Multiplex PCR rapid detection kit and primer group for common feline viruses
A detection kit and detection primer technology, which is applied in the field of cat respiratory and enterovirus nucleic acid detection, can solve public health threats and increase the risk of global pandemics, etc.
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Embodiment 1
[0025] Example 1, kit assembly and use
[0026] To facilitate use, the reagent used in the present invention is assembled into four kinds of common viral multiple RT-PCR rapid detection kits, mainly composed of PCR standard, positive control, negative reference, primers, and casing. A corresponding container hole is provided in the box for each reagent.
[0027] The primers include the four pairs of PCR primers shown in Table 1 (Synthesis from Beijing Oyster New Industry Biotechnology Co., Ltd., the synthesis amount is 1OD per tube), which are common for FCV, FPV, FIV, FHV-1 four cats, They have the base sequence of sequence list seq.id.no.1 to seq.id.no.8, respectively. All upstream primers in PCR primers are co-packaging, all downstream primers.
[0028] Table 1 Multiple PCR primers designed in the present invention
[0029]
[0030] The primer design of the present invention is large and the existing PCR detection is large: on the one hand, due to the NS1 gene of cat films an...
Embodiment 2
[0042] Example 2, the present invention detects specific experiments of five viruses
[0043] According to the multi-PCR reaction system, Green Taq Mix (Vazyme) 14 μL, Fcv, FPV and FiV upper primer mixture 1.5 μL, downstream primer mixture 1.5 μL, FHV-1 upper prism, 0.75 μl, downstream primer, DDH 2 O 1 μL, 5.5 μl of sample DNA or cDNA template, including 1: ultrapure water negative control; 2: Fcv, FPV, FIV and FHV-1; 3: Fcv; 4: FPV; 5: FIV; 6: FHV- 1; 7: FCOV.
[0044] The reaction was carried out at the Life Proflex PCR amplifier, and the reaction procedure was: Pre-deformation of 95 ° C for 5 min, and the reaction conditions of 40 cycles were 95 ° C degeneration 35s, 56 ° C for 1 min, 72 ° C, and finally 72 ° C for 10 min. Take 10 μl of PCR product, in order to 2% agar gel electro-bore, 140V voltage, electrophoresis is about 20 min, and the photographic determination after the gel imager ultraviolet imaging, the results see Figure 5 The corresponding PCR product size is comple...
Embodiment 3
[0045] Example 3, the present invention detects sensitivity experiment of five viruses
[0046] Multi-PCR sensitivity detection reaction system: Green Taq Mix (Vazyme) 14 μL, Fcv, FPV and FIV upper primer mixture 1.5 μL, downstream primer mixture 1.5 μL, FHV-1 upper hopper primer 0.75 μL, downstream primer 0.75 μL, DDH 2 O1 μL, add 5.5 μL of 10 times series diluted FCV, FPV, FIV and FHV-1 yang-type mixtures (4.45 × 10 10 ~ 4.45 × 10 1 COPIES / μL) template.
[0047] The reaction was carried out at the Life Proflex PCR amplifier, and the reaction procedure was: Pre-deformation of 95 ° C for 5 min, and the reaction conditions of 40 cycles were 95 ° C degeneration 35s, 56 ° C for 1 min, 72 ° C, and finally 72 ° C for 10 min. Take 10 μl of PCR product, 2% agar gel electrophoresis.
[0048] The experimental results show that the minimum detection amount of multiple PCR reactions against FCV, FPV, FIV and FHV-1 virus is 24.48copies, which shows that the sensitivity of this detection met...
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