Fluorescent RPA (recombinase polymerase amplification) primer, kit and detection method for detecting Jaagsiekte sheep retrovirus
A detection method and kit technology, applied in biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of high cost of ELISA, unsuitable for large-scale detection in farms, and complicated operation.
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Embodiment 1
[0042] Preparation and Identification of Positive Control in Example 1 Kit
[0043] (1) Extraction of total RNA: RNA was extracted from JSRV-positive lung tissue with a rapid total RNA extraction kit.
[0044] (2) Reverse transcription: the above-mentioned extracted total RNA was reverse-transcribed into cDNA using a method commonly used in the art.
[0045] (3) Use the PCR primers in Table 1 for PCR identification, the target fragment is 1848bp, and the amplification result is consistent with the size of the target fragment (see figure 1 ).
Embodiment 2
[0046] Design and screening of embodiment 2 primers and probes
[0047] RPA is a new type of isothermal amplification technology. Compared with the above-mentioned detection methods, RPA has the advantages of simple operation, short reaction time (detection results can be observed within 10-20 minutes), low operating temperature (37-42°C), and commercially available. Freeze-dried reagents are available, and the method has been used for point-of-care diagnostics outside of the laboratory. One of the keys of the present invention is to design primers and probes based on recombinase polymerase amplification technology, but the core and difficulty of the RPA method is that the design and screening of primers and probes cannot be assisted by software, and can only rely on manual design.
[0048] According to the env gene sequence downloaded from Genbank with accession numbers AF105220 and JQ837489, DNAMAN was used to analyze the highly conserved region of env, and 4 pairs of specif...
Embodiment 3
[0057] The determination of embodiment 3 fluorescent RPA reaction system and reaction conditions
[0058] Use TwistAmpexo Kit for fluorescent RPA amplification, the total reaction volume is 50 μL, including RehydrationBuffer 29.5 μL, template 1 μL, forward and reverse primers 2.1 μL (10 μM), probe 0.6 μL (10 μM), add DNase / RNase-Free Make up to 47.5 μL with Deionized Water, mix well and transfer to RPA lyophilized enzyme powder reaction tube, then add 2.5 μL of MgAc solution with a concentration of 280 mmol / L. After mixing, put the reaction tube into a constant temperature nucleic acid amplification instrument and react for 20 minutes (take out the reaction tube at 4 minutes and mix it well before putting it back into the instrument), and collect FAM fluorescence signals during the entire reaction process. The reaction temperature was set to 37°C, 38°C, 39°C, 40°C, 41°C, 42°C respectively and the optimal reaction temperature was selected. An exponentially increasing amplifica...
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