Development of real time PCR for detection and quantitation of Dengue Viruses
Development of real time PCR for detection and quantitation of Dengue Viruses
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DOI：
10.1186/1743-422x-6-10
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发表时间：
2009-01-23
期刊：
影响因子：
4.8
通讯作者：
Cecilia, D.
中科院分区：
文献类型：
--
作者：
Gurukumar, K. R.;Priyadarshini, D.;Cecilia, D.
背景资料：登革病毒（DENV）是一种蚊媒黄病毒，是一种重要的病原体，每年在全世界引起5000多万人感染。登革热的诊断依赖于血清学，而血清学在疾病的早期阶段和病毒分离中没有用处，而病毒分离是费力和耗时的。需要一种快速、灵敏和高通量的方法来在疾病的早期阶段检测DENV。已经描述了几种用于登革热病毒的实时PCR测定，但仍有改进的余地。本研究采用新一代TaqMan小沟结合（MGB）探针技术，建立了一种改良的登革病毒（DENV）真实的实时定量RT-PCR（qRT-PCR）方法。在所有四种血清型中保守的区域用于靶向qRT-PCR的引物和探针。设计了一个单一的MGB探针和一个单一的引物对所有四种血清型的登革病毒。两步qRT-PCR测定的灵敏度为每个反应10个拷贝的RNA分子。当用一组39份已知阳性和阴性样本进行检测时，该检测试剂盒的特异性和灵敏度为100%。病毒RNA可以在感染的小鼠脑、细胞培养物、蚊子和临床样品中检测和定量。甚至在血清转换后直到感染后10天，在患者中也可以检测到病毒RNA。结论：建立了一种高灵敏度、高特异性的登革病毒qRT-PCR检测方法。该检测方法将是一个有用的工具，用于鉴别诊断登革热的情况下，一些其他临床上难以区分的传染病，如疟疾，基孔肯雅热，立克次体和钩端螺旋体的发生。该测定法检测接种蚊子中的DENV-2的能力使其成为检测现场捕获的蚊子中的DENV的潜在工具。
Background: Dengue virus (DENV), a mosquito borne flavivirus is an important pathogen causing more than 50 million infections every year around the world. Dengue diagnosis depends on serology, which is not useful in the early phase of the disease and virus isolation, which is laborious and time consuming. There is need for a rapid, sensitive and high throughput method for detection of DENV in the early stages of the disease. Several real-time PCR assays have been described for dengue viruses, but there is scope for improvement. The new generation TaqMan Minor Groove Binding (MGB) probe approach was used to develop an improved real time RT-PCR (qRT-PCR) for DENV in this study.Results: The 3'UTR of thirteen Indian strains of DENV was sequenced and aligned with 41 representative sequences from GenBank. A region conserved in all four serotypes was used to target primers and probes for the qRT-PCR. A single MGB probe and a single primer pair for all the four serotypes of DENV were designed. The sensitivity of the two step qRT-PCR assay was 10 copies of RNA molecules per reaction. The specificity and sensitivity of the assay was 100% when tested with a panel of 39 known positive and negative samples. Viral RNA could be detected and quantitated in infected mouse brain, cell cultures, mosquitoes and clinical samples. Viral RNA could be detected in patients even after seroconversion till 10 days post onset of infection. There was no signal with Japanese Encephalitis (JE), West Nile (WN), Chikungunya (CHK) viruses or with Leptospira, Plasmodium vivax, Plasmodium falciparum and Rickettsia positive clinical samples.Conclusion: We have developed a highly sensitive and specific qRT-PCR for detection and quantitation of dengue viruses. The assay will be a useful tool for differential diagnosis of dengue fever in a situation where a number of other clinically indistinguishable infectious diseases like malaria, Chikungunya, rickettsia and leptospira occur. The ability of the assay to detect DENV-2 in inoculated mosquitoes makes it a potential tool for detecting DENV in field-caught mosquitoes.