Kit and method for rapidly detecting human bocavirus based on isothermal amplification
An isothermal amplification, Boca virus technology, applied in the field of molecular biology, to achieve the effect of simple operation, good specificity and rapid diagnosis
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Embodiment 1
[0030] Example 1: Selection and preparation of targeting sequences
[0031] Find the published human bocavirus NP1 gene sequence from NCBI, and use the nucleotide sequence between positions 10-279 of the human bocavirus NP1 gene as a template through multiple alignments. The template nucleotide sequence was provided by Shanghai Sangon Biotechnology Synthesized by Engineering Technology Services Co., Ltd. and integrated into the EcoRI site of pBluescriptⅡSK(+) of the plasmid, then transformed the recombinant plasmid into E. coli TOP 10, and inoculated it into LB medium supplemented with 100 μg / mL ampicillin;
[0032] After incubating at 37° C. for 8 h, the plasmid was extracted according to the instructions of the plasmid mini-prep kit (TIANprep Mini Plasmid Kit, purchased from TIANGEN, catalog number DP103). The concentration of the extracted plasmid was measured by ultra-micro spectrophotometer to be 1024ng / μL, and then the plasmid was cleaved. The enzyme digestion system: DN...
Embodiment 2
[0033] Example 2: Design and optimization of primers
[0034] In this example, a series of RPA primers were designed for the conserved region of the human bocavirus NP1 gene, and 4 pairs of primers were synthesized by Shanghai Sangon Bioengineering Technology Service Co., Ltd., see Table 1 for details.
[0035] Table 1: RPA primers designed for the conserved region of the human bocavirus NP1 gene
[0036]
[0037] The primers in Table 1 were then screened by PCR amplification reaction.
[0038] The linearized plasmid frozen in Example 1 was used as a template for PCR amplification. The amplification system and conditions are shown in Table 2.
[0039] Table 2: PCR amplification conditions for human bocavirus NP1 gene with different primer pairs
[0040]
[0041]
[0042] Perform 2% agarose gel electrophoresis on the amplified product, load 6 μL of sample, 110V, 40min. The electrophoresis result is as follows figure 2 As shown, the brightness of the band amplified by...
Embodiment 3
[0055] Embodiment 3: specificity experiment
[0056] Using the same target sequence preparation method as in Example 1, adenovirus Hexon gene, human metapneumovirus N gene, and syncytial virus N gene were synthesized as templates respectively, and RPA amplification was performed using the preferred primer P3 in Example 2, For specific operations, refer to the instructions of the RPA amplification kit (RAA Nucleic Acid Amplification Kit, purchased from Jiangsu Qitian Gene Biotechnology Co., Ltd., product number B00000), react at 39°C for 30 minutes, and detect by gel electrophoresis. Experimental results such as image 3 As shown, only human bocavirus is positive, and the others are all negative, indicating that the RPA detection system of the present invention has good specificity.
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