Universal primer set for amplifying whole genome sequence of canine distemper viruses and use thereof
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A canine distemper virus and whole genome technology, applied in the field of universal primer sets, can solve problems such as virulence decrease, and achieve the effect of improving efficiency and shortening time
Pending Publication Date: 2018-12-14
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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However, it has not been reported whether the virulence of canine distemper virus wild isolates will decrease after serial passage on Vero-SLAM cells
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Embodiment 1
[0036] Example 1 Design and synthesis of a universal primer set for amplifying the whole genome sequence of canine distemper virus
[0037] According to the 23 canine distemper virus genome sequences published in the online GenBank, the specific strains and GenBank numbers are as follows: 01-2689 (AY649446), 007Lm / B (AB490680), Onderstepoort (AF378705), CDV SY (KJ466106), LN (10)1(KP765764), 98-2654(AY466011), 00-2601(AY443350), SD(14)7(KP765763), HLJ1-06(JX681125), 98-2646(AY542312), 98-2645(AY445077) ), CDV2784-2013 (KF914669), A75 17 (AF164967), Hebei (KC427278), CDV3 (EU726268), M25CR / H (AB490681), 50CBI / H (AB490678), 011C / H (AB490674), PS (JN896331) , Snyder Hill (GU138403), 164071 (EU716337), CDV-RD-JL (KJ848781), CDV-ZC (KJ994343). The whole genome sequence of all the above-mentioned strains was compared and analyzed, and 11 pairs of specific universal primers were designed using the software Primer Premier 5.0 (see Table 1 for the sequence), which were used for the ampli...
Embodiment 2
[0042] Example 2 Application of universal primers in the amplification of the whole genome sequence of canine distemper virus
[0043] 1Materials and methods
[0044] 1.1 Material
[0045] 1.1.1 Strain
[0046] Canine distemper virus wild virus HBF-1 strain, SD14(7) strain, and canine distemper virus vaccine virus CDV3 strain, OR12 strain, CDV / R-20 / 8 strain, all are controlled by the Institute of Special Products, Chinese Academy of Agricultural Sciences Research room preservation.
[0047] 1.1.2 Main reagents and instruments
[0048] GeneAmp PCR System 2700 and Nanodrop 2000 spectrophotometer were purchased from Thermo; RNeasy Mini Kit was purchased from Qiagen; SuperScript III First-Strand Synthesis SuperMix was purchased from Invitrogen; Plasmid mini-extraction kit was purchased from Axygen; General PCR kit Fast Pfu PCR SuperMix was purchased from Transgen; DNA Marker 5000 and DNA Marker 2000 were purchased from Takara Company.
[0049] 1.2 method
[0050] 1.2.1 RNA extraction of dif...
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Abstract
The invention discloses a universal primer set for amplifying a whole genome sequence of canine distemper viruses and a use thereof. The primer set is composed of 11 pairs of primers. Each pair of theprimers are composed of an upstream primer and a downstream primer. The nucleotide sequences of the 11 pairs of primers are respectively shown in the formulas of SEQ ID NO. 1-22. The whole genome sequences of 23 canine distemper virus strains published in GenBank are compared and 11 pairs of specific universal primers are designed according to a conserved region. The research result shows that the 11 pairs of universal primers can conveniently and accurately amplify the whole genome sequences of a wild canine distemper virus strain and a vaccine strain, significantly improve the efficiency ofacquiring whole genome sequences of different strains of canine distemper viruses, and shorten the acquisition time of the whole genome sequences of other unknown canine distemper virus strains. Through the universal primer set, the viral nucleotide variation before and after the adaptation of wild canine distemper viruses to cells is determined and the technological means for understanding the pathogenic mechanism of wild canine distemper viruses and finding a key virulence site for wild canine distemper viruses are provided.
Description
Technical field [0001] The invention relates to a universal primer set for amplifying the whole genome sequence of canine distemper virus and its application. The invention belongs to the field of biotechnology. Background technique [0002] Canine Distemper (Canine Distemper) is an acute febrile, highly contagious infectious disease caused by Canine Distemper Virus (Canine Distemper Virus) infecting predators such as dogs, raccoon dogs and minks. The disease has a high morbidity rate, diverse clinical symptoms, mixed infections in the later stages of infection, and a mortality rate as high as 30%-100%. In recent years, the natural infection host of CDV has continued to increase. With the increase in human possession of dogs and cats and other pets, the canine distemper virus has brought great losses and harm to the animal breeding industry and biodiversity, but also threatens the society. Public safety. [0003] CDV is a member of the measles virus genus of the Paramyxoviridae ...
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