Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti.
Sensitive multiplex PCR assay to differentiate Lyme spirochetes and emerging pathogens Anaplasma phagocytophilum and Babesia microti.
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DOI：
10.1186/1471-2180-13-295
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发表时间：
2013-12-20
期刊：
影响因子：
4.2
通讯作者：
Parveen N
中科院分区：
文献类型：
--
作者：
Chan K;Marras SA;Parveen N
伯氏疏螺旋体感染可导致急性到慢性莱姆病。此外，在美国和欧洲的流行地区，蜱传病原体、巴贝斯虫和嗜吞噬细胞无形体的共同感染一直在增加。目前使用的血清学诊断测试往往难以解释，此外，针对病原体的抗体存在很长时间，因此难以确认该疾病的治愈。此外，在适应性免疫反应建立之前，这些检测不能用于早期疾病状态的诊断。由于病原体的核酸在治愈后不存在，基于dna的诊断测试在检测传染病方面变得非常有用。在这项研究中，我们描述了一种实时多重PCR方法，可以同时检测伯氏疏螺旋体、微螺旋体和嗜吞噬细胞芽胞杆菌的存在，即使它们的拷贝数很低。有趣的是，这种定量PCR技术还能够通过PCR后变性谱分析和单分子信标探针来区分所有三种主要的莱姆病螺旋体，即伯氏螺旋体、阿夫泽利螺旋体和加里尼螺旋体。这对于在所有三种莱姆病螺旋体普遍存在的欧洲国家诊断和区分各种莱姆病螺旋体可能非常有用。作为对患者样本原理的证明，我们使用我们的检测方法检测到少量莱姆病螺旋体在人类血液中出现。最后，无论样品中每种病原体的含量如何，我们的多重检测方法都能以敏感和特定的方式检测出所有三种蜱传病原体。我们预计，这种新的诊断方法将能够同时诊断早期到慢性莱姆病，巴贝斯虫病和无形体病使用患者的血液样本。本研究所选扩增子采用特异性引物和分子信标探针进行实时定量PCR，可同时准确检测三种蜱传病原体。
The infection with Borrelia burgdorferi can result in acute to chronic Lyme disease. In addition, coinfection with tick-borne pathogens, Babesia species and Anaplasma phagocytophilum has been increasing in endemic regions of the USA and Europe. The currently used serological diagnostic tests are often difficult to interpret and, moreover, antibodies against the pathogens persist for a long time making it difficult to confirm the cure of the disease. In addition, these tests cannot be used for diagnosis of early disease state before the adaptive immune response is established. Since nucleic acids of the pathogens do not persist after the cure, DNA-based diagnostic tests are becoming highly useful for detecting infectious diseases. In this study, we describe a real-time multiplex PCR assay to detect the presence of B. burgdorferi, B. microti and A. phagocytophilum simultaneously even when they are present in very low copy numbers. Interestingly, this quantitative PCR technique is also able to differentiate all three major Lyme spirochete species, B. burgdorferi, B. afzelii, and B. garinii by utilizing a post-PCR denaturation profile analysis and a single molecular beacon probe. This could be very useful for diagnosis and discrimination of various Lyme spirochetes in European countries where all three Lyme spirochete species are prevalent. As proof of the principle for patient samples, we detected the presence of low number of Lyme spirochetes spiked in the human blood using our assay. Finally, our multiplex assay can detect all three tick-borne pathogens in a sensitive and specific manner irrespective of the level of each pathogen present in the sample. We anticipate that this novel diagnostic method will be able to simultaneously diagnose early to chronic stages of Lyme disease, babesiosis and anaplasmosis using the patients’ blood samples. Real-time quantitative PCR using specific primers and molecular beacon probes for the selected amplicon described in this study can detect three tick-borne pathogens simultaneously in an accurate manner.
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影响因子：
1.9
作者：
Franke, J.;Meier, F.;Hildebrandt, A.
通讯作者：
Hildebrandt, A.
影响因子：
3.7
作者：
Eshoo, Mark W.;Crowder, Christopher C.;Aucott, John N.
通讯作者：
Aucott, John N.
影响因子：
9.4
作者：
Banada, Padmapriya P.;Sivasubramani, Satheesh K.;Alland, David
通讯作者：
Alland, David
影响因子：
1
作者：
Chandrashekar, Ramaswamy;Mainville, Celine A.;Breitschwerdt, Edward B.
通讯作者：
Breitschwerdt, Edward B.
DOI：
10.1016/s0732-8893(03)00111-1
发表时间：
2003-09-01
影响因子：
2.9
作者：
Hernández-Novoa, B;Orduña, A;Rodríguez-Torres, A
通讯作者：
Rodríguez-Torres, A