| Viral infectious diseases have long been the focus of global public health. There are no effective vaccines and drugs for the most of viral infectious diseases. Therefore, establishment of accurate, rapid and reliable detection methods has become more and more crucial for the prevention and control of infectious diseases. Ebola viruses are the causative agents of Ebola hemorrhagic fever (EHF) which can cause bleeding symptoms. The 2014 Ebola virus (EBOV) outbreak was the largest to date in West African countries. Currently, nucleic acid-based detection methods can be classified into two categories:temperature-switch nucleic acid amplification and isothermal nucleic acid amplification techniques.The temperature-switch nucleic acid amplification assays such as polymerase chain reaction (PCR)-based assays have been used widespread. However, those PCR-based assays need expensive and fine instruments as well as long reaction time. In comparison, isothermal nucleic acid amplification techniques such as loop mediated isothermal amplification (LAMP) and isothermal multiple-self-matching-initiated amplification (IMSA) reveal the characteristics of rapidity, sensitivity, high efficiency and high specificity. They have the ability to be performed with simple equipment and are more applicable to primary clinical settings.In the first part of the present study, we established two isothermal multiple-self-matching-initiated amplification (IMSA) assays targeting nuclear protein (NP) gene and Glycoprotein (GP) gene of Zaire Ebola virus (ZEBOV) respectively, and compared in parallel with reverse transcription quantitative polymerase chain reaction (RT-qPCR) assays. The isothermal amplification assays employed six primers and were carried out at 63 ℃, the products were detected by visual inspection of color change or by collecting fluorescence. The specificities of the assays were validated by detecting dengue virus and Japanese encephalitis virus and human plasma and no cross-reactivity was observed. Within vitro transcribed RNA and virus like particles containing the ZEBOV NP gene and GP gene as templates, the sensitivity of each assay was evaluated and compared to RT-qPCR assay proposed by Li Aqian. The results showed that the real time RT-IMSA (NP) and RT-qPCR (NP) had equal sensitivity (103 copies/reaction) while the sensitivity of RT-IMSA (GP) and RT-qPCR (GP) were both 102 copies/reaction. The RT-IMSA (NP) was further evaluated and in parallel compared to the commercial RT-qPCR kit in detection of the Ebola suspected clinical blood samples in Sierra Leone. The clinical performance demonstrated the sensitivity and specificity of the RT-IMSA was 91.4% and 100%, respectively. Therefore, the established isothermal amplification methods have the potential to be widely adopted as platforms for rapid detection of EBOV infections and are suitable to be used in remote areas.In the second part, we explored whether parallel complementary DNA amplification could happen as proposed in two published papers. First, we repeated exactly the experimental protocols as described in those two papers, then set up additional experiments to further test whether the base pairing of parallel complementary DNA could be formed. We conclude that parallel complementary DNA amplification is impossible under either isothermal working condition or conventional PCR condition.In conclusion, isothermal multiple-self-matching-initiated amplification (IMSA) assays-RT-IMSA(NP) and RT-IMSA(GP) are established to detect the Ebola virus. IMSA assays provide rapid and effective methods for the detection of EBOV and are important alternatives to RT-qPCR assays in preparation to deal with possible outbreaks in China. In addition, we attempt to testify the possibility of the parallel complementary DNA amplification and provide direct evidence that parallel complementary DNA amplification is unlikely happen under our experimental protocols. |
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