Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR.
Rapid diagnostic tests as a source of DNA for Plasmodium species-specific real-time PCR.
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DOI：
10.1186/1475-2875-10-67
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发表时间：
2011-03-24
期刊：
影响因子：
3
通讯作者：
Jacobs J
中科院分区：
文献类型：
--
作者：
Cnops L;Boderie M;Gillet P;Van Esbroeck M;Jacobs J
本研究描述了使用疟疾快速诊断测试（RDTs）作为疟原虫种特异性实时PCR的DNA来源。首先，从RDT回收DNA的最佳方法进行了研究，然后这种DNA提取方法的适用性进行了评估，对12个不同的RDT品牌。最后，两个RDT品牌（OptiMAL快速疟疾测试和SDFK 60疟疾Ag恶性疟原虫/潘测试）在一组提交给ITM进行常规疟疾诊断的临床样本上进行了全面评估。用18 S rRNA实时PCR进行DNA扩增，靶向四种疟原虫。将RDT的PCR结果与全血样品的PCR结果进行比较。通过用简单的DNA洗脱方法从RDT条的硝酸纤维素组分的近端部分分离DNA获得最佳结果。RDT的PCR检测限为0.02个/μl，与全血PCR检测结果一致。对于测试的所有12个RDT品牌，除了一个品牌在应用低寄生虫密度样本时检测到DNA。在用塑料密封覆盖硝化纤维素条的RDT中，DNA提取受到阻碍。对临床RDT样本的PCR分析表明，对所有RDT样本的恶性疟原虫（n = 60）、间日疟原虫（n = 10）、卵形疟原虫（n = 10）和三日疟原虫（n = 10）的无性寄生虫的单一种属感染的鉴定是正确的。在所有OptiMAL和11次SDFK 60检测中的10次中均检测到仅含配子母细胞的样本。阴性样本（n = 20）在RDT上通过PCR均未给出信号。采用RDT PCR，观察到的Ct值高于全血PCR，OptiMAL和SDFK 60的平均差异分别为2.68和3.53。在4/5次OptiMAL检测和2/5次SDFK 60检测中，通过RDT上的PCR正确鉴定了混合感染。RDT是疟原虫实时PCR的可靠DNA来源。这项研究证明了最好的方法，RDT片段取样的广泛的RDT品牌结合一个简单的和低成本的提取方法，允许RDT质量控制。
This study describes the use of malaria rapid diagnostic tests (RDTs) as a source of DNA for Plasmodium species-specific real-time PCR. First, the best method to recover DNA from RDTs was investigated and then the applicability of this DNA extraction method was assessed on 12 different RDT brands. Finally, two RDT brands (OptiMAL Rapid Malaria Test and SDFK60 malaria Ag Plasmodium falciparum/Pan test) were comprehensively evaluated on a panel of clinical samples submitted for routine malaria diagnosis at ITM. DNA amplification was done with the 18S rRNA real-time PCR targeting the four Plasmodium species. Results of PCR on RDT were compared to those obtained by PCR on whole blood samples. Best results were obtained by isolating DNA from the proximal part of the nitrocellulose component of the RDT strip with a simple DNA elution method. The PCR on RDT showed a detection limit of 0.02 asexual parasites/μl, which was identical to the same PCR on whole blood. For all 12 RDT brands tested, DNA was detected except for one brand when a low parasite density sample was applied. In RDTs with a plastic seal covering the nitrocellulose strip, DNA extraction was hampered. PCR analysis on clinical RDT samples demonstrated correct identification for single species infections for all RDT samples with asexual parasites of P. falciparum (n = 60), Plasmodium vivax (n = 10), Plasmodium ovale (n = 10) and Plasmodium malariae (n = 10). Samples with only gametocytes were detected in all OptiMAL and in 10 of the 11 SDFK60 tests. None of the negative samples (n = 20) gave a signal by PCR on RDT. With PCR on RDT, higher Ct-values were observed than with PCR on whole blood, with a mean difference of 2.68 for OptiMAL and 3.53 for SDFK60. Mixed infections were correctly identified with PCR on RDT in 4/5 OptiMAL tests and 2/5 SDFK60 tests. RDTs are a reliable source of DNA for Plasmodium real-time PCR. This study demonstrates the best method of RDT fragment sampling for a wide range of RDT brands in combination with a simple and low cost extraction method, allowing RDT quality control.
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DOI：
10.4269/ajtmh.2005.73.850
发表时间：
2005-11-01
影响因子：
3.3
作者：
Swan, H;Sloan, L;Rosenblatt, J
通讯作者：
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影响因子：
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作者：
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Jacobs J