A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction.
A simple and rapid method for detection of Trypanosoma evansi in the dromedary camel using a nested polymerase chain reaction.
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DOI：
10.1186/1475-9292-5-2
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发表时间：
2006-05-20
期刊：
影响因子：
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通讯作者：
Majid, Ali A
中科院分区：
文献类型：
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作者：
Aradaib, Imadeldin E;Majid, Ali A
建立了一种基于巢式聚合酶链反应（nPCR）的检测方法，并对该方法用于实验感染小鼠和自然感染骆驼（骆驼）中伊氏锥虫的快速检测进行了评价。从T. evansi），并用于PCR扩增。第一次扩增，使用一对外部引物TE 1和TE 2，从T.伊文思的DNA第二次扩增，使用巢式（内部）引物对TE 3和TE 4，产生270-bp PCR产物。t.从实验感染的小鼠和自然感染的苏丹单峰骆驼品种的血液样品中提取的伊氏病毒DNA通过这种基于巢式PCR的测定进行检测。巢式引物TE 3和TE 4提高了PCR检测的灵敏度，且T.扩增伊氏病毒DNA（相当于寄生虫推定基因的单拷贝）并在溴化乙锭染色的琼脂糖凝胶上显现。当基于PCR的测定应用于来自其他血液寄生虫（包括环形梭线虫、二联Babeleria bigemina或无核酸样品）的DNA时，未检测到扩增产物。将这种基于nPCR的检测方法应用于临床样本，可直接检测T。从实验感染的小鼠和自然感染的骆驼的血液中收集的各种组织样品中提取伊文思。所描述的基于nPCR的检测方法为研究T.骆驼和其他易感动物群体中的伊氏病毒感染。
A nested polymerase chain reaction (nPCR)-based assay, was developed and evaluated for rapid detection of Trypanosoma evansi in experimentally infected mice and naturally infected camels (Camelus dromedarius). Four oligonucleotide primers (TE1, TE2, TE3 and TE4), selected from nuclear repetitive gene of T. evansi, were designed and used for PCR amplifications. The first amplification, using a pair of outer primers TE1 and TE2, produced a 821-bp primary PCR product from T. evansi DNA. The second amplification, using nested (internal) pair of primers TE3 and TE4, produced a 270-bp PCR product. T. evansi DNAs extracted from blood samples of experimentally infected mice and naturally infected Sudanese breed of dromedary camels were detected by this nested PCR-based assay. The nested primers TE3 and TE4 increased the sensitivity of the PCR assay and as little as 10 fg of T. evansi DNA (equivalent to a single copy of the putative gene of the parasite) was amplified and visualized onto ethidium bromide-stained agarose gels. Amplification products were not detected when the PCR-based assay was applied to DNA from other blood parasites including Thieleria annulata, Babesia bigemina or nucleic acid free samples. Application of this nPCR-based assay to clinical samples resulted in direct detection of T. evansi from a variety of tissue samples collected from experimentally infected mice and blood from naturally infected camels. The described nPCR-based assay provides a valuable tool to study the epidemiology of T. evansi infection in camels and other susceptible animal populations.