Primer composition for recombinase-polymerase amplification reaction for rapid diagnosis of Israeli acute bee paralysis virus and use thereof
Proposed is a primer composition for a recombinase-polymerase amplification reaction for rapid diagnosis of Israeli acute paralysis virus and use thereof. A genomic RNA of Israeli acute paralysis virus (IAPV) is isolated from honey bees to analyze a nucleotide sequence of IAPV, and a primer pair capable of being detected by a recombinase-polymerase amplification method was prepared based on the IAPV RNA sequence. In addition, the optimal conditions of the prepared primer pair were confirmed, and it was confirmed that the genomic RNA of IAPV could be rapidly detected and diagnosed.
1 . A primer set for detecting Israeli acute paralysis virus (IAPV), the primer set consisting of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2,
wherein the primer set generates an amplification product having a length of 191 base pairs from a target nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11 in a recombinase polymerase amplification reaction performed at a temperature of 24° C. for 45 minutes.
2 . A composition for detecting Israeli acute paralysis virus (IAPV), the composition comprising:
a primer set consisting of a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2,
wherein the primer set generates an amplification product having a length of 191 base pairs from a target nucleic acid comprising the nucleotide sequence of SEQ ID NO: 11 in a recombinase polymerase amplification reaction performed at a temperature of 24° C. for 45 minutes.
3 . The composition of claim 2 , comprising 0.5 to 3.5 μl of the primer set.
4 . The composition of claim 2 , comprising 0.5 to 4.0 μl of MgOAc.
5 . The composition of claim 2 , further comprising a DNA polymerase for a recombinase-polymerase amplification reaction, dNTPs, a reaction buffer, and distilled water.
6 . A method for detecting Israeli acute paralysis virus (IAPV), the method comprising:
providing a bee specimen;
grinding the bee specimen to form a ground bee specimen;
diluting the ground bee specimen in sterilized distilled water to form a diluted bee specimen;
mixing the diluted bee specimen with a DNA extraction solution at a volume ratio of 1:1 to form an extraction mixture;
heating the extraction mixture at 95° C. for 5 minutes to form a heated mixture;
separating a supernatant from the heated mixture;
amplifying Israeli acute paralysis virus RNA contained in the supernatant in a recombinase polymerase amplification reaction performed at 24° C. for 45 minutes using a forward primer consisting of the nucleotide sequence of SEQ ID NO: 1 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 2 to generate an amplification product having a length of 191 base pairs; and
confirming the amplification product by gel electrophoresis.