The invention discloses a method for preparing lentiviral particle packaged EBOV 
RNA as a positive reference substance for EBOV 
nucleic acid detection. The preparation method comprises the following steps: connecting part of segments of three genes NP, GP and L of EBOV in series and then integrating the segments into an 
expression vector of 
lentivirus, separately amplifying the constructed lentiviral expression 
plasmid and packaging 
plasmid in an 
engineering escherichia coli strain to obtain a large number of plasmids, then jointly transfecting the constructed lentiviral expression 
plasmid andpackaging plasmid into HEK293T cells, and carrying out 
cell expression to obtain a large number of lentiviral particles packaged with 
RNA genes of EBOV. The invention relates to the technical field of 
gene engineering. According to the method for preparing lentiviral particle packaged EBOV 
RNA as a positive reference substance for EBOV 
nucleic acid detection. The obtained particles are used as apositive reference substance for EBOV 
nucleic acid detection and have the 
advantage of well simulating the whole process from 
virus nucleic acid extraction to 
nucleic acid detection in an actual sample, and freeze-dried 
powder obtained by freeze-
drying pseudovirus particles of EBOV through a freeze-
drying protective agent is very stable.