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实时SYBR Green登革热检测法与实时TaqMan RT-PCR登革热检测法及传统巢式PCR用于诊断原发性和继发性登革热感染的比较。

Comparison of real-time SYBR green dengue assay with real-time taqman RT-PCR dengue assay and the conventional nested PCR for diagnosis of primary and secondary dengue infection.

作者信息

Paudel Damodar, Jarman Richard, Limkittikul Kriengsak, Klungthong Chonticha, Chamnanchanunt Supat, Nisalak Ananda, Gibbons Robert, Chokejindachai Watcharee

机构信息

Department of Internal Medicine Nepal Police Hospital, Maharagjung, Kathmandu, Nepal.

出版信息

N Am J Med Sci. 2011 Oct;3(10):478-85. doi: 10.4297/najms.2011.3478.

DOI:10.4297/najms.2011.3478
PMID:22363089
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3271430/
Abstract

BACKGROUND

Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman(®) assay and conventional nested PCR assay.

AIMS

To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti).

MATERIALS AND METHODS

Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR(®) Green assay, real time Taqman(®) assay to compare the sensitivity and specificity.

RESULTS

Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR(®) green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay.

CONCLUSION

We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus.

摘要

背景

登革热和登革出血热由登革病毒引起。登革热感染仍是许多国家的一个紧迫问题。为了在早期诊断急性登革热，我们改进了低成本、快速的SYBR Green实时检测方法，并将其敏感性和特异性与实时Taqman检测方法和传统巢式PCR检测方法进行比较。

目的

开发低成本、快速且可靠的实时SYBR Green登革热诊断检测方法，并与Taqman实时检测方法和传统巢式PCR（改良的Lanciotti法）进行比较。

材料与方法

将8种培养的病毒株以十倍稀释至PCR检测不到的水平，以优化引物、温度（退火和延伸）并检测该检测方法的检测限。用实时SYBR Green检测方法、实时Taqman检测方法对193份经ELISA和PCR证实的登革热临床样本进行检测，以比较敏感性和特异性。

结果

实时SYBR Green登革热检测方法的敏感性和特异性（分别为84%和66%）与Taqman实时PCR登革热检测方法的敏感性和特异性（81%和74%）几乎相当。实时SYBR Green RT-PCR在初次感染和二次感染中同样敏感，而实时Taqman在二次感染中敏感性较低。实时Taqman对DENV3的敏感性（87%）与SYBR Green实时PCR登革热检测方法相同。

结论

我们开发了低成本快速诊断的SYBR Green登革热检测方法。需要进一步研究以制备用于登革病毒血清分型的双重引物检测方法。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c9/3271430/61282bf653f7/NAJMS-3-478-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c9/3271430/c9fb90d77ccf/NAJMS-3-478-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c9/3271430/61282bf653f7/NAJMS-3-478-g006.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c9/3271430/c9fb90d77ccf/NAJMS-3-478-g005.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/19c9/3271430/61282bf653f7/NAJMS-3-478-g006.jpg

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