微小按蚊种群成员分子变异与系统发育的关系

目的：研究微小按蚊与种群之间（乌头按蚊、吉甫按蚊、微小按蚊A／C型）线粒体DNA的分子变异及系统发育关系。方法：PCR扩增和mtDNA测序。结果：在云南发现两个微小按蚊隐形种,即微小按蚊A／D型;证实了微小按蚊种团之间遗传差异,微小按蚊A／C分歧时间稍晚于乌头按蚊,吉甫按蚊分歧时间远早于前2种。结论：地理环境对mtDNA遗传差异有一定影响,但不同地理的群体之间微小按蚊的遗传没有明显变化。     

16SrRNA基因探针与ctx A基因探针用于霍乱弧菌分型

以地高辛为标记物,利用ＰＣＲ方法制备１６ＳｒＲＮＡａ基因探针与ｃｔｘＡ基因探针,经Ｓｏｕｔｈｅｒｎ杂交,对霍乱弧菌进行分型,对广东地区分离自病人的６０株埃尔托型（ＥＶＣ）、１０株Ｏ１３９群和２株标准株进行研究,经１６ＳｒＲＮＡ基因在探针共分为４个类型（Ａ、Ｂ、Ｃ和Ｄ）,ＥＶＣ和Ｏ１３９群均以Ｄ型为优势分为４个类型（Ａ、Ｂ、Ｃ和Ｄ）,ＥＶＣ和Ｏ１３９群均以Ｄ型我隆,经ｃｔｘＡ基因探针Ｓｏｕｔｈｅｒｍ     

云南不同地株微小按蚊酯酶同工酶比较研究

笔者应用聚丙烯酰胺凝胶电泳（ＰＡＧＥ）技术，比较分析了云南５个（元阳、潞西、镇康、思茅、昭通）地株微小按蚊酯酶（Ｅｓｔ）同工酶，并对各酶谱进行扫描分析。结果：Ｅｓｔ同工酶均出现Ⅰ、Ⅱ两组区带，各自具有相对稳定的酶谱，同时也存在遗传上的多态现象，各地株间的酶谱中存在着一定的差异。     

应用形态和分子特征对我国海南省按蚊的鉴定研究

目的利用形态特征与分子特征相结合对我国海南省按蚊进行种类鉴定。方法研究样本为海南省4个不同地理环境所采集的按蚊成虫和幼虫,依据形态特征鉴定成虫后,测定和分析部分成虫和幼虫的rDNA？ITS2和28S？D3序列以进行分子鉴定。结果对采集的336只按蚊成虫进行形态鉴定,结果显示总共分为9种,其中迷走按蚊所占比例最高,为81.55%。测序获得成虫和幼虫的ITS2序列37条,D3序列41条,Blast对比的结果显示,形态鉴定的成虫中,1只乌头按蚊错定为微小按蚊;分子鉴定的幼虫包括中华按蚊、迷走按蚊、腹簇按蚊和未定名种（与圣代克按蚊和浅色按蚊同源性高）;序列分析发现在ITS2序列中迷走按蚊和微小按蚊分别存在1处（G/A）和2处（A/T）单碱基双峰,D3序列中迷走按蚊和腹簇按蚊存在3处（G/A）和1处（G/A）单碱基双峰。结论应用形态特征结合分子特征鉴定按蚊的可靠性和实用性强;分子特征提示迷走按蚊、微小按蚊和腹簇按蚊等处于隐种的分化中。     

保定地区肾综合征出血热病人血清PCR分型及核苷酸序列测定

「目的」明确保定肾综合征出血热疫区型别。「方法」采用ＲＴ－ＰＣＲ法检测急性期肾综合征出血热病人血清汉垣病毒ＲＮＡ,并进行分型及部分核苷酸序列测定和与国内外汉垣病毒的代表析进行比较。「结果」３１份急性期病人血清经分型,２４份为家鼠型（ＳＥＯ）型,２份为野鼠型（ＴＨＮ）型,测得ＳＥＯ型Ｃ３株Ｍ基因（１９８５到２３１４位）,ＨＴＮ型ＬＢＹ株Ｍ基因（１９９６到２３１４位）核苷酸序列与国内外代表株比较：Ｃ３     

七日热型,澳洲型钩端螺旋体野生株rRMA基因MRS …

目的：用两对物地异引物扩增１２株七日热型、澳洲型钩端螺旋体野生株及相应的２株参考株的１６Ｓ、２３ＳｒＲＮＡ基因,用Ｈｉｎｆ内切酶对扩增产物作ＭＲＳＰ分析。方法：应用ｒＲＮＡ基因ＭＲＳＰ分析技术。结果;不同宿主来源（钩端螺旋体病人、耕牛）的同一血清型钩端螺旋体野生株之间具有相同的ＭＲＳＰ型,同一血甭型钩端螺旋体野生株与相应的参与株的ＭＲＳＰ型也相同,故七日热型、奥洲型钩端螺旋体属同一ＭＲＳＰ型。结论     

肾综合征出血热患者病原基因的检测分析

目的：探讨肾综合征出血热基因诊断的方法。方法：采用肾综合征出血热（ＨＦＲＳ）Ⅰ型病毒ＨＴＮ株,Ⅱ型病毒Ｒ２２株的Ｍ基因序列为参照,设计出引物Ａ１￣Ａ７,用ＲＴ－ＰＣＲ技术,直接从ＨＦＲＳ患者外周血单个核细胞（ＰＢＭＣ）中检测ＨＦＲＳ－ＲＮＡ。（２）２０例病人中Ⅰ型引物扩增阳性者５例,Ⅱ型引物阳性者１９例,二者重叠阳性者４例。结论：以ＨＦＲＳ病毒Ｍ片段Ｇ１区核苷酸序列设计的引物可以用于ＨＦＲＳ临床病     

柯达M-35自动洗片机故障分析及修理

故障表现：送片前按下复位开关（ＳＴＡＮＤＢＹＲＥＳＥＴＳＷＩＴＣＨ）Ｓ１或Ｓ２,此时主驱动电机（Ｂ１）、风机（Ｂ２）和胶片传动系统启动工作,但一松开上述开关系统就停止工作,不能进行洗片。故障分析：分析该系统的控制电路由逻辑、时序数字电路组成。主要电路...     

HIV—1感染相关基因的多态性研究进展

近年来，通过对人免疫缺陷病毒Ｉ型（ＨＩＶ－１）协同受体（如ＣＣＲ５，ＣＸＣＲ４，ＣＣＲ２，ＣＣＲ３和ＣＸＣＲ４的天然配基ＳＤＦ１等）的基因多态性分析，发现ＣＣＲ５－Δ３２等位基因型的个体对Ｍ－ｔｒｏｐｉｃ病毒株感染有天然的抵抗力；ＣＣＲ５－Δ３２和ＣＣＲ２－６４Ｉ等位基因型分别对艾滋病（ＡＩＤＳ）的发病进程有显著的延缓作用，ＳＤＦ１－３‘Ａ等位基因型也直接影响到ＡＩＤＳ的预后。因此，协同受体的发现     

二硫化碳对邻苯三酚自氧化化学发光体系及超氧阴离子的影响

目的 探讨二硫化碳（ＣＳ２）对邻苯三酚－鲁米诺（ＰＡ－Ｌ）自氧化化学发光体系以及对超氧阴离子产生的影响。方法 应用ＰＡ－Ｌ化学发光体系观察ＣＳ２的发光动力学曲线及超氧化物歧化酶（ＳＯＤ）对发光强度的抑制作用。结果 不同浓度ＣＳ２（１０、４０、８０ｍｇ／ｍｌ）与乙醇溶剂对照组比较,均能使ＰＡ－Ｌ体系化学发光强度增强,峰时延迟,呈明显的剂量－效应关系,其中ＣＳ２浓度为８０ｍｇ／ｍｌ时化学发光峰值高于无     

核糖体28S-D3等位基因特异扩增鉴别微小按蚊亲缘种A和C

目的 建立微小按蚊复合体不同亲缘种A和C的分子鉴别方法。方法 用单蚊蚊腿消化提取基因组DNA，PCR特异扩增28S第3结构域(D3)基因，对PCR产物进行纯化、克隆、测序和序列分析；基于序列差异设计特异引物，进行等位基因特异扩增(PCR—ASA)，根据扩增片段大小区分微小按蚊不同亲缘种。结果 发现微小按蚊不同亲缘种A和C(GeneBank登录号：AF416782，AF425594)，根据二者序列差异设计的特异引物能在普通琼脂糖凝胶上直观地区分两亲缘种，两者除在376bp处有一共同条带外，分别在294bp和112bp处出现各自的特异扩增带。结论 我们建立的ASA分子鉴别方法能有效地将我国微小按蚊不同亲缘种A和C区分开来。     

马尔尼菲青霉菌DNA检测及序列分析

目的设计马尔尼菲青霉菌种特异性引物,探讨马尔尼菲青霉病早期诊断的方法. 方法采用真菌通用引物ITS1和ITS4 PCR扩增我科保存的2株和广西医科大学附一院惠赠的1株马尔尼菲青霉菌rDNA ITS,扩增产物纯化后直接测序,测序结果在国际基因库核酸序列数据库进行同源序列搜索、比对、分析. 结果 3株临床分离的马尔尼菲青霉菌rDNA ITS序列相同;与来源于美国、印度尼西亚、法国、澳大利亚的马尔尼菲青霉菌的rDNA ITS序列一致;马尔尼菲青霉菌与荚膜组织胞浆菌、新生隐球菌、念珠菌以及曲霉和青霉属间的rDNA ITS序列差异较大,而青霉种间的rDNA ITS序列差异不大. 结论不同来源的马尔尼菲青霉菌种间的rDNA ITS序列具有高度的同源性;提示该区不适合作为靶基因来设计该菌的种特异性引物或探针,应考虑在别的区域继续寻找.     

Evidence for a new sibling species of Anopheles minimus from the Ryukyu Archipelago, Japan

The Anopheles minimus complex is known to comprise at least 2 sibling species (A and C) in Thailand and Vietnam. This study investigated the specific status of An. minimus on Ishigaki Island, the Ryukyu Archipelago, Japan using morphological and genetic analyses. Morphological studies revealed that almost all (99.5%) of the adult mosquitoes are characterized by the humeral pale spot on the costa of their wings, a character that partially differentiates species A and C elsewhere. A high frequency (81.4%) have a pale fringe spot at the tip of vein 1A, a character rarely observed in other An. minimus populations. Significant seasonal variation in the size of wild An. minimus mosquitoes on the island was observed, with the largest size in the winter. Scanning micrographs of the cibarial armature of females from Ishigaki Island revealed that over 90% had cone filaments clearly differing in shape from those of species A or C. The Giemsa-stained metaphase karyotypes of larval brain cells were somewhat similar to those of species A, with a few exceptions, but were very different from those reported for species C. Crossing experiments between species A (CM strain) from Thailand and the progeny of An. minimus from Ishigaki Island (ISG strain) revealed postzygotic genetic incompatibility, although no prezygotic isolation. Hybrid progeny were only obtained from CM female x ISG male. F2 hybrid progeny were not obtained, since the hybrid males were sterile or almost sterile with atrophied testes or abnormal spermatozoa, although the polytene chromosomes of hybrid larvae showed synapsis. The hybrid females backcrossed with either CM or ISG males laid eggs with significantly lowered fertility and viability. The sequence for the D3 region of the 28S gene of ribosomal DNA of the ISG strain differed from those of species A and C. In addition, sequence data from Vietnamese mosquitoes suggest that the An. minimus complex may contain additional species. The morphological, cytogenetic, molecular, and hybridization evidence together suggest the existence of another sibling species of the An. minimus complex on Ishigaki Island, which is provisionally designated An. minimus species E.     

广西微小按蚊种群密度地理分布及种类分子鉴定研究

目的研究20世纪以来广西壮族自治区(广西)微小按蚊的种群密度和地理分布,为疟疾防治提供参考。方法收集广西20世纪50-90年代微小按蚊资料及疟疾发病率,于2004-2010年在不同经纬度原以微小按蚊为主要传播媒介的疟疾流行区收集该蚊成蚊,采用形态学和PCR方法鉴定采集的微小按蚊样品。结果1957-1998年对全自治区不同经纬度媒介按蚊调查,92个县中有56个县存在微小按蚊;2004-2007年在该蚊活动频繁的36个县的乡村收集按蚊,仅在20个县40个媒介点采集到微小按蚊244只;采用种类分子鉴定,除百色市旺甸村有微小按蚊种类A存在外,其他地区均为微小按蚊种类C。2008年后全自治区各县疟疾疫情发病率已降至0.1/万。结论目前在广西存在微小按蚊种类A和C两种,以种类C为主;全自治区不同经纬度的微小按蚊种群密度和分布范围已经明显减少,该蚊有可能不再是该区域疟疾传播的主要媒介。     

How reliable is the humeral pale spot for identification of cryptic species of the Minimus Complex?

The Anopheles minimus Complex Theobald (Diptera: Culicidae) is composed of the 3 sibling species A, C, and E. The malaria vectors An. minimus A and C are distributed over the Southeast Asian region, whereas species E is restricted to the Ryukyu Japanese Islands. Because species A and C can be sympatric and present specific behaviors and have a role in malaria transmission, it is important to differentiate them. The literature mentioned the presence of a presector pale spot on the wing costa of An. minimus A, whereas species C may exhibit both presector and humeral pale spots. However, the reliability of their diagnostic power has not been established over large temporal and geographic surveys. From the analyses of 9 populations throughout Southeast Asia, including published data and field populations from 2 sites in Thailand, we showed that the wing patterns present spatial and temporal variations that make these two morphological characters unreliable for the precise identification of An. minimus A and C. Therefore, molecular identification remains the most efficient method to obtain an unambiguous differentiation of these 2 species. Correct species identification is essential and mandatory for any relevant study on the Minimus Complex and for the application of successful control strategies.     

嗜人按蚊和中华按蚊相关细菌差异的初步探讨

目的通过对河南省桐柏地区嗜人按蚊和中华按蚊的DNA提取物中细菌DNA的拷贝数和种类是否存在差异进行初步探讨，为进一步深入开展蚊媒中肠细菌与蚊媒传播疟疾能力之间的关系提供理论基础。方法以保存于75％乙醇中的成蚊为研究对象，首先应用聚合酶链反应（PCR）和聚合酶链反应-限制性片段长度多态性（PCR—PFLP）方法确定蚊种，在此基础上，用PCR比较蚊虫细菌拷贝数的异同，同时克隆并酶切4只按蚊中细菌的16SrDNA序列，对酶切结果进行统计分析。结果共明确23只按蚊的种类。不同蚊种DNA中所含细菌拷贝数存在差异，但与按蚊的种类没有关系。4只按蚊DNA中共发现19种细菌16SrDNA的酶切类型。不同中华按蚊个体间存在共同的细菌DNA酶切类型；嗜人按蚊仅呈现2种细菌DNA酶切类型，均与中华按蚊不同。结论嗜人按蚊和中华按蚊中含有的细菌种类存在差异，该项研究为我们进一步研究按蚊中肠细菌是否会影响按蚊对疟原虫的感染性奠定了基础。     

First record of Anopheles minimus C and significant decrease of An. minimus A in central Vietnam

Before August 1998, in the Khanh Phu commune (central Vietnam), Anopheles minimus s.l. individuals were identified as species A and showed the typical species A wing form. After a significant decrease over the 3 years 1999-2001, an increase in 2002 of An. minimus s.l. possessing a different wing pattern was observed. To determine the specific status of the An. minimus species collected in 2002 and to follow changes in the species composition, an allele-specific polymerase chain reaction was applied to samples collected from 1993 to 2002. This study reports the first record of An. minimus C in central Vietnam and, since 1998, a significant reduction of An. minimus A that coincided with the wide use of permethrin-treated bednets. This change in anopheline composition may have important consequences on malaria transmission. This work shows that the geographic distribution of malaria vectors in southeast Asia is only partially known and highlights the importance of species identification for understanding changes in the vector composition as a result of selective vector control.     

Study of the 16S–23S ribosomal DNA internal spacer of Coxiella burnetii

The complete 16S–23S ribosomal DNA (rDNA) internal transcribed spacer (ITS) of 22 isolates of the obligate intracellular bacterium Coxiella burnetii, the agent of Q fever, were amplified by the polymerase chain reaction (PCR) and sequenced using an a utomated laser fluorescent DNA sequencer. The ITS measured 497 base pairs (bp) and encoded isoleucine-tRNA and alanine-tRNA. The comparison of the sequence alignments of the 22 C. burnetii strains revealed very high levels of sequence similitary (< 99%) although they had different geographic origins and phenotypic characteristics. Sequencing of the 16S–23S rDNA ITS of C. burnetii could be utilized for identification of the bacterium but is not applicable to studies of epidemiology, virulence and taxonomy.     

Genetic diversity and molecular identification of mosquito species in the Anopheles maculatus group using the ITS2 region of rDNA.

The species diversity and genetic structure of mosquitoes belonging to the Anopheles maculatus group in Southeast Asia were investigated using the internal transcribed spacer 2 (ITS2) of ribosomal DNA (rDNA). A molecular phylogeny indicates the presence of at least one hitherto unrecognised species. Mosquitoes of chromosomal form K from eastern Thailand have a unique ITS2 sequence that is 3.7% divergent from the next most closely related taxon (An. sawadwongporni) in the group. In the context of negligible intraspecific variation at ITS2, this suggests that chromosomal form K is most probably a distinct species. Although An. maculatus sensu stricto from northern Thailand and southern Thailand/peninsular Malaysia differ from each other in chromosomal banding pattern and vectorial capacity, no intraspecific variation was observed in the ITS2 sequences of this species over this entire geographic area despite an extensive survey. A PCR-based identification method was developed to distinguish five species of the group (An. maculatus, An. dravidicus, An. pseudowillmori, An. sawadwongporni and chromosomal form K) to assist field-based studies in northwestern Thailand. Sequences from 187 mosquitoes (mostly An. maculatus and An. sawadwongporni) revealed no intraspecific variation in specimens from Thailand, Cambodia, mainland China, Malaysia, Taiwan and Vietnam, suggesting that this identification method will be widely applicable in Southeast Asia. The lack of detectable genetic structure also suggests that populations of these species are either connected by gene flow and/or share a recent common history.     

Internal transcribed spacers (ITS) of Trypanosoma rangeli ribosomal DNA (rDNA): a useful marker for inter-specific differentiation.

The internal transcribed spacers (ITS) flanking the 5.8S subunit of the ribosomal RNA genes (rDNA) of Trypanosoma rangeli strains isolated from distinct geographical regions and hosts were studied. The results revealed the sequence variability of the ITS spacers showing the presence of microsatellite repeats and single nucleotide polymorphisms (SNP), which were also observed within the 5.8S rDNA sequence. ITS-2 spacer was the most phylogenetically informative region due the presence of a higher number of parsimonious sites in both inter- and intra-specific analysis. Sequence analysis of both ITS spacers plus the 5.8S rDNA of T. rangeli strains allowed a clear inter-specific differentiation from Trypanosoma cruzi strains representative of the parasite zymodemes.