明颤菌血红蛋白天然启动子下游,转化大肠杆菌DH5α,构建改组文库。以限氧培养条件下菌体沉淀的颜色为指标进行试管初筛,再以限氧和极端限氧条件下菌体湿重为指标进行摇瓶复筛,最终得到一个高活性突变蛋白VHb'042506。该蛋白使宿主的菌体湿重在限氧和极端限氧条件下较原基因转化子分别提高了31.25%和58.75%。经测序和比对,该基因与原基因相比发生了11处碱基点突变,致氨基酸4处错义突变。CO差光谱实验显示该蛋白具有更强的特征吸收。     

相关序列扩增多态性(SRAP)标记及其应用研究进展

SRAP是一项基于PCR技术的分子标记技术,利用其独特的引物设计对基因组的开放阅读框(ORFs)进行特异扩增,利用个体以及物种的内含子、启动子和间隔序列的不同,产生基于内含子和外显子的SRAP多态性。阐述了SRAP的原理和流程,详细论述了SRAP标记目前在植物遗传多样性、作物品种鉴定、遗传图谱构建等方面的研究进展及应用前景。     

Enumeration and detection of acetic acid bacteria by real-time PCR and nested PCR

Acetic acid bacteria play a negative role in wine making because they increase the volatile acidity of wines. They can survive in the various phases of alcoholic fermentation and it is very important to control their presence and ulterior development. The main objective of the present work is to test fast, sensitive and reliable techniques such as real-time PCR (rt-PCR) and nested PCR for enumerating and detecting the presence of this bacterial group without plating. Primers were designed on the basis of the available 16S rRNA gene sequences and tested successfully with reference acetic acid bacteria strains. The usefulness of rt-PCR was demonstrated by comparing the results with traditional techniques (colony and microscope counting). The results were similar with all the techniques. Optimized rt-PCR enabled numbers between 10(7) and 10(1) cells mL(-1) to be enumerated, while nested PCR detected less than 10 cells mL(-1). Although this latter technique cannot be used for enumeration, it has several advantages in routine laboratory analysis.     

Sperm Mediated Transfer of Genes into Goldfish and Detection by Polymerase Chain Reaction

Goldfish sperms were mixed with eggs for fertilization after incubation with antifreeze protein gene(AFP)from ocean pout for 30 min.A number of embryos and 145 adult goldfish were obtained.DNA from adult goldfish and embryos was extracted separately.Results of the amplification by PCRand Southern blot molecular hybridization indicate the integration of exogenous antifreeze gene intothe genome of a part of the recipient goldfish.Of the 45 samples detected by PCR,twelve showedpositive reaction with distinct hybridization band.The positive rate was 26%.     

冻干腮腺炎活疫苗细胞培养生产工艺研究

报告了冻干腮腺炎活疫苗细胞培养生产工艺研究结果,通过对鸡胚疫苗株的适应培养及对细胞培养生产工艺的试验优化,建立了连续细胞培养多次收获疫苗生产工艺并制备出了冻干细胞培养腮腺炎活疫苗制剂。本生产工艺切实可行,生产成本低、投入产出率高,所用原材料规范、质量易于控制,具有明显的技术优势。生产的冻干疫苗制剂质量稳定可靠,符合中国生物制品规程要求,有利于预防腮腺炎的规模化推广使用     

Detection of Genomic Polymorphism in Plasmodium falciparum Using an Arbitrarily Primed PCR Assay

ABSTRACT. Modifications of the arbitrarily primed polymerase chain reaction assay (i.e. a low annealing temperature and a very slow increase in the temperature during the elongation steps during the amplification cycles) allowed it to be used with the AT-rich Plasmodium falciparum DNA. The analysis of the products by polyacrylamide—urea gels, after silver staining, resulted in high resolution and sensitivity. Eighteen single and six combined pairs of arbitrary primers were tested. Two produced polymorphic patterns complex enough to differentiate between close Colombian isolates in a single assay. This method may be useful in studying the distribution and migration of strains in endemic areas, and for identifying intralaboratory cross-contamination of cultures.     

海参微卫星DNA的多态性

微卫星DNA(microsatellite DNA)广泛存在于真核生物的基因组中。由于其具有突变频率快、多态性丰富、呈共显性遗传、通用性等特点,已成为近年来被广泛应用的分子遗传标记。本研究对10个海参微卫星DNA进行了克隆与测序。结果表明:90%的微卫星DNA序列存在长度多态性,这为进一步研究海参的分子标记辅助育种奠定了基础。