Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis.
Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis.
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DOI：
10.1186/1475-2875-13-99
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发表时间：
2014-03-15
期刊：
影响因子：
3
通讯作者：
von Nickisch-Rosenegk M
中科院分区：
文献类型：
--
作者：
Kersting S;Rausch V;Bier FF;von Nickisch-Rosenegk M
核酸扩增是目前检测恶性疟原虫最敏感、特异的方法。然而，聚合酶链反应仍然是基于实验室的，必须由受过训练的人员进行。此外，热循环过程的功率依赖性和读出所需的昂贵设备在资源有限的环境中难以覆盖。本研究的目的是开发和评估一种结合等温核酸扩增和简单的侧向流试纸检测疟疾寄生虫的即时检测。采用等温重组酶聚合酶扩增（RPA）法，在38°C恒温条件下10 min内扩增出恶性疟原虫18 S rRNA基因的特异性片段。通过将独特的探针系统添加到反应溶液中，扩增产物可以在简单的横向流动条上可视化，而无需进一步标记。使用各种疟原虫和其他原生动物/细菌菌株以及人类DNA测试了这些方法组合的灵敏度和特异性。进行了其他研究，以分析该试验的最佳温度、反应速度和耐用性。侧流RPA（LF-RPA）检测具有较高的灵敏度和特异性。实验证实，恶性疟原虫基因组DNA的检测限低至100 fg，相当于每个反应约4个寄生虫的灵敏度。所有被调查的恶性疟原虫菌株（n = 77）均检测为阳性，而所有11份非疟原虫样品的检测结果均为阴性。酶促反应可以在30-45°C的宽范围条件下进行，具有高抑制浓度的已知PCR抑制剂。确定从反应开始到读出的15分钟的结果时间。结合等温RPA和侧流检测是一种在资源有限的环境中提高恶性疟原虫分子诊断的方法。该系统不需要或仅需要很少的用于核酸扩增反应的仪器，并且可以用肉眼读出。该方法显示出与可比诊断方法相同的灵敏度和特异性，但同时提高了反应速度并大大降低了检测要求，因此有可能成为疟疾寄生虫的真正即时检测方法。
Nucleic acid amplification is the most sensitive and specific method to detect Plasmodium falciparum. However the polymerase chain reaction remains laboratory-based and has to be conducted by trained personnel. Furthermore, the power dependency for the thermocycling process and the costly equipment necessary for the read-out are difficult to cover in resource-limited settings. This study aims to develop and evaluate a combination of isothermal nucleic acid amplification and simple lateral flow dipstick detection of the malaria parasite for point-of-care testing. A specific fragment of the 18S rRNA gene of P. falciparum was amplified in 10 min at a constant 38°C using the isothermal recombinase polymerase amplification (RPA) method. With a unique probe system added to the reaction solution, the amplification product can be visualized on a simple lateral flow strip without further labelling. The combination of these methods was tested for sensitivity and specificity with various Plasmodium and other protozoa/bacterial strains, as well as with human DNA. Additional investigations were conducted to analyse the temperature optimum, reaction speed and robustness of this assay. The lateral flow RPA (LF-RPA) assay exhibited a high sensitivity and specificity. Experiments confirmed a detection limit as low as 100 fg of genomic P. falciparum DNA, corresponding to a sensitivity of approximately four parasites per reaction. All investigated P. falciparum strains (n = 77) were positively tested while all of the total 11 non-Plasmodium samples, showed a negative test result. The enzymatic reaction can be conducted under a broad range of conditions from 30-45°C with high inhibitory concentration of known PCR inhibitors. A time to result of 15 min from start of the reaction to read-out was determined. Combining the isothermal RPA and the lateral flow detection is an approach to improve molecular diagnostic for P. falciparum in resource-limited settings. The system requires none or only little instrumentation for the nucleic acid amplification reaction and the read-out is possible with the naked eye. Showing the same sensitivity and specificity as comparable diagnostic methods but simultaneously increasing reaction speed and dramatically reducing assay requirements, the method has potential to become a true point-of-care test for the malaria parasite.
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影响因子：
4.1
作者：
Krolov, Katrin;Frolova, Jekaterina;Langel, Olo, I
通讯作者：
Langel, Olo, I
影响因子：
7.4
作者：
Shen, Feng;Davydova, Elena K.;Du, Wenbin;Kreutz, Jason E.;Piepenburg, Olaf;Ismagilov, Rustem F.
通讯作者：
Ismagilov, Rustem F.
影响因子：
1.5
作者：
SNOUNOU, G;VIRIYAKOSOL, S;BROWN, KN
通讯作者：
BROWN, KN
DOI：
10.4269/ajtmh.1999.60.183
发表时间：
1999-02-01
影响因子：
3.3
作者：
Rubio, JM;Benito, A;Alvar, J
通讯作者：
Alvar, J
影响因子：
9.4
作者：
Polley, Spencer D.;Mori, Yasuyoshi;Sutherland, Colin J.
通讯作者：
Sutherland, Colin J.