Establishment And Application Of Sequence Independent Single Primer Amplification Technology Of Unknown RNA Viral Pathogens

Objective: To establish a detection method of unknown viral pathogens-sequence independent single primer amplification technology（SISPA）,and provide a scientific basis for clinical diagnosis,treatment,prevention and control of infectious diseases especially the emerging ones.Methods:1 The strain of Severe Fever with Thrombocytopenic Syndrome Bunyavirus（SFTSV）was taken as the research object.After filtering and digestive treatment by DNase I,the total RNA of the strain of SFTSV was extracted;2 Using random primer containing known fragment to reverse transcribe the extracted RNA,and double chain cDNA was synthetic and purify;3 The known fragment in the random primer was taken as single primer to nonspecifically amplify（SISPA-PCR）pathogen genes;4 SISPA-PCR amplification products were Analyzed and recycled,and performed TA clone;5 The result of the clone was indentified,and the recombinant plasmids containing insert segments were sequenced,and the BLAST software was applied to search homologous sequences in the nucleic acid database to judge their species;6 The extracted RNA nucleic acids of SFTSV strain are diluted by 10 times more continuous grades,then take the diluted samples as research objects to evaluate the sensitivity of the SISPA technology;7 The strains of intestinal virus CoxA16 and EV71,influenza A virus strain H1N1,and the mixture which contains these three kinds of virus were taken as the research objects to evaluate the specificity of the SISPA technology;8 The SISPA method is applied to detect 76 cerebrospinal fluid specimens which are diagnosed with viral encephalitis in clinic,and these specimens are collected from Pingshan country in Hebei Province.Results: 1 The establishment of sequence independent single primer amplification technology（SISPA-PCR）: the nucleic acid of RNA viral pathogens of SFTSV strain was amplified by SISPA,and a number of DNA fragments were acquired.After being recycled,cloned and sequenced,all PCR products were analyzed by BLAST software.The homology is up to 98% with SFTSV-sequence in the GeneBank,so we can judge that the detected pathogen is SFTSV;2 The sensitivity analysis: as shown in Fig 6 and Fig 7,the concentration of the diluted nucleic acids of SFTSV was between 10⁶ and 10¹ TCID₅₀/ml.After SISPA-PCR amplifying,classical SISPA amplified bands were detected in the blot of the diluted samples whose concentration was between 10⁶ and 10² TCID₅₀/ml.And we got the same results in at least 3 tests.So we can conclude that this method was highly sensitive and can detect the viral nucleic acids whose concentration is down to 10² TCID₅₀/ml;3 The specificity analysis: the intestinal virus CoxA16,EV71,influenza A virus H1N1 and the mixture which contains these three virus were amplified by SISPA-PCR,and a number of DNA fragments were acquired.After TA cloning,sequencing and BLAST analysis,the sequence homologies of CoxA16,EV71 and H1N1 in the GeneBank were up to 98%,97% and 99% respectively.And we got the same results in at least 3 tests.So we can judge that the SISPA method has high specificity as it can detect a variety of virus in a reaction;4 The application of SISPA method: The SISPA-PCR method was applied to detect 76 cerebrospinal fluid specimens,among which 11 cerebrospinal fluid specimens were detected positive for Echo18 virus.All the specimens were collected from Pingshan country in Hebei Province and diagnosed with viral encephalitis in clinic.Conclusions:1 A technology platform which can apply the SISPA technology to detect unknown RNA viral pathogen was successfully established.2 The SISPA method established in this study does not depend on the separated culture of the virus,which shorten the time to detect unknown viruses and can identify viral pathogens quickly.3 The SISPA method established in this study can detect the virus with 10²TCID₅₀/ml copy number.It can be used to detect low-concentration viral nucleic acid of unknowing its sequence.4 The SISPA method established in this study can detect the known viruses and the unknown ones.So this method has a broad scope of application.