A Preliminary Study Of Random Amplification Based Methods For High-throughput Detection Of Hemorrhagic Fever Virus

Viral hemorrhagic fever (VHFs) is a group of acute infectious diseases with high case fatality rates up to 90%;the clinical symptoms of these life-threatening diseases are often accompanied by fever and bleeding,which leads to shock, multi-organ failure and other dangerous clinical process.With deepening of the globalization process,global warming and destruction of ecological environment, the imported outbreak and new virus infectious diseases may be rising sharply. So establishing a rapid, sensitive, specific and high flux detection method for hemorrhagic fever virus and timely conducting the viral sequencing play an important role in clinical treatment and epidemiological investigation. This study is to establish a fast accurate and high flux detection method for hemorrhagic fever virus.1 Preliminary study of specific purification and enrichment for hemorrhagic fever virus genome based on nucleic acid hybridizationOn the detection or sequencing process of the samples based on random amplification and other nonspecific amplification, the detection and sequencing effect are influenced by non-target nucleic acid or background nucleic acid.The enrichment and purification of samples before the nonspecific amplification can reduce background noise on the samples and make the results more sensitive and accurate.For this purpose, we developed a specific purification and enrichment method for hemorrhagic fever virus genome based on nucleic acid hybridization.Flavivirus, Hantavirus and Phlebovirus were studied, blasted the genome conservative district and designed the hybridization probes, the probes were modified with biotin at 5'ends to covalent bond with streptavidin magnetic beads. By comparing the composition proportion of nucleic acid in samples and effective reads in the second generation sequencing before and after purification to evaluate the enrichment and purification effect. The results showed that the ratio of effective nucleic acid mass proportion of the four subtypes of Dengue virus in samples after the purification compared to samples before the purification are 2.3,2.6,2.4,8.3 respectively.In the same way,the ratio of effective nucleic acid mass proportion of yellow fever virus is 8.5, the ratio of effective Nucleic acid mass proportion of Hantaan virus(HTNV), Seoul virus (SEOV),Puumala virus(PUUV) are 1.5,4.0,1.9 respectively, and the ratio of effective Nucleic acid mass proportion of Severe Feverwith Thrombocytopenic Syndrome Bunyavirus(SFTSV) is 1.5. Specific enrichment and purification for viral genome before sequencing can significantly increase the proportion of valid data and whole genome coverage. The valid data proportions of the groups purified before or after whole transcriptome amplification (WTA) were improved significantly compared to group without purifying. The valid data proportion respectively were 5.57%,6.39%,2.71%, and the difference was significant (x2=9799.8, P<0.001)). And the effect of the group purified after whole transcriptome amplification was better than group purified before wholetranscriptome amplification (P<0.001).So this enrichment and purification method based on nucleic acid hybridization could be successfully capture the viral nucleic acid, improve the effective components of nucleic acid, and effectively reduce the nonsense nucleic acid composition or background in second generation sequencing.2 The preliminary study of detection methods for the hemorrhagic fever virus by liquichip after random PCR.To deal with the sudden outbreak of viral hemorrhagic fevers, a fast, accurate and high flux detection method is particularly important. This study combined the random PCR and tem-PCR to amplify the sample nonspecifically, and then detected the PCR products by liquichip technology for high throughput detection. The method can breaks the limitations of specific primer amplification.That is the random amplification was carried out on the template nucleic acid to amplify a wide range of different sizes and concentration fragments, and then the specific detection probes will capture these fragments. This can improve the number of virus in one detection, in theory that can detect 100 different pathogens one time. This can greatly reduce the time for a variety of pathogens screening, improving the detection efficiency. Results show that there are some nonspecific reaction, the establishment of this method needs further experimental studies.