Site-specific gene mass spectrometric detection based duck-origin virus detection kit and detection method
A virus detection and mass spectrometry detection technology, which can be used in the determination/inspection of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc. Low rate, good specificity
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Embodiment 1
[0054] Example 1 sample and virus strain
[0055] The samples used in this embodiment are respectively: duck hepatitis A virus type 1 (DHAV-1) C83 strain, duck hepatitis A virus type 3 (DHAV-3) C-GY strain, duck astrovirus type 1 (DAstV-1) D17 strain , duck astrovirus type 2 (DAstV-2) SL4 strain, duck reovirus type 1 (DRV-1) 815-12 strain, duck reovirus type 2 (DRV-2) 091 strain, Tembusu Virus (TMUV) PS strain, gosling plague virus (GPV) JS1 strain, duck enteritis virus (DEV), avian influenza virus (AIV) H9N2 subtype, the above samples were all donated by China Agricultural University.
Embodiment 2
[0056] Embodiment 2 primers and quality probe design
[0057] The VP1 gene of DHAVs and GPV, the ORF2 gene of DAstVs, the S1 fragment of DRVs, the NS5 gene of TMUV, and the UL6 gene of DEV were selected as target genes, and β-actin was selected as an internal reference, and the primers, multiple PCR primers and quality probe. The molecular weight of the mass probe and the molecular weight after single base extension are shown in Table 1 and Table 2 below, and the primer sequence for constructing the recombinant plasmid is shown in Table 3. In order to avoid the interference of multiple PCR primers on the detection, a 10 nt base sequence ACGTTGGATG (as shown in SEQ ID No.56) can be modified at the 5' end of the multiple PCR primers. According to SEQ ID No.1-SEQ ID No.56, the sequences were sent to Sangon Bioengineering (Shanghai) Co., Ltd. for synthesis.
[0058] Table 1 PCR primers
[0059]
[0060] Table 2 Quality Probes
[0061]
[0062]
[0063] Table 3 Primers...
Embodiment 3
[0065] The construction of embodiment 3 recombinant plasmids
[0066] 3.1 Nucleic acid extraction
[0067] Viral genomic nucleic acid was extracted using a viral genome DNA / RNA extraction kit (Tiangen Biochemical Technology (Beijing) Co., Ltd.). Take 0.3g tissue sample, cut it into pieces and place it in a 2ml grinding tube, add 5 glass beads and 1ml sterilized physiological saline (containing a mixture of 100U / ml penicillin and 10mg / ml streptomycin), place it in a grinder (Mini Beadbeater- 16) Grinding in medium for 1 min, ice bathing for 1 min, repeated 3 times to make a homogenate, set the volume to 1.5 ml, place in a 4°C centrifuge at 12,000×g for 15 min, absorb the supernatant for later use. According to the instructions of the instructions, the nucleic acid was extracted through the steps of lysis, precipitation, washing and elution, and the concentration of the nucleic acid was determined using Nanodrop 2000, and the nucleic acid was diluted to 20-50ng / μl for use.
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