A highly sensitive, PCR-based method for the detection of Plasmodium falciparum clones in microtiter plates.
A highly sensitive, PCR-based method for the detection of Plasmodium falciparum clones in microtiter plates.
复制标题
DOI：
10.1186/1475-2875-7-222
复制
发表时间：
2008-10-29
期刊：
影响因子：
3
通讯作者：
Adams JH
中科院分区：
文献类型：
--
作者：
Maher SP;Balu B;Shoue DA;Weissenbach ME;Adams JH
通过有限稀释克隆寄生虫是疟疾研究许多方面（包括基因组和基因操作研究）中必不可少的限速步骤。用于检测寄生虫的标准吉姆萨染色血涂片非常耗时，而更灵敏的寄生虫乳酸脱氢酶测定涉及多个步骤并且需要新鲜试剂。因此，测试了一种基于 PCR 的简单方法，用于寄生虫检测，该方法可适用于高通量研究。微量滴定克隆板每孔中约 1 μL 的浓缩红细胞直接用作模板 DNA，使用寄生虫 18s rRNA 基因的引物进行 PCR 反应。通过凝胶电泳快速分离 PCR 产物后，鉴定出含有寄生虫的阳性孔。基于PCR的方法可以一致地检测出低至0.0005%的寄生虫血症，相当于96孔板的单个孔中的30个寄生虫基因组。使用该方法可以很容易地从克隆板上检测到寄生虫克隆，并且将PCR结果与吉姆萨染色的血涂片进行比较表明，PCR不仅检测到涂片中识别出的所有阳性孔，而且还检测到了其他方式未识别出的孔，从而证实了其敏感性。本文报道的基于 PCR 的方法是一种简单、灵敏且有效的检测培养物中寄生虫克隆的方法。该方法需要很少的体力劳动，并且可以完全自动化以进行高通量研究。该方法足够灵敏，可以在吉姆萨涂片中看到寄生虫之前一周检测到寄生虫，并且在识别生长缓慢的寄生虫克隆方面非常有效。
Cloning of parasites by limiting dilution is an essential and rate-limiting step in many aspects of malaria research including genomic and genetic manipulation studies. The standard Giemsa-stained blood smears to detect parasites is time-consuming, whereas the more sensitive parasite lactate dehydrogenase assay involves multiple steps and requires fresh reagents. A simple PCR-based method was therefore tested for parasite detection that can be adapted to high throughput studies. Approximately 1 μL of packed erythrocytes from each well of a microtiter cloning plate was directly used as template DNA for a PCR reaction with primers for the parasite 18s rRNA gene. Positive wells containing parasites were identified after rapid separation of PCR products by gel electrophoresis. The PCR-based method can consistently detect a parasitaemia as low as 0.0005%, which is equivalent to 30 parasite genomes in a single well of a 96-well plate. Parasite clones were easily detected from cloning plates using this method and a comparison of PCR results with Giemsa-stained blood smears showed that PCR not only detected all the positive wells identified in smears, but also detected wells not identified otherwise, thereby confirming its sensitivity. The PCR-based method reported here is a simple, sensitive and efficient method for detecting parasite clones in culture. This method requires very little manual labor and can be completely automated for high throughput studies. The method is sensitive enough to detect parasites a week before they can be seen in Giemsa smears and is highly effective in identifying slow growing parasite clones.
登录
查看更多内容
DOI：
10.1073/pnas.0504679102
发表时间：
2005-11-08
影响因子：
11.1
作者：
Balu, B;Shoue, DA;Adams, JH
通讯作者：
Adams, JH
影响因子：
3.3
作者：
Ang, HH;Cheang, HS
通讯作者：
Cheang, HS
影响因子：
2.1
作者：
Goodyer, ID;Taraschi, TF
通讯作者：
Taraschi, TF
DOI：
10.1073/pnas.93.3.1130
发表时间：
1996-02-06
影响因子：
11.1
作者：
Wu, YM;Kirkman, LA;Wellems, TE
通讯作者：
Wellems, TE
DOI：
10.4269/ajtmh.1993.48.739
发表时间：
1993-06-01
影响因子：
3.3
作者：
MAKLER, MT;RIES, JM;HINRICHS, DJ
通讯作者：
HINRICHS, DJ