Establishment Of Multiplex Real Time RT-PCR Assays For Hemorrhagic Fever Viruses Detection

Viral hemorrhagic fevers (VHFs) refer to a group of acute infectious diseases with high case fatality rates up to90%;the clinical symptoms of these life-threatening diseases are often accompanied by fever and bleeding. A wide variety of viruses can cause VHFs, and novel viruses are constantly being discovered. VHFs are associated with dozens of pathogens belonging to four distinct RNA virus families of bunyaviridae, flaviviridae, filoviridae and arenaviridae, some of which have the potent to be used as biological weapons. With a few noteworthy exceptions, there is no specific drug treatment for VHFs. In the early phase of VHFs, the clinical symptoms are similar to influenza. Therefore, the establishment of a fast, simple, sensitive and specific laboratory test would be of great significance to timely clinical diagnosis and treatment, epidemiological investigation and controlling the spread of this kind of diseases.Molecular diagnosis has become a trend, more and more applications based on polymerase chain reaction (PCR), such as nestedPCR, multiplePCR, and fluorescence quantitative PCR have been developed and utilized in detection of infectious diseases. The advantages of real-time PCR technology are as follows:firstly, high sensitivity is provided and better specificitycould be obtained with the adoption of fluorescent probes; secondy, relative or abosolute quantification could be achievedusing target gene referencestandards; thirdly, low contaminationexists for the reason that the reaction system for gene amplification and analysis is carried out in a full closure;fourthly, multiple detection could be emplyed using probes with different fluorescent wavelength;finally,it isconvenient and time-saving because there is no electrophoresis or image processing after PCR reaction.TaqMan probe is currently the most widely used labeling technology in quantitative PCR.Viral nucleic acid detection for infectious diseases is always carried out in a separate reaction system, where nucleic acid of one kind of virus or a group of related viruses derived from clinical specimens were amplified and detected. However, when a new or rare emerging infectious disease breaks out, investigation and identification of multiple pathogens are often required, and the problem of insufficient sample size in actual testing process mayappear. Therefore, in order to meet the requirements of multiplex pathogens screening, it is necessary to establish a high-fidelity and non-specific amplification method for nucleic acids of pathogens from limited clinical specimens, in the laboratory diagnosis process of viral diseases, especially new or rare emerging infectious diseases.Multiple displacement amplification (MDA) is an in vitro isothermal amplification technology based on random primers and Phi29DNA polymerase, which has been mainly used in human and bacterial whole genome amplification(WGA). There are fewer reports about this method applied in viral genome amplification, particularly in RNA viruses, for the reason that a reverse transcription reaction is always needed in the process, while the nucleotide length of cDNA fragment produced by random primer varies and may affect amplification efficiency.Thepurpose of this study is to establish a TaqMan multiplex real time RT-PCR system to detect the major hemorrhagic fever viruses, and explore RNA virus genome amplification method using multiple displacement amplification technology.1. Establishment of TaqMan multiplex real time RT-PCR assays for detection and absolute quantification of hemorrhagic fever viruses Full genomic nucleic sequences of30hemorrhagic fever viruses and chikungunya fever virus were obtained through the GenBank database, conserved regions were determined by Bioedit software analysis, RNA templates of target genes were synthesized by in vitro transcription, primers and TaqMan probes were designed under the premise of the unified PCR reaction condition, and mutual impactsamong primers and probes undermultiple combinationcondition were also evaluated, blast program was used to preliminary analyzethe specificity of each primer-probe pair.RNA standardswere used to evaluate each singleplex real time RT-PCR assay, standard curveswere establishedand sensitivities werecalculated, the detection limitsranged from50to150copies/PCR. Cross-reactivity of primer-probe sets for detection of these viruses belonging the same virus family were checked, the results showed there were no significant amplification curves or positive signals, which suggested high specificities of all these designed primer-probe sets. Standard curvesand sensitivities were also established and calculated under multiplexcombination conditions, and there were no significant differences compared with singleplex condition for eachassay. Repeated experiments under multiplexconditions were employed to verify the stabilities of the assays and coefficient of variation were calculated. Part of clinical serum samples and animal tissue samples were detected by some of these assays.2. Investigation of RNA viral genome amplification by multiple displacement amplification techniqueAnegative-strand RNA virus, severe fever with thrombocytopenia syndrome bunyavirus, and a positive-strand RNA virus,dengue virus, were used to investigate RNA viral genome unspecific amplification method by multiple displacement amplification technique. Series of10-fold diluted purified viral RNA were utilized as analog samples with different pathogen loads, after a series of reactions were sequentially processed, single-strand cDNA, double-strand cDNA, double-strand cDNA treated with ligation without or with supplemental RNA were generated, then a Phi29DNA polymerase depended isothermal amplification was employed, and finally the target gene copies were detected by· real time PCR assays to evaluate the amplification efficiencies of various methods. The results showed that multiple displacement amplification effects of single-strand or double-strand cDNA templates were limited, while the fold increases of double-strand cDNA templates treated with ligation could be up to6×103, even2×105when supplemental RNA existed, and better results were obtained when viral RNA loads were lower.