A rapid pcr-hrm detection method for distinguishing prrsv vaccine strain gdr180 from other strains
A PCR-HRM, detection method technology, applied in the directions of microorganism-based methods, biochemical equipment and methods, and microorganism determination/inspection, etc., can solve the problems of inability to obtain melting curve probe peaks, inability to achieve the purpose of differentiation, etc. Achieve the effect of low synthesis price, shorten the time required for typing and low cost
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Embodiment 1
[0043] Example 1 PCR-HRM primer design
[0044] The method of the present invention combines PCR and high-resolution melting curve technology to screen the specific base sites of the GDr180 strain. By adding a 3'-end blocked oligonucleotide chain, the melting curve probe is made according to the difference in the sequence matching of the target site The peaks appear to have a difference in melting temperature to achieve the purpose of distinction.
[0045] Through repeated screening, the inventors selected highly specific primers and probes, and their nucleotide sequences are as follows:
[0046] P1: 5'-TTCTTCTTGCCTTTTCTATGCTTC-3' (SEQ ID NO:1);
[0047] P2: 5'-CAACTGTGTCAAGGAAATGGCT-3' (SEQ ID NO: 2);
[0048] P3: 5'-CAGGATAGGGCATGACCGAT-3' (SEQ ID NO: 3);
[0049] P4: 5'-CCAAATATCTCGGGATGGAACT-3' (SEQ ID NO: 4);
[0050] Probe 1: 5'-AATGTGTCAGGCACTGTGGCTGTGTG-C3-3' (SEQ ID NO: 5).
[0051] One end of the probe Probe 1 is modified and blocked with modifiers, including amino-modified C6, ...
Embodiment 2
[0053] Example 2 Preparation of standard samples and PCR-HRM analysis
[0054] 1) Extract viral RNA from the sample:
[0055] Fully homogenize it on ice in a tissue homogenizer, freeze-thaw repeatedly for 3 times, centrifuge at 10,000 rpm for 5 minutes, and take the supernatant for later use (filter sterilization if possible). The tissue supernatant or serum was extracted with the instructions of Tiangen Trizol reagent (Tiangen Biochemical).
[0056] 2) Preparation of positive standard samples:
[0057] In order to verify the feasibility and reliability of the method of the present invention, while constructing standard positive samples to provide HRM positive control for subsequent clinical sample testing, the present invention preferentially prepares PRRSV GDr180 strain and the other two non-GDr180 strain standard samples (including HuN4-F112, MLV), using NTC as a negative blank control.
[0058] The preparation steps of the standard samples are as follows: take the plasmid DNAs of ...
Embodiment 3
[0067] Example 3 PCR-HRM detection of clinical samples
[0068] 1) Extract viral RNA from the sample: the method is the same as the RNA extraction method in Example 2;
[0069] 2) Using the extracted RNA as a template, reverse transcription into cDNA, PCR pre-amplification, and the pre-amplification reaction system:
[0070]
[0071] Pre-denaturation at 94°C for 5min; denaturation at 94°C for 30s, annealing at 55°C for 30s, extension at 72°C for 35s; 35 cycles; final extension at 72°C for 5min.
[0072] 3) Using the pre-amplified PCR product as a template, the amplification reaction system is:
[0073]
[0074] Denaturation at 98°C for 10sec, annealing at 55°C for 30sec, extension at 72°C for 20sec, 45 cycles. The HRM heating steps are 92°C for 1 min, 40°C for 2 minutes; 50°C to 90°C, and the heating rate is 0.5°C / step. The results of HRM experiments were analyzed with Rotor-Gene QTM software.
[0075] Among the asymmetric PCR amplification products of 10 samples (including 3 standard ...
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