A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen, Norway.
A novel, single-amplification PCR targeting mitochondrial genome highly sensitive and specific in diagnosing malaria among returned travellers in Bergen, Norway.
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一种针对线粒体基因组的新型单扩增 PCR 在诊断挪威卑尔根回国旅行者的疟疾方面具有高度敏感性和特异性。
DOI：
10.1186/1475-2875-12-26
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发表时间：
2013-01-22
期刊：
影响因子：
3
通讯作者：
Hanevik K
中科院分区：
文献类型：
--
作者：
Haanshuus CG;Mohn SC;Mørch K;Langeland N;Blomberg B;Hanevik K
套式聚合酶链式反应具有较高的敏感性和特异性，是诊断疟疾的常用方法。然而，它很耗时，容易受到相当大的污染风险，而且成本效益低。使用存在于多个拷贝中的扩增靶标，如rRNA18S，或具有更高拷贝数的线粒体靶标，可能会提高敏感性。根据已发表的针对18S和线粒体基因组的引物，设计了两种新设计的针对疟原虫属特异性单轮扩增的PCR程序，并与广泛使用的套式18S PCR程序进行了比较。对132例发热疑似输入性疟疾患者进行了恶性疟原虫标准物质稀释系列分析，并对常规镜检135份血样进行了回顾性分析。对220bp的线粒体聚合酶链式反应产物进行测序。在阈值稀释度为0.5Parasites/μ时，线粒体法的敏感性为97%(29/30个平行)，单轮18S法为93%，参照套式18S法为87%。三种检测方法的检测结果都低至0.05p/μL，但并不一致。在患者队列中，21%(28/135)的样本被诊断为疟疾，至少有两种方法定义为阳性。两种单轮扩增方法均能检出套式聚合酶链式反应阳性的疟疾患者，其敏感性为96%(27/28)。线粒体聚合酶链式反应检测到另一例标本，镜检也呈阳性，是唯一一种具有100%敏感性的方法(28/28)。线粒体聚合酶链式反应的敏感性和特异性在统计学上不逊于参考套式聚合酶链式反应。显微镜检查漏掉了所有聚合酶链式反应检测到的两种感染。对属特有的线粒体聚合酶链式反应产物进行测序，发现了不同的单核苷酸多态性，这使得物种鉴定的28个序列如下：20个恶性疟原虫，6个间日疟原虫，1个卵形疟原虫和1个疟疾原虫。在本研究中，通过设计合适的参数和优化的聚合酶链式反应程序，可以得到更简单、更快速的单轮扩增分析。新的线粒体聚合酶链式反应的灵敏度和特异度均为100%，且不逊于参考的套式聚合酶链式反应。属特异性线粒体聚合酶链式反应产物的测序可用于物种鉴定。
Nested PCR is a commonly used technique in diagnosis of malaria owing to its high sensitivity and specificity. However, it is time-consuming, open to considerable risk of contamination and has low cost-efficiency. Using amplification targets presented in multiple copies, such as rRNA 18S, or mitochondrial targets with an even higher copy number, might increase sensitivity. The sensitivity and specificity of two newly designed Plasmodium genus-specific single-round amplification PCR programmes, based on previously published primers targeting 18S and mitochondrial genome, were compared with a widely used nested 18S PCR. Analyses of dilution series from Plasmodium falciparum reference material were performed, as well as retrospective analyses of 135 blood samples, evaluated by routine microscopy, from 132 fever patients with potential imported malaria. Sequencing of the 220 bp mitochondrial PCR products was performed. At the threshold dilution 0.5 parasites/μl, the sensitivity of the mitochondrial PCR was 97% (29/30 parallels), that of the single-round 18S PCR 93% and the reference nested 18S PCR 87%. All three assays detected as low as 0.05 p/μl, though not consistently. In the patient cohort, malaria was diagnosed in 21% (28/135) samples, defined as positive by at least two methods. Both single-round amplification assays identified all malaria positives diagnosed by nested PCR that had sensitivity of 96% (27/28). The mitochondrial PCR detected one additional sample, also positive by microscopy, and was the only method with 100% sensitivity (28/28). The sensitivity and specificity of the mitochondrial PCR were statistically non-inferior to that of the reference nested PCR. Microscopy missed two infections detected by all PCR assays. Sequencing of the genus-specific mitochondrial PCR products revealed different single nucleotide polymorphisms which allowed species identification of the 28 sequences with following distribution; 20 P. falciparum, six Plasmodium vivax, one Plasmodium ovale and one Plasmodium malariae. In this study, design of PCR programmes with suitable parameters and optimization resulted in simpler and faster single-round amplification assays. Both sensitivity and specificity of the novel mitochondrial PCR was 100% and proved non-inferior to that of the reference nested PCR. Sequencing of genus-specific mitochondrial PCR products could be used for species determination.
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影响因子：
2.9
作者：
Berry, A.;Benoit-Vical, F.;Magnaval, J. F.
通讯作者：
Magnaval, J. F.
影响因子：
64.8
作者：
SMALLEY, ME
通讯作者：
SMALLEY, ME
影响因子：
9.4
作者：
Polley, Spencer D.;Mori, Yasuyoshi;Sutherland, Colin J.
通讯作者：
Sutherland, Colin J.
影响因子：
14.2
作者：
H채nscheid, T
通讯作者：
H채nscheid, T
影响因子：
9.4
作者：
Ndao, M;Bandyayera, E;Ward, BJ
通讯作者：
Ward, BJ