Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies.
Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies.
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基于寡核苷酸的磁珠捕获OnChocerca volvulus DNA用于PCR泳池筛选矢量黑蝇。
DOI：
10.1371/journal.pntd.0001712
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发表时间：
2012
影响因子：
3.8
通讯作者：
Unnasch TR
中科院分区：
文献类型：
--
作者：
Gopal H;Hassan HK;Rodríguez-Pérez MA;Toé LD;Lustigman S;Unnasch TR
在用于确定盘尾丝虫的传播是否已被阻断以及寄生虫是否已被局部消灭的标准中，对Simuerca病媒的昆虫学调查是一个重要组成部分。然而，由于在控制成功的地区，病媒种群中的感染是相当罕见的，因此需要对大量苍蝇进行检查，以证明传播中断。目前，这是通过PCR池筛选大量的苍蝇。该过程的效率受到可以筛选的池的大小的限制，而池的大小又由测定的生物化学施加的约束决定。目前从媒介黑蝇池中纯化DNA的方法依赖于二氧化硅吸附。该方法可用于筛选最多含有50个个体（来自拉丁美洲媒介）或100个个体（来自非洲媒介）的样本库。我们已经评估了另一种方法的DNA纯化池筛选黑蝇依赖于寡核苷酸捕获盘尾丝虫基因组DNA制备的匀浆从池的拉丁美洲和非洲的载体。寡核苷酸捕获试验显示可靠地检测一个O。在含有200只非洲或拉丁美洲苍蝇的池中检测肠扭转感染性幼虫，比常规测定提高了2 - 4倍。捕获测定需要与常规测定等量的技术时间来进行，导致测定的每只昆虫的劳动力成本降低2 - 4倍，并且将试剂成本降低至每200只苍蝇的池3.81美元，或每只测定的昆虫低于0.02美元。寡核苷酸捕获试验是用于检测媒介人群中寄生虫流行率的程序的重大改进，这是证明消除盘尾丝虫病过程中采用的一个主要指标。在盘尾丝虫的黑蝇媒介中不存在盘尾丝虫的感染性幼虫是用于证明在一个焦点中传播已被消除的一个主要标准。这个过程需要筛选大量的苍蝇。目前，这是通过使用基于PCR的测定筛选苍蝇库来实现的。由于DNA纯化过程的限制，目前每个库中可能包括的蝇类数量为拉丁美洲病媒50只，非洲病媒100只。在这里，我们描述了一种新的DNA纯化方法，该方法依赖于一种特定的寡核苷酸来捕获和吸附磁珠上的寄生虫DNA。该方法能可靠地检测单个感染性O.在含有多达200只苍蝇的水池中扭转。本文所述的方法将大大提高病媒黑蝇池筛选的效率，使消除认证过程更容易实施，成本更低。
Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors). We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed. The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis. The absence of infective larvae of Onchocerca volvulus in the black fly vector of this parasite is a major criterion used to certify that transmission has been eliminated in a focus. This process requires screening large numbers of flies. Currently, this is accomplished by screening pools of flies using a PCR-based assay. The number of flies that may be included in each pool is currently limited by the DNA purification process to 50 flies for Latin American vectors and 100 flies for African vectors. Here, we describe a new method for DNA purification that relies upon a specific oligonucleotide to capture and immobilize the parasite DNA on a magnetic bead. This method permits the reliable detection of a single infective larva of O. volvulus in pools containing up to 200 individual flies. The method described here will dramatically improve the efficiency of pool screening of vector black flies, making the process of elimination certification easier and less expensive to implement.
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影响因子：
4.4
作者：
Foddai, Antonio;Elliott, Christopher T.;Grant, Irene R.
通讯作者：
Grant, Irene R.
DOI：
10.1016/j.diagmicrobio.2010.09.022
发表时间：
2011-03-01
影响因子：
2.9
作者：
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Bang, Dang Duong
DOI：
10.4269/ajtmh.2003.68.222
发表时间：
2003-02-01
影响因子：
3.3
作者：
Guevara, AG;Vieira, JC;Unnasch, TR
通讯作者：
Unnasch, TR
DOI：
10.4269/ajtmh.1994.51.676
发表时间：
1994-11-01
影响因子：
3.3
作者：
TOE, L;MERRIWEATHER, A;UNNASCH, TR
通讯作者：
UNNASCH, TR
DOI：
10.4269/ajtmh.2006.74.779
发表时间：
2006-05-01
影响因子：
3.3
作者：
Katholi, Charles R.;Unnasch, Thomas R.
通讯作者：
Unnasch, Thomas R.