应用多重PCR法鉴定蚊胃血血源的研究

目的建立多重PCR法鉴定蚊胃血来源。方法依据常见蚊吸血对象（人、牛、猪和犬）的线粒体DNA细胞色素b序列的差异,设计种特异引物,建立多重PCR法,并应用该法检测现场按蚊标本。结果应用多重PCR法共检测249只按蚊,血源来自牛和猪的共91只和63只,未检出吸人血按蚊。立即处死并干燥保存的现场按蚊标本检测成功率最高,为92.50%。结论多重PCR法鉴定蚊胃血血源快速、灵敏,结果客观、可靠。     

聚合酶链式反应(PCR)扩增基因鉴定蚊胃血血源

目的探寻鉴定蚊胃血来源的实验室快速检测方法。方法对抗人血红蛋白胶体金试纸条(简称试纸条)检测有人血成分的按蚊干血痕标本,依据常见蚊吸血对象(人、牛、猪)的DNA序列的差异,设计特异DNA基因引物,运用聚合酶链式反应(PCR)方法对按蚊胃内干血痕标本进行DNA基因扩增,1.5%琼脂糖电泳检测扩增产物,并对扩增产物进行序列测定后查询其同源性匹配。结果试纸条共检测87只按蚊,血源来自牛和猪的共83只,检出仅含人血痕4份。该4份人血痕蚊标本经PCR扩增检测和基因序列匹配查询,与人基因序列同源性为100%,而无牛和猪基因。结论PCR法鉴定蚊胃血血源快速、灵敏、特异。     

应用多重PCR法分析西藏察隅疟疾流行区按蚊吸血习性

目的用多重PCR法分析西藏疟疾流行区察隅县常见按蚊的吸血习性,为下一步研究传疟媒介提供参考。方法2011年7~8月选择察隅县不同生态环境的3个自然村(日玛村、塔玛村和京都村),每个村在人房和畜舍选择8个点,采用诱蚊灯全通宵(20∶00至次日08∶00)诱捕法捕捉按蚊,次日清晨收集诱捕的蚊虫,经形态学鉴定蚊种,分析按蚊组成。收集饱血按蚊,分别提取单只蚊胃血的DNA,采用基于不同动物mtDNA-cytb序列差异的多重PCR法鉴定各蚊胃血源,计算人血指数,分析按蚊的吸血习性。结果共捕获按蚊1 442只,经形态学鉴定,多斑按蚊种团占99.6%(1 436/1 442),带足按蚊和腹簇按蚊占0.4%(6/1442)。多斑按蚊种团中,伪威氏按蚊占85.5%(1228/1436),威氏按蚊占14.5%(208/1 436)。用多重PCR检测202只多斑按蚊种团(伪威氏按蚊188只和威氏按蚊14只)的饱血蚊胃血,结果显示,伪威氏按蚊兼吸牛/猪血和人血,人血指数为0.35,威氏按蚊吸食猪血和人血,人血指数为0.29。结论西藏察隅县2种常见按蚊(伪威氏按蚊和威氏按蚊)均兼吸人畜血,伪威氏按蚊的人血指数较高。     

醋酸纤维膜微量免疫扩散法鉴定按蚊胃血的实验研究

本文用醋酸纤维膜微量扩散法鉴定按蚊胃血,具有简便、经济和准确等优点。为了解敏感性和特异性,应用醋纤法和沉淀法对2～3期按蚊胃血标本129份作鉴定,其阳性率分别为98.4％和89.9％,对4～5期按蚊胃血标本121份的阳性率分别为93.4％和19.8％,对采自牛、猪房的102价标本,醋纤法全部阳性,沉淀法阳性85份。表明醋纤法的敏感性和特异性高于沉淀性.     

鉴定蚊胃血的一种快速方法

蚊胃血血源的鉴定是虫媒疾病流行病学调查研究的一个重要内容,本文介绍一种可在现场鉴定蚊胃血血源的琼脂扩散试验方法。按照Templis等(1963)方法制备抗下列3组动物的混台免疫血清,Ⅰ组为抗牛、羊、猪等免疫血清,Ⅱ组为抗人、猴、狗、马、鼠、兔等     

直接免疫荧光抗体试验鉴定蚊胃血血源的研究

近年来,国内学者在改进蚊胃血血源鉴定方法方面进行了不少的研究。为探讨直接免疫荧光抗体试验(下称直接法)鉴定蚊胃血血源的效果,而进行此项研究,同时以胃血环状沉淀试验(下称沉淀法)对照比较,研究结果如下。材科与方法一、材料 1.蚊胃血标本:实验室饲养羽化4～6d饥饿24h大劣按蚊叮刺志愿者前臂吸饱血,然后在不同时间制备胃血滤纸标本;于海口市郊人、牛房采集吸饱血的三带喙库蚊制胃血滤纸标本。装入塑料袋,4℃密封保存。     

云南省边境地区景洪市疟疾媒介调查

目的了解云南省边境地区景洪市疟疾重要传播媒介的按蚊种类、生态习性及其疟原虫子孢子感染情况。方法于2015年在景洪市选取1个历史上疟疾流行高发村曼辉龙村为调查点,6-10月每月捕蚊3 d。在调查点东、西、南、北、中5个方位各选择1户人房和畜房,采用灯诱法通宵捕捉按蚊,调查按蚊种群密度;多重PCR检测按蚊胃血,观察其嗜血习性。选择村内和村外各1处采用帐诱法捕捉按蚊,观察按蚊夜间活动规律;采用巢式PCR检测按蚊疟原虫子孢子感染情况。结果共捕获1 286只按蚊,属13种,其中中华按蚊936只(占72.8%),微小按蚊188只(占14.6%),为当地按蚊优势蚊种。按蚊的捕获来源地主要为畜房,占85.6%(1 101只)。125份中华按蚊胃血多重PCR检测结果显示,人血指数为0,猪血指数为100。中华按蚊村内叮人率为0.6只/(人·h),村外的叮人率为0.2只/(人·h),夜间活动高峰期为20∶00-22∶00。101只微小按蚊、 517只中华按蚊和40只多斑按蚊巢式PCR检测,均未发现疟原虫子孢子感染。结论景洪市按蚊媒介种群以中华按蚊为主,其次为微小按蚊。中华按蚊以嗜家畜血为主。捕获的按蚊中未检测到疟原虫子孢子感染。     

中缅边境(西段)传疟媒介的初步调查

目的了解中缅边境(西段)传疟媒介的分布与构成。方法 2008年8～9月,在中缅边境的中国云南省盈江县及其相邻的缅甸昔懂县6个自然村,用诱蚊灯在人房和牛棚共进行20次通宵诱捕。将捕获的蚊虫以传统方法进行形态学鉴定,然后用复合PCR法鉴别微小按蚊、乌头按蚊和杰普按蚊。同时,抽提部分蚊虫标本总基因组DNA,以巢式PCR方法检测蚊体内的疟原虫感染情况。结果共捕获各类蚊虫4 571只,隶属9属50种,其中按蚊属是优势蚊种,占总量的54.32%(2 483/4 571)。人房和牛棚的按蚊蚊种构成差异有统计学意义,其中人房以腹簇按蚊、微小按蚊和中华按蚊为主,而牛棚以腹簇按蚊(223只)、环纹按蚊(184只)、迷走按蚊(131只)和杰普按蚊(129只)为主。对比有牛村和无牛村中人房的蚊种构成发现,有牛村的人房以微小按蚊(260只)和腹簇按蚊(49只)为主,而无牛村人房则以腹簇按蚊(481只)和中华按蚊(124只)为主。巢式PCR检测1 075只按蚊,其中9只检出疟原虫阳性,分别为微小按蚊(7/408)、乌头按蚊(1/125)和伪威氏按蚊(1/101)。经测序鉴定均为恶性疟原虫感染,目的条带长204 bp。结论中缅边境(西段)...     

醋酸纤维膜对流免疫电泳鉴定蚊胃血血源的研究

以醋酸纤维膜作对流免疫电泳,试用于鉴定蚊胃血,经试验选择的工作浓度:抗血清为1:8,抗原1:500,与常用的环状沉淀试验法同时对比鉴定离体刺吸人血或猪血的中华按蚊胃血标本,吸血后4、12及24小时制作的标本,于4℃冰箱贮存6、16及29个月的标本以及现场采集的蚊胃血标本,两法鉴定结果一致,反应特异性相似,初步证明此法可试用于蚊胃血的鉴定。     

海南岛残存微小按蚊调查

微小按蚊仍为海南岛传疟媒介之一。经研究从其形态特征及酯酶同工酶的比较,可分为两个不同类型,即微小按蚊A型和B型。人工感染实验证明两型微小按蚊对间日疟原虫均为易感。我们选择海南岛西南部石碌镇牙营自然村为观察点,在该村的边缘微小按蚊幼虫孳生地附近,建两间茅草实验小屋,采用人、牛为诱饵,从日落开始至日出为止,观察两型微小按蚊的活动情况。在不同场所采集吸血的微小按蚊,经鉴定后制作胃血标本,用醋酸纤维膜对流免疫电泳和环状沉淀法,鉴别两型微小按蚊的胃血血源。 1986年4～6月在牙营村两间实验小屋观察     

Identification of mosquito avian-derived blood meals by polymerase chain reaction-heteroduplex analysis

A polymerase chain reaction (PCR) heteroduplex assay (HDA) was developed to identify avian derived mosquito blood meals to the species level. The assay used primers amplifying a fragment of the cytochrome B gene from vertebrate but not invertebrate species. In Culex tarsalis fed on quail, PCR products derived from the quail cytochrome B gene were detected seven days post-engorgement. In an analysis of wild-caught mosquitoes, 85% of blood-fed mosquitoes produced detectable PCR products. Heteroduplex patterns obtained from bird-derived PCR products were found to permit the unambiguous identification of all species examined. No intraspecific variation in HDA patterns was found. The PCR-HDA was used to characterize blood meals in wild caught Cx. tarsalis. Of the 67 blood meals analyzed, 60% were derived from avian sources. Of the avian blood meals, 65% were derived from a single host, the common grackle.     

Blood meals of mosquito vectors of filariasis in Guizhou Province, China

The blood meals of mosquito vectors of W. bancrofti and B. malayi were determined by performing CIEP of the eluant of crushed mosquitoes in filter paper against rabbit anti-human, cow and pig sera. The mosquitoes were collected from houses, cowsheds and pigpens in two counties in Guizhou Province. It was shown that all three species fed on blood from humans, cows and pigs with different preference. While An sinensis fed more on cows, Cx. fatigans and An. lesteri fed on the hosts that were nearby, i.e., Cx. fatigans caught from households fed more on humans, and those collected from cowsheds or pigpens fed more on cows and pigs, respectively.     

我国赫坎按蚊种团的分子鉴别及中华按蚊的区系分布研究

目的改进赫坎按蚊种团部分成员种的多重PCR鉴别方法,鉴别赫坎按蚊种团中的中华按蚊,研究我国中华按蚊的区系分布及其影响因素。方法依据赫坎按蚊种团成员种中华按蚊、八代按蚊、雷氏按蚊、比伦按蚊和克莱按蚊的核糖体DNA内转录间隔区2 (rDNA-ITS2)序列差异设计种特异引物,改进多重PCR分子鉴别方法。2013-2018年,在我国8个省(直辖市、自治区)共18个地点,以诱虫灯和人工吸取相结合的方法采集按蚊,依据形态特征初步鉴定为赫坎按蚊种团的成员种。提取单只蚊虫基因组DNA,使用改进的多重PCR方法鉴定种类。对多重PCR法无扩增产物的个体分析rDNA-ITS2序列,在GenBank上进行BLAST比对,确定其种类。查找我国以及韩国和俄罗斯远东地区用分子特征鉴别为中华按蚊的文献,结合本研究结果,汇总中华按蚊采集地的地理位置和气候数据,使用地理探测器模型计算影响决定力q值,分析经度、纬度、年平均气温和年平均降雨量对中华按蚊分布的影响。结果改进的多重PCR法一次扩增即可依据扩增片段大小鉴别赫坎按蚊种团的5个成员种:中华按蚊(490 bp)、雷氏按蚊(313 bp)、八代按蚊(216 bp)、克莱按蚊(386 bp)和比伦按蚊(165 bp)。在我国18个采集点共捕获赫坎按蚊种团按蚊365只,多重PCR鉴别为中华按蚊114只(来自陕西、安徽与山东的采集点)、八代按蚊34只、雷氏按蚊9只、克莱按蚊181只和比伦按蚊5只。22只多重PCR无扩增产物的蚊虫中,经rDNA-ITS2序列分析鉴定为贵阳按蚊2只、类中华按蚊1只、朝鲜按蚊8只、林氏按蚊7只和帕氏按蚊4只。获取用分子特征鉴别为中华按蚊的文献17篇,共汇总了101个采集地信息,其中有中华按蚊分布的采集点80个,无中华按蚊分布的21个地点。地理探测器软件计算获得的q值,从大到小依次为0.592 0(年平均气温)、0.507 2 (纬度)、0.351 2 (经度)和0.214 4 (年平均降雨量)。中华按蚊的分布与年平均气温关系最为密切,其次是纬度。综合分析分布地的纬度和年平均温度等结果显示,年平均气温10℃可以作为划分中华按蚊在我国分布北界线的依据。我国中华按蚊的分布范围包括云南、贵州、重庆、河南、山东、天津、江苏、安徽、湖北、浙江、上海、福建、江西、广西、广东、海南等省(直辖市、自治区)及台湾、香港、澳门特别行政区的全境,以及西藏、四川、甘肃、陕西、山西、河北、北京、辽宁等省(直辖市、自治区)的南部部分地区。结论改进的赫坎按蚊种团多重PCR分子鉴定方法快速简单、客观可靠。综合分析显示划分中华按蚊在我国分布北界线的依据是年平均气温10℃线,中华按蚊在我国的分布应小于之前记载的范围。     

Evaluation of multiplex nested polymerase chain reaction for routine hepatitis C virus genotyping in egyptian patients

Background

At least six HCV (hepatitis C virus) genotypes are unequally distributed worldwide. HCV genotyping guides the selection of treatment regimens and provides important epidemiological markers that enable the outbreak source to be traced and the spread of disease to be controlled. In Egypt, there is an increasing need for cost-effective, fast, and easily performable HCV genotyping assays.Recently, a multiplex PCR assay was developed to determine HCV genotypes. It employs genotype-specific primers, based on sequences of the entire core region and part of the 5’UTR of the genome.

Objectives

In this study, we compared a simple, new, modified multiplex PCR system for HCV genotyping with a commercially available line probe assay (INNO-LiPA) that is based on reverse hybridization.

Patients and Methods

Serum samples from chronic HCV Egyptian patients (n = 73) were genotyped using the modified multiplex PCR assay, and genotypes were verified using the INNO-LiPA HCV II assay.

Results

The modified multiplex PCR method was able to type HCV-4 in 65 of 70 typeable samples (92.86%) and had 100% concordance with the INNO-LiPA assay.

Conclusions

Genotype 4 was the most prevalent genotype in our study. Based on our results, the modified multiplex nested PCR assay is a sensitive and inexpensive alternative for HCV genotyping and can be used in routine diagnostic laboratories. INNO-LiPA may be useful as a second-line assay for genotyping samples that are indeterminate by multiplex PCR. This approach will effect better treatment optimization and a reduction of the spread of HCV.     

Diagnosis of meningococcal meningitis in Brazil by use of PCR

Fever and a petechial rash are strongly associated with meningococcal disease in the city of Rio de Janeiro. Early antibiotic therapy is indicated and, consequently, a reduction of confirmed cases by culture, Gram stain, and latex agglutination test is expected. We evaluated a multiplex PCR assay to identify Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae in biological samples from cases of non-culture proven meningitis with a petechial rash at presentation. To detect DNA in cerebrospinal fluid (n = 71) or blood (n = 5), a PCR screen was performed, based on the crgA, ply and bexA targets, respectively. Of the total, 70 CSF and 3 blood samples (96%) were positive by PCR for the presence of N. meningitidis DNA. Another PCR assay predicted in 82% of these samples N. meningitidis serogroups A (2%), B (60%), C (7%), X (3%), Y (2%), 29E (2%) or W135 (24%). In non-culture proven meningitis, PCR was found to be a valuable adjunct for the demonstration of meningococcal aetiology.     

Identification of mosquito blood meals by enzyme-linked immunosorbent assay

A micro enzyme-linked immunosorbent assay for identifying mosquito blood meals is described. Antisera for the assay were prepared in rabbits and guinea pigs and purified by precipitation with cross-reacting blood sera. Positive blood source identifications could be made to the generic level using as little as 58 micrograms of ingested blood serum. The blood meals of 37 of 38 laboratory mosquitoes fed on seven host animals were correctly identified, including a mosquito that contained a double feed.     

Multiplex PCR assay for the detection of Anopheles fluviatilis species complex, human host preference, and Plasmodium falciparum sporozoite presence, using a unique mosquito processing method

A multiplex PCR assay has been developed for detection of Anopheles fluviatilis cryptic species, their human host preference, and Plasmodium falciparum presence in the mosquito. PCR conditions were optimized using primer sets specific for A. fluviatilis cryptic species, Homo sapiens, and P. falciparum and evaluated with field-collected mosquitoes. A unique mosquito processing method was used for screening P. falciparum carrying capacity and human host preference of A. fluviatilis mosquitoes in first-round multiplex PCR. The vectorial status of the mosquito for P. falciparum parasite was confirmed in second-round PCR. Of the 121 collected mosquitoes, 92 were of S type, 26 of T type, and 3 were of other types. Human host preference was dominant in S type, of which 4% were P. falciparum sporozoite positive. This assay and processing method can also be used to evaluate vector competence of other anophelines.     

Identification in triatomine vectors of feeding sources and Trypanosoma cruzi variants by heteroduplex assay and a multiplex miniexon polymerase chain reaction

Feeding sources of triatomine vectors (Triatoma longipennis) collected in peridomiciles in Mexico were identified by a heteroduplex assay developed with triatomine blood meals. Trypanosoma cruzi parasites were also characterized in the same blood meal samples by multiplex-polymerase chain reaction assay of mini-exon gene inter-genic regions. The main blood meal source was from rats, but the bugs were able to feed on a wide variety of hosts, and human blood meals were identified. Trypanosoma cruzi was the only flagellate species identified in the blood meals. All populations belong to the T. cruzi I lineage, a result that is consistent with the previously assumed predominance of this lineage in Mexico. This combination of blood meal and T. cruzi lineage identification provides a powerful tool for understanding T. cruzi transmission cycles.     

Detection of host virus-reactive antibodies in blood meals of naturally engorged mosquitoes

Although serosurvey in human or animals is a useful and straightforward strategy routinely used for public health, it often faces different types of impediments: ethics, beliefs, limitation by animal owners, hazard of access to wild animals. To survey virus circulation, we applied the enzyme-linked immunosorbent assay (ELISA) technique to detect Dengue and Japanese encephalitis (JE) virus-reactive antibodies in blood meals collected from mosquitoes without regard to the potential of mosquito species to be a virus vector. ELISA was performed on mosquito colonies and wild specimens collected from farms and urban areas. Blood meals from Aedes aegypti freshly fed on naturally infected volunteers showed the same levels of dengue immunoglobulin (Ig)G and IgM as the sera directly collected from volunteers. A significant clearance of antibodies during the digestion process started from 13 hours after blood meal, and a negative baseline was reached after 30 hours. The ELISA test performed on wild mosquitoes showed that 37% of Culex quinquefasciatus mosquitoes that engorged on humans in a dengue urban endemic area tested positive for dengue IgG, and in a JE virus-endemic area, 88% of Culex tritaeniorhynchus mosquitoes that engorged on pigs from a large pig farm tested positive for JE virus antibodies versus 11% in a small farm. The main limitation of the ELISA method is the antibody cross-reactivity among flaviviruses; also, sampling strategy should be adjusted to take into account that the actual host from which the blood meal was taken may not be determined. Nevertheless, ELISA performed on recently (1-2 days) engorged mosquito, or any other hematophagous arthropod species, could potentially be used as a "wild phlebotomist" to monitor the prevalence or emergence of a variety of pathogens, with less of the practical, ethical, or risk limitations due to direct blood collection from humans and wild or domestic animals.     

Evaluation of quantitative buffy coat (QBC) assay and polymerase chain reaction (PCR) for diagnosis of malaria

A prospective study was undertaken to compare the Polymerase Chain Reaction (PCR) and Quantitative Buffy Coat (QBC) assay with conventional Giemsa technique for diagnosis of malaria. A total of 104 samples were taken for the purpose. They comprised of fever cases suggestive of malaria (n=74) and control group, fever cases other than malaria (n=30). Peripheral blood smears were prepared by Giemsa staining and QBC assay was performed as per standard protocol. From the stored blood samples, parasite DNA was extracted and PCR was performed using P. falciparum and P. vivax specific sets of primers. The QBC assay was 100% in agreement with the Giemsa stain. Specificity of the PCR detection of P. falciparum parasites was 100%. However, sensitivity for detection of P. falciparum and P. vivax by PCR was 64.28% and 82.35% respectively. In mixed cases of malaria (n=2), PCR results were in 100% agreement with that of Giemsa. The lower sensitivity of PCR for P. falciparum could probably be due to inaccessibility of target DNA, presence of PCR inhibitors in samples and parasite strain variations.