One-Step Reverse Transcription-Polymerase Chain Reaction for Ebola and Marburg Viruses

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• 作者：Sun-Whan Park ; Ye-Ji Lee ; Won-Ja Lee ; Youngmee Jee ; WooYoung Choi ; ^{wychoi65@korea.kr" class="auth_mail" title="E-mail the corresponding author}
• 关键词：Ebola ; Filoviridae ; Marburg ; reverse transcription-polymerase chain reaction
• 刊名：Osong Public Health and Research Perspectives
• 出版年：2016
• 出版时间：June 2016
• 年：2016
• 卷：7
• 期：3
• 页码：205-209
• 全文大小：798 K

文摘

Ebola and Marburg viruses (EBOVs and MARVs, respectively) are causative agents of severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. In 2014, there was a major Ebola outbreak in various countries in West Africa, including Guinea, Liberia, Republic of Sierra Leone, and Nigeria. EBOV and MARV are clinically difficult to diagnose and distinguish from other African epidemic diseases. Therefore, in this study, we aimed to develop a method for rapid identification of the virus to prevent the spread of infection.

Methods

We established a conventional one-step reverse transcription-polymerase chain reaction (RT-PCR) assay for these pathogens based on the Superscript Reverse Transcriptase-Platinum Taq polymerase enzyme mixture. All assays were thoroughly optimized using in vitro-transcribed RNA.

Results

We designed seven primer sets of nucleocapsid protein (NP) genes based on sequences from seven filoviruses, including five EBOVs and two MARVs. To evaluate the sensitivity of the RT-PCR assay for each filovirus, 10-fold serial dilutions of synthetic viral RNA transcripts of EBOV or MARV NP genes were used to assess detection limits of viral RNA copies. The potential for these primers to cross react with other filoviruses was also examined. The results showed that the primers were specific for individual genotype detection in the examined filoviruses.

Conclusion

The assay established in this study may facilitate rapid, reliable laboratory diagnosis in suspected cases of Ebola and Marburg hemorrhagic fevers.