我们感谢博士。安东尼山茱萸和劳拉·诺里斯在加州大学戴维斯分校和卡塔琳娜Kreppel博士在格拉斯哥大学从坦桑尼亚提供蚊子标本。我们感谢Smita达斯女士和道格拉斯·诺里斯博士公共卫生约翰霍普金斯学院分享蚊虫样本赞比亚。我们也感谢李五米永先生在兽医遗传学实验室对实验设计培训。这项工作是支持由美国国立卫生研究院授予R01AI 078183和R21AI062929。

1. PCR扩增<ol><li>混合所有的PCR引物（见补充表S1）的1.5ml离心管。每个SNP具有两个引物（正向和反向）。确保原料浓度每种PCR引物被保持在100微米。使足够引物混合物为500-1000的反应，以减少在工作台上移液误差和时间（参见补充表S2）。如果需要的话，制备等分试样，每管并储存100-200反应在-20℃。 </li><li>制备的PCR鸡尾酒如生产商的方案（表1）中所描述。 96反应的体积包括20％的突出端。涡包含PCR鸡尾酒轻轻离心30秒，2000×g的管子。 </li><li>分配3微升的PCR鸡尾酒成的每个孔中的非裙边96孔板中。中继器吸管可以节省时间，这个过程中。 </li><li>添加2微升的蚊子的适当的基因组DNA中的每个孔中。 注:获得从米的基因组DNA的方法<html> osquito组织（头部/胸部，腹部或全身），在其他文献29-32描述。选择一个合适的DNA提取方法来区分那些与疟原虫在血液（腹部）从受感染蚊子（ 疟原虫的头部/胸部）。我们通常使用DNA从蚊子的身体部位（头部/胸部或腹部）中提取，因此从0.05-2ng /μl的DNA浓度变化。虽然总的DNA输入比制造商的建议（5-10ng / RXN）下，我们通常得自98％的DNA样本的没有全基因组扩增的SNP健壮呼叫。 </li><li>封板，轻轻拌匀或涡流，并在370×g的1分钟离心机。 </li><li>使用以下热循环协议来进行PCR扩增:（1）94℃2分钟，（2）94℃，30秒，（3）56℃，30秒，（4）72℃，1分钟，（5）重复步骤（2） - （4）45个循环，（6）72℃，1分钟，4℃保持。 </li></ol>_title"&gt; 2。SAP治疗<ol><li>准备在96孔板在SAP酶溶液（见表2）。处理在一个板96个样品。 96反应的体积包括20％的突出端。 </li><li>涡SAP酶液轻轻离心管30秒，2000×g的。分配2微升的SAP酶溶液加入到每个孔中。再次，一个转发器移液管，可以节省时间用于此过程。 </li><li>封板，轻轻拌匀或涡流，并在370×g的1分钟离心机。孵育板如下:（1）37℃下40分钟，（2）85℃下进行5分钟，以使酶失活，（3）4℃保持。在该步骤完成后，在-20存储板℃下再继续。 2个星期内处理存储的板块。 </li></ol> 3. SNP扩展<ol><li>如果样品板的SAP处理后冷冻，允许它继续之前解冻至室温。 </li><li>制备所述延伸引物混合物（补充表S2）。每个SNP有一个EXTEnsion底漆。保持股票集中每个延伸引物在500微米。任选地，对等分试样每管并储存100-200反应在-20℃。 </li><li>如表3中96的反应体积，包括20％的悬垂描述制备延伸组合。 </li><li>涡延伸鸡尾酒轻轻离心管30秒，2000×g的。分配2微升的延伸鸡尾酒到每个孔中。再次，一个转发器移液管，可以节省时间用于此过程。 </li><li>封板，轻轻拌匀或涡流，并在370×g的1分钟离心机。热循环， 如图1中的板。在这一点上，进行质谱法或板储存在-20℃达2周。 </li></ol> 4.空调的SNP扩展产品优化质谱分析<ol><li>如果样品板SNP后延冻结，允许它继续之前解冻到室温。 </li><li>从CONTA转移树脂为15mg核能研究所使用上的长勺酒窝板。使用刮刀蔓延树脂进入凹坑板的孔中。从一边扫刮板跨越酒窝板到另一边蔓延树脂。确保有树脂每口井。 </li><li>刮去多余的树脂脱使用刮板酒窝板。让树脂站在凹坑板为至少20分钟。同时让树脂站在凹坑板中，添加41微升的HPLC级水到样品板的各孔中。 </li><li>轻轻将样品板倒置，到酒窝板。确保样品板复位靠在凹坑板的小圆形交，该对齐的孔中的样品板与孔中的凹坑板。 </li><li>保持样品板和凹坑板一起，轻轻翻转它们在这样的树脂脱落凹坑板的进入样品板的孔中。点击酒窝板，使树脂脱落到样品板。确保所有的树脂在迪mple板脱落到样品板的孔中。离心板在3200 XG为30秒。 </li><li>旋转旋转器上的样品板5分钟，在室温。旋转器必须旋转样品板约长轴360°。 </li><li>离心样品板在3200×g离心5分钟。此步骤之后，进行到下一步骤之前，长达2周存储的样品板在-20℃下。 </li></ol> 5. MALDI-TOF质谱<ol><li>转移条件的SNP延伸产物到生物芯片诸如SpectroCHIP按照制造商的说明。我们使用MassArray纳米分配器用于此步骤。 </li><li>一旦转移完成，确定测定法和板如何被设置在测定数据库中。命名法和板使得每个板块都有唯一的标识符，链接到特定的试验设计（参考表三）。 </li><li>法和板已被定义后，获取利用质谱仪光谱。 </li></ol><ol><li>使用实时PCR分析数据解释软件，如打字员分析器或TyperViewer（Sequenom公司）进行数据分析。在包含样本的布局，质谱图每个井.xml格式获得的数据，该标记信息包括名称，用于对应的SNP的每种变体预期质量，并且任何错误或与每个反应相关的警告消息。参见图2所产生的质谱图。 </li><li>由该检测阈值设定（幅度或信噪比），并允许每个基因型公差/方差进行的SNP基因型的聚类分析。 图3示出了04707-KDR＃2的SNP基因型簇的例子。 </li><li>导出所得的SNP基因型的呼叫到电子表格程序。 </li></ol>

The authors have nothing to disclose.

该MDA是由五个主要步骤:PCR扩增，虾碱性磷酸酶（SAP）的反应，SNP延伸，延伸产品的调理和基质辅助激光解吸/电离-飞行（MALDI-TOF）质谱33-37的时间。第一次PCR扩增步骤扩增的DNA的侧翼每个SNP，使得足够的模板DNA将是可利用的SNP的延伸步骤。在SAP反应中和未使用的dNTP其可以用下面的SNP延伸步骤干涉。 SNP扩展涉及每个​​SNP位点和大众修饰的核苷酸终止一个延伸引物。 3&#39;-末端修饰的核苷?...

阿拉伯按蚊，按蚊coluzzii和冈比亚按蚊负责非洲1疟疾传播的主要媒介。这三种物种在形态区分2，并且只能通过分子测定法3-9进行区分。此外，还有许多下游实验经常进行，以帮助流行病学和群体遗传学的研究。这些包括（1）为形态岛屿10-12基因分型测定法中，（2）一种基因分型测定法识别在1014的非同义的SNP 个 段基因的氨基酸的密码子的位置（ 击倒抗性或KDR，SNP）13 -18，（3）的寄生虫检测的PCR 19-23，和（4）筛选在蚊子中肠24,25宿主特异的DNA。 我们开发了将所有这些测定与一个目标的单个多重反应的多检测分析（MDA）的alyzing在非洲疟疾向量的流行病学重要特征。 MDA的测定法包括:多个标记，用于检测（1）种（A.阿拉伯按蚊，冈比亚按蚊，A coluzzii，或其它（所有的3）的），（2）杀虫剂中KDR的SNP表示电阻（两者L1014F 和L1014S） ，（3）存在的两个主要的疟原虫，恶性疟原虫和P.间日疟原虫 ，和（4）从一禽和六个哺乳动物宿主血液来源。 传统上，这些测定法在分开的聚合酶链式反应进行的。当使用常规PCR平台完成所有这些测定中，它需要进行8-10 PCR反应检测和伴随凝胶电泳步骤。从准备每个PCR反应，记录结果需要4-5小时，而MDA方法，这里介绍需要大约5小时的总和。这相当于节省了单独的劳动力成本的90％。在MDA这里介绍的价格为5美元每个样品基因型的所有33个SNP。这是相当便宜比单琼脂糖凝胶为基础的分析，其中的成本约为1.50美元样本。测定法检测所涵盖的所有的MDA的特性将需要至少8-10分开琼脂糖凝胶为基础的测定以$ 12-15每一个样品的成本。此外，MDA的大大减少产生假阳性或阴性通过利用至少三个标记为每个寄生虫或主机的源检测的机会。 我们所使用的平台不限于疟疾的载 ​​体，但可以在各种各样的应用，如医药，兽药和基础生物学26-28中使用。深入涉及的样品大（为了100S）的数量关联研究或群体遗传学研究需要经济高效的检测筛选同时为多个标记。利用两个或更多个单独的PCR测定大多数研究可以实现MDA的用于以较低的成本更快的结果。 </p&gt;

<table><tbody><tr><td>MassARRAY Analyzer Compact</td><td>Sequenom</td><td>MT9</td><td>MALDI-TOF mass spectrometry for genomic applications to analyze nucleic acids.</td></tr><tr><td>MassARRAY Nanodispenser</td><td>Sequenom</td><td>RS1000</td><td>Transfers completed iPLEX reaction products to the SpectroCHIP</td></tr><tr><td>iPLEX Gold Genotyping Reagent Set</td><td>Sequenom</td><td>10158</td><td>Reagents used for iPLEX assay including SAP kit.</td></tr></tbody></table>

a multi-detection assay for malaria transmitting mosquitoes

在冈比亚按蚊种类复杂，包括主要的疟疾在非洲发射蚊子。由于这些品种都是这样的医疗重要性，一些特质的典型特点采用分子检测的流行病学研究提供帮助。这些特征包括物种鉴定，抗药性，寄生虫感染状态，和主机偏好。由于冈比亚按蚊复杂的群体在形态上没有区别，聚合酶链式反应（PCR）是传统上用于识别物种。一旦种是已知的，一些下游的测定是进行例行阐明进一步的特点。例如，在一对位基因被称为KDR突变赋予对DDT和拟除虫菊酯类杀虫剂抗性。此外，酶联免疫测定（ELISA）或疟原虫寄生虫的DNA检测的PCR测定法是用于检测存在于蚊子组织寄生虫。最后，一​​个combinatioÑPCR和限制酶消化的可用于阐明宿主偏好（ 例如 ，人类与动物的血液）通过筛选蚊子吸血寄宿特异性DNA。我们已经开发了在一个时刻将所有的上述分析成单个多重反应的基因分型33SNPs为96或384个样品的多检测分析（MDA）。因为MDA包括多个标记的物种， 疟原虫的检测，和主机血液鉴定，产生假阳性或阴性的可能性大大从以前的测定法，其中包括每性状只有一个标记降低。这个强大而简单的分析可以经济高效地在现有的实验的一小部分时间检测到这些关键的蚊子特点。

物种鉴定: 以下5个SNP一起识别3种（A.阿拉伯按蚊，A coluzzii和冈比亚按蚊 ）（ 见表4）。如果样品是不是3种之一，三个单核苷酸多态性（01073-213，04679-157和10313 -052）无法放大。 KDR基因型推断抗药性: 对位电压-门控钠通道的1014 个密码子对应于KDR。两个单核苷酸多态...

Watch this Scientific Journal Video about A Multi-detection Assay for Malaria Transmitting Mosquitoes at JoVE.com

传播疟疾的蚊子的多重检测分析|生物学 |视频

A Multi-detection Assay for Malaria Transmitting Mosquitoes

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

一种多检测试验为疟疾蚊子发射

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, ...

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SCIENTISTS IN ACTION

13.1K 次观看.  School of Veterinary Medicine, University of California - Davis. Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

视频: A Multi-detection Assay for Malaria Transmitting Mosquitoes

Protocol for Production of a Genetic Cross of the Rodent Malaria Parasites

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to successful identification of genes or loci underlying various traits in malaria parasites of rodents1-3 and humans4-6. The malaria parasite Plasmodium yoelii is one of many malaria species isolated from wild African rodents and has been adapted to grow in laboratories. This species reproduces many of the biologic characteristics of the human malaria parasites; genetic markers such as microsatellite and amplified fragment length polymorphism (AFLP) markers have also been developed for the parasite7-9. Thus, genetic studies in rodent malaria parasites can be performed to complement research on Plasmodium falciparum. Here, we demonstrate the techniques for producing a genetic cross in P. yoelii that were first pioneered by Drs. David Walliker, Richard Carter, and colleagues at the University of Edinburgh10.

Genetic crosses in P. yoelii and other rodent malaria parasites are conducted by infecting mice Mus musculus with an inoculum containing gametocytes of two genetically distinct clones that differ in phenotypes of interest and by allowing mosquitoes to feed on the infected mice 4 days after infection. The presence of male and female gametocytes in the mouse blood is microscopically confirmed before feeding. Within 48 hrs after feeding, in the midgut of the mosquito, the haploid gametocytes differentiate into male and female gametes, fertilize, and form a diploid zygote (Fig. 1). During development of a zygote into an ookinete, meiosis appears to occur11. If the zygote is derived through cross-fertilization between gametes of the two genetically distinct parasites, genetic exchanges (chromosomal reassortment and cross-overs between the non-sister chromatids of a pair of homologous chromosomes; Fig. 2) may occur, resulting in recombination of genetic material at homologous loci. Each zygote undergoes two successive nuclear divisions, leading to four haploid nuclei. An ookinete further develops into an oocyst. Once the oocyst matures, thousands of sporozoites (the progeny of the cross) are formed and released into mosquito hemoceal. Sporozoites are harvested from the salivary glands and injected into a new murine host, where pre-erythrocytic and erythrocytic stage development takes place. Erythrocytic forms are cloned and classified with regard to the characters distinguishing the parental lines prior to genetic linkage mapping. Control infections of individual parental clones are performed in the same way as the production of a genetic cross.

Genetic crosses of rodent malaria parasites are performed by feeding two genetically distinct parasites to mosquitoes. Recombinant progeny are cloned from mouse blood after allowing mosquitoes to bite infected mice. This video shows how to produce genetic crosses of Plasmodium yoelii and is applicable to other rodent malaria parasites.

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

Variation in response to antimalarial drugs and in pathogenicity of malaria parasites is of biologic and medical importance. Linkage mapping has led to ...

科研

免疫与感染

Protocol for Plasmodium falciparum Infections in Mosquitoes and Infection Phenotype Determination

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito.    This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed.  The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the mosquito. This protocol illustrates how the extent of plasmodium infections of mosquitoes can be assayed. The techniques for preparing the gametocyte culture, membrane feeding mosquitoes human blood, and assaying viral titers in the mosquito midgut are demonstrated.

恶性疟原虫在蚊子和病毒感染的表型测定感染的议定书

Once a gene is identified as potentially refractory for malaria, it must be evaluated for its role in preventing Plasmodium infections within the ...

生物学

Multiplexed Isothermal Amplification Based Diagnostic Platform to Detect Zika, Chikungunya, and Dengue 1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different downstream patient-to-patient transmission potentials, and require very different patient treatments. Thus, recent Zika outbreaks make it urgent to develop tools that rapidly discriminate these viruses in patients and trapped mosquitoes, to select the correct patient treatment, and to understand and manage their epidemiology in real time.
Unfortunately, current diagnostic tests, including those receiving 2016 emergency use authorizations and fast-track status, detect viral RNA by reverse transcription polymerase chain reaction (RT-PCR), which requires instrumentation, trained users, and considerable sample preparation. Thus, they must be sent to &#34;approved&#34; reference laboratories, requiring time. Indeed, in August 2016, the Center for Disease Control (CDC) was asking pregnant women who had been bitten by a mosquito and developed a Zika-indicating rash to wait an unacceptable 2 to 4 weeks before learning whether they were infected. We very much need tests that can be done on site, with few resources, and by trained but not necessarily licensed personnel.
This video demonstrates an assay that meets these specifications, working with urine or serum (for patients) or crushed mosquito carcasses (for environmental surveillance), all without much sample preparation. Mosquito carcasses are captured on paper carrying quaternary ammonium groups (Q-paper) followed by ammonia treatment to manage biohazards. These are then directly, without RNA isolation, put into assay tubes containing freeze-dried reagents that need no chain of refrigeration. A modified form of reverse transcription loop-mediated isothermal amplification with target-specific fluorescently tagged displaceable probes produces readout, in 30 min, as a three-color fluorescence signal. This is visualized with a handheld, battery-powered device with an orange filter. Forward contamination is prevented with sealed tubes, and the use of thermolabile uracil DNA glycosylase (UDG) in the presence of dUTP in the amplification mixture.

Current multiplexed diagnostics to detect Zika, chikungunya, and dengue viruses require complex sample preparation and expensive instrumentation, and are difficult to use in low resource environments. We show a diagnostic that uses isothermal amplification with target-specific strand displaceable probes to detect and differentiate these viruses with high sensitivity and specificity.

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

Zika, dengue, and chikungunya viruses are transmitted by mosquitoes, causing diseases with similar patient symptoms. However, they have different ...

生物化学

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectrometer in conjunction with Multivariate Data Analysis (MVDA). 3D7 P. falciparum were cultured to 10% parasitemia ring stage parasites and used to spike fresh donor isolated RBCs to create a dilution series between 0&#8211;1%. 10 &#181;L of each sample were placed onto the center of the ATR diamond window to acquire the spectrum. The sample data was treated to improve the signal to noise ratio and to remove the contribution of water, and then the second derivative was applied to resolve spectral features. The data were then analyzed using two types of MVDA: first Principal Component Analysis (PCA) to determine any outliers and then Partial Least Squares Regression (PLS-R) to build the quantification model.

Detection and Quantification of Plasmodium falciparum in Aqueous Red Blood Cells by Attenuated Total Reflection Infrared Spectroscopy and Multivariate Data Analysis

Here, we present a protocol for the detection and quantification of Plasmodium falciparum in infected aqueous red blood cells using an attenuated total reflection infrared spectrometer and multivariate data analysis.

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

We demonstrate a method of quantification and detection of parasites in aqueous red blood cells (RBCs) by using a simple benchtop Attenuated Total ...

化学

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

数字液滴PCR检测转基因按蚊种群中的Indels突变

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

遗传学

Methods to Increase the Sensitivity of High Resolution Melting Single Nucleotide Polymorphism Genotyping in Malaria

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been accompanied by increased genomic information about Plasmodium parasite species responsible for malaria, including characteristics of geographical populations as well as variations associated with reduced susceptibility to anti-malarial drugs.
One common genetic variation, single-nucleotide polymorphisms (SNPs), offers attractive targets for parasite genotyping. These markers are useful not only for tracking drug resistance markers but also for tracking parasite populations using markers not under drug or other selective pressures.
SNP genotyping methods offer the ability to track drug resistance as well as to fingerprint individual parasites for population surveillance, particularly in response to malaria control efforts in regions nearing elimination status.
While informative SNPs have been identified that are agnostic to specific genotyping technologies, high-resolution melting (HRM) analysis is particularly suited to field-based studies. Compared to standard fluorescent-probe based methods that require individual SNPs in a single labeled probe and offer at best 10% sensitivity to detect SNPs in samples that contain multiple genomes (polygenomic), HRM offers 2-5% sensitivity. Modifications to HRM, such as blocked probes and asymmetric primer concentrations as well as optimization of amplification annealing temperatures to bias PCR towards amplification of the minor allele, further increase the sensitivity of HRM. While the sensitivity improvement depends on the specific assay, we have increased detection sensitivities to less than 1% of the minor allele.
In regions approaching malaria eradication, early detection of emerging or imported drug resistance is essential for prompt response. Similarly, the ability to detect polygenomic infections and differentiate imported parasite types from cryptic local reservoirs can inform control programs.
This manuscript describes modifications to high resolution melting technology that further increase its sensitivity to identify polygenomic infections in patient samples.

While high resolution melting analysis offers the ability to differentiate between single nucleotide polymorphisms in a heterogeneous population, mutant allele amplification bias can increase its ability to detect alleles present at relatively low percentages within a sample. This protocol describes improvements that improve the sensitivity of high resolution melting analysis.

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

Despite decades of eradication efforts, malaria remains a global burden. Recent renewed interest in regional elimination and global eradication has been ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. However, effective malaria parasite targets for vaccine and chemotherapy development are still limited. This is largely due to the unavailability of relevant and practical in vivo infection models for human Plasmodium species, most notably for P.&#160;falciparum and P. vivax. Therefore, rodent malaria species have been extensively used as practical alternative in vivo models for malaria vaccine, drug targeting, immune response, and functional characterization studies of conserved Plasmodiumspp. genes. Indeed, rodent malaria models have proven to be invaluable, especially for exploring mosquito transmission and liver stage biology, and were indispensable for immunological studies. However, there are discrepancies in the methods used to evaluate the phenotypes of transgenic and wild-type asexual and sexual blood-stage parasites.&#160;Examples of these discrepancies are the choice of an intravenous vs. intraperitoneal infection of rodents with blood-stage parasites and the evaluation of male gamete exflagellation. Herein, we detail standardized experimental methods to evaluate the phenotypes of asexual and sexual blood stages in transgenic parasites expressing reporter-gene or wild-type rodent malaria parasite species. We also detail the methods to evaluate the phenotypes of malaria parasite mosquito stages (gametes, ookinetes, oocysts, and sporozoites) inside Anopheles mosquito vectors. These methods are detailed and simplified here for the lethal and non-lethal strains of P. berghei and P. yoelii but can also be applied with some adjustments to P. chabaudi and P. vinckei rodent malaria species.

Due to the striking similarities of the life cycle and biology of rodent malaria parasites to human malaria parasites, rodent malaria models have become indispensable for malaria research. Herein, we standardized some of the most important techniques used in the phenotypic analysis of wild-type and transgenic rodent malaria species.

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

使用寨卡病毒对 埃及伊 蚊的载体能力分析

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This parasitic disease requires a protozoan parasite, an Anopheles mosquito vector, and a vertebrate host. The Anopheles genus comprises more than 500 species, of which 60 are known as vectors of the parasite. The Plasmodium parasite genus consists of 250 species, and 48 of these are involved in disease transmission. Furthermore, the Plasmodium falciparum parasite has contributed toward an estimated 99.7% of malaria cases in sub-Saharan Africa in recent years. 
Gametocytes form part of the sexual stage of the parasite and are ingested by the female mosquito upon feeding on an infected human host. Further development of the parasite within the mosquito is enhanced by favorable environmental conditions in the midgut of the mosquito. Here, the fusion of the female and male gametes takes place, and the motile ookinetes originate. The ookinetes enter the midgut epithelium of the mosquito, and mature ookinetes form oocysts, which, in turn, produce motile sporozoites. These sporozoites migrate to the mosquito's salivary glands and are injected as a mosquito takes a blood meal. 
For drug discovery purposes, mosquitoes were artificially infected with gametocyte-infected blood in the standard membrane feeding assay (SMFA). To detect infection within the mosquito and/or to assess the efficacy of antimalarial compounds, the midguts of the female mosquitoes were removed post infection and were stained with mercurochrome. This method was used to enhance the visual detection of oocysts under the microscope for the accurate determination of oocyst prevalence and intensity.

Standard Membrane Feeding Assay for the Detection of Plasmodium falciparum Infection in Anopheles Mosquito Vectors

The standard membrane feeding assay (SMFA) is regarded as the gold standard for the assessment and identification of potential antimalarial compounds. This artificial feeding system is used to infect mosquitoes to further evaluate the effects of such compounds on the intensity and prevalence of the Plasmodium falciparum parasite.

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

Malaria remains one of the most devastating diseases worldwide and, to date, the African region is still responsible for 94% of all cases worldwide. This ...

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

Plasmodium sporozoites are the infective stage of malaria parasites that infect humans. The sporozoites residing in the salivary glands of female Anopheles mosquitoes are transmitted to humans via mosquito bites during blood feeding. The presence of sporozoites in the mosquito salivary glands thus defines mosquito infectiousness. To determine whether an Anopheles mosquito carries Plasmodium sporozoites, the enzyme-linked immunosorbent assay (ELISA) method has been the standard tool to detect the Plasmodium circumsporozoite protein (CSP), the major surface protein of the sporozoites. In this method, the head along with the thorax of each mosquito is separated from the abdomen, homogenized, and subjected to a sandwich ELISA to detect the presence of CSP specific to Plasmodium falciparum and each of the two subtypes, VK210 and VK247, of Plasmodium vivax.This method has been used to study malaria transmission, including the seasonal dynamics of mosquito infection and the species of the major malaria vectors in the study sites.

Detection of Plasmodium Sporozoites in Anopheles Mosquitoes using an Enzyme-linked Immunosorbent Assay

This protocol describes a sandwich enzyme-linked immunosorbent assay to detect salivary gland sporozoites in mosquitoes. Using easily available monoclonal antibodies, the method enables cost-effective, high-throughput detection of mosquitoes carrying Plasmodium falciparum or Plasmodium vivax. The method is suitable for malaria transmission research, including vector surveys.

一种多检测试验为疟疾蚊子发射

摘要

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此视频中的章节

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

恶性疟原虫在蚊子和病毒感染的表型测定感染的议定书

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

数字液滴PCR检测转基因按蚊种群中的Indels突变

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

使用寨卡病毒对埃及伊蚊的载体能力分析

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

使用酶联免疫吸附测定法检测按蚊中的疟原虫孢子

一种多检测试验为疟疾蚊子发射

摘要

探索更多视频

此视频中的章节

生产的灭鼠疟疾寄生虫的遗传交叉议定书“

恶性疟原虫在蚊子和病毒感染的表型测定感染的议定书

基于复用等温放大诊断平台检测 Zika、基孔肯亚和登革热1

衰减全反射红外光谱和多变量数据分析检测和定量水红血球中恶性疟原虫

数字液滴PCR检测转基因按蚊种群中的Indels突变

方法来提高高分辨率的敏感性疟疾熔单核苷酸多态性基因分型

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

使用寨卡病毒对 埃及伊 蚊的载体能力分析

用于检测按蚊媒介中恶性疟原虫感染的标准膜喂养测定

使用酶联免疫吸附测定法检测按蚊中的疟原虫孢子

使用寨卡病毒对埃及伊蚊的载体能力分析