HRM detection primers, kit and method for rapidly identifying porcine circovirus type 1, type 2 and type 3
A porcine circovirus, a rapid technology, applied in the directions of biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of reducing the immune effect of vaccines, and achieve rapid high-throughput analysis and high detection sensitivity. , the effect of low cost
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Embodiment 1
[0038] The present invention is a kind of HRM detection primer for rapid identification of porcine circovirus type 1, type 2 and type 3. The nucleotide sequence of its upstream primer P1 is shown in SEQ ID NO: 1, and its downstream primer P1 nucleotide sequence is SEQ ID NO: 2 shows:
[0039] The above primers were designed based on the conserved sequences of the rep genes of these three viruses, as shown in the following table
[0040]
[0041] Among them: PK-15 cells (ATCCCCL-33) were purchased from ATCC in the United States, PCV-2, CSFV, PRRSV, PRV, and PPV were enough from commercial vaccines, and the full gene sequence synthesized by PCV3.
Embodiment 2
[0043] A kind of HRM detection method for rapidly distinguishing porcine circovirus type 1, type 2 and type 3 provided by the invention specifically comprises the following steps:
[0044] 1) Viral nucleic acid extraction
[0045] Lymph nodes, spleen and other tissue samples collected from chicken farms were put into centrifuge tubes, added with appropriate amount of sterilized PBS, and homogenized; samples such as vaccines, cytotoxicity, and tissue homogenate in PBS solution were extracted with Tiangen nucleic acid automatic extractor, and operated Instructions are carried out.
[0046] 2) Preparation of 3 kinds of pathogenic plasmid standard products
[0047] Use Tiangen's automatic nucleic acid extractor to extract PCV-1, PCV-2 pathogenic DNA, and PCV-3 synthetic gene as templates, use the upstream primer P1 nucleotide sequence such as SEQ ID NO: 1, and the downstream primer P1 nucleotide The primers shown in the sequence of SEQ ID NO: 2 were amplified by PCR, and the amp...
experiment example
[0060] Testing of Clinical Samples
[0061] 1. From 23 samples of lymph nodes and spleens collected from chicken farms, DNA was extracted with Tiangen nucleic acid automatic extractor, amplified using takara's Premix Extaq, using DNA as a template, and using the PCR-HRM detection method established in Example 2 Detect, M: DL2000Mark; 1: PCV1; 2: PCV2; 3: PCV3; 4: CSFV; 5: PRRSV; 6: PRV; 7: PPV; 8: negative control 1; the result is as follows image 3 As shown, PCV1, PCV2, PCV3, and no electrophoresis bands appeared in other samples, indicating that the designed primers have high specificity and are suitable for HRM analysis.
[0062] 2. Detect 23 clinical samples, circovirus type 1, type 2, type 3 standard samples HRM standardized melting curve of clinical samples refer to Figure 4 For circovirus type 1, type 2, and type 3 standard samples, refer to the HRM peak melting curve of clinical samples Figure 5 , As a result, 18 parts were PCV2, 3 parts were PCV3, 2 parts were mi...
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