Quantitative detection of Schistosoma japonicum cercariae in water by real-time PCR.
Quantitative detection of Schistosoma japonicum cercariae in water by real-time PCR.
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DOI：
10.1371/journal.pntd.0000337
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发表时间：
2008
影响因子：
3.8
通讯作者：
Remais, Justin
中科院分区：
文献类型：
--
作者：
Hung, Yuen Wai;Remais, Justin
仅在中国，估计就有 3000 万人面临由日本血吸虫寄生虫引起的血吸虫病的危险。疾病在先前已实现传播控制的几个地区重新出现，加强了主动监测的必要性。众所周知，寄生虫的环境阶段表现出高度的空间和时间变异性，当前的检测技术依赖于哨兵小鼠方法，该方法在获取时间和空间数据方面存在严重限制。在这里，我们描述了一种实时 PCR 测定法，用于定量检测实验室样品和已添加已知数量的日本血吸虫的天然水中的日本血吸虫尾蚴。设计并评估了多个引物，并使用性能最佳的引物组和 TaqMan 探针来定量日本血吸虫。由此产生的检测是选择性的，没有检测到曼氏血吸虫、埃及血吸虫、禽类血吸虫或非地方性地表水样本中存在的生物体的扩增。含有不同浓度日本血吸虫尾蚴的重复样品表明，实时PCR方法与光镜计数具有很强的线性相关性（R2 = 0.921），检测限低于半个尾蚴的DNA当量。将各种尾蚴浓度添加到 1 升天然水中，然后进行过滤，93% 的分析样品均呈阳性检测。实时 PCR 方法在量化各种加标样品的相对浓度方面表现良好，尽管绝对浓度估计值在重复样品之间表现出很大的差异。总体而言，该方法有潜力应用于环境水样，以对流行地区的尾蚴位置进行快速、可靠的测定。就感染人数和高危人数而言，血吸虫病在寄生虫病中仅次于疟疾。日本血吸虫是在中国、菲律宾以及印度尼西亚造成人类和动物疾病的物种。最近中国血吸虫病重新出现的证据表明，在这些地区进行积极的疾病监测的必要性。血吸虫病感染是通过接触被日本血吸虫尾蚴污染的水而发生的，日本血吸虫尾蚴是中间宿主蜗牛的寄生虫的自由生活阶段。目前检测环境中尾蚴的做法是使用哨兵小鼠，这种方法有严重的局限性，其中小鼠被暴露在环境水中，然后被维持六周，然后被解剖以计数蠕虫。该方法是劳动密集型的，并且在时间和资源方面成本高昂，因此在后勤上无法定期或全面监测水接触点。在这里，我们开发了一种定量 PCR 测定法来测量水中日本血吸虫尾蚴的浓度，为现场快速可靠的数据收集提供了一种潜在的方法，有可能取代活体动物模型的使用。
In China alone, an estimated 30 million people are at risk of schistosomiasis, caused by the Schistosoma japonicum parasite. Disease has re-emerged in several regions that had previously attained transmission control, reinforcing the need for active surveillance. The environmental stage of the parasite is known to exhibit high spatial and temporal variability, and current detection techniques rely on a sentinel mouse method which has serious limitations in obtaining data in both time and space. Here we describe a real-time PCR assay to quantitatively detect S. japonicum cercariae in laboratory samples and in natural water that has been spiked with known numbers of S. japonicum. Multiple primers were designed and assessed, and the best performing set, along with a TaqMan probe, was used to quantify S. japonicum. The resulting assay was selective, with no amplification detected for Schistosoma mansoni, Schistosoma haematobium, avian schistosomes nor organisms present in non-endemic surface water samples. Repeated samples containing various concentrations of S. japonicum cercariae showed that the real-time PCR method had a strong linear correlation (R2 = 0.921) with light microscopy counts, and the detection limit was below the DNA equivalent of half of one cercaria. Various cercarial concentrations spiked in 1 liter of natural water followed by a filtration process produced positive detection from 93% of samples analyzed. The real-time PCR method performed well quantifying the relative concentrations of various spiked samples, although the absolute concentration estimates exhibited high variance across replicated samples. Overall, the method has the potential to be applied to environmental water samples to produce a rapid, reliable assay for cercarial location in endemic areas. Schistosomiasis ranks second only to malaria among parasitic diseases with regard to the number of people infected and those at risk. Schistosoma japonicum is the species that causes human and animal disease in China, the Philippines, and to a lesser extent, Indonesia. Recent evidence of schistosomiasis re-emergence in China has reinforced the need for active disease surveillance in these areas. Schistosomiasis infection occurs through contact with water contaminated with S. japonicum cercariae, the free-living stage of the parasite shed from intermediate host snails. Current practice of detecting cercariae in the environment uses sentinel mice, a method with serious limitations in which mice are exposed to environmental water and then maintained for 6 weeks before being dissected to count worms. The method is labor intensive and costly in terms of time and resources, making it logistically prohibitive to monitor water contact sites regularly or comprehensively. Here we develop a quantitative PCR assay to measure S. japonicum cercariae concentration in water, providing a potential method for rapid and reliable data collection in the field, potentially replacing the use of live animal models.
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DOI：
10.4269/ajtmh.2004.71.554
发表时间：
2004-11-01
影响因子：
3.3
作者：
Spear, RC;Zhong, B;Qiu, DC
通讯作者：
Qiu, DC
DOI：
10.1590/s0074-02762006000900021
发表时间：
2006-10-01
期刊：
Memórias do Instituto Oswaldo Cruz
影响因子：
--
作者：
Gomes, Ana Lisa do Vale;Melo, Fábio L;Abath, Frederico GC
通讯作者：
Abath, Frederico GC
影响因子：
1.4
作者：
Hallett, Sascha L.;Bartholomew, Jerri L.
通讯作者：
Bartholomew, Jerri L.
影响因子：
1.3
作者：
Hanelt, B;Adema, CM;Loker, ES
通讯作者：
Loker, ES
影响因子：
168.9
作者：
King, CH;Dickman, K;Tisch, DJ
通讯作者：
Tisch, DJ