Universal porcine reproductive and respiratory syndrome virus triple-nested RT-PCR detection primers and method
A technology of RT-PCR and porcine blue-ear virus, which is applied in the field of general-purpose porcine blue-ear virus triple-nested RT-PCR detection primers, can solve the problems of not being able to understand the characteristics of PRRS genes in time, long detection time, cumbersome operations, etc., and achieve Conducive to promotion and popularization, wide application range, good interference effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0030] Example 1: Establishment of triple nested RT-PCR detection method for porcine blue ear virus
[0031] 1. Design of primers
[0032] According to the sequence analysis of porcine PRRS virus, the NSP2 protein amino acid of popular strains is highly conserved, which can be used as a target antigen for clinical diagnosis. A base sequence encoding the NSP2 protein is designed to obtain a pair of outer primers and a pair of inner primers, and the inner primers are inside the amplified sequence of the outer primers. The two pairs of primers contain degenerate bases, so that different bands of classical strains, highly pathogenic variant strains and NADC-30 can be amplified, and the purpose of one-time detection and differentiation of these three subtypes can be achieved. Wherein, the sequences of the two pairs of primers are respectively:
[0033] PRRSV-O-F: AATCTTGARGAATGCTTGGC;
[0034] PRRSV-O-R: GCTGAGTAYTTTGGGCGTG;
[0035] PRRSV-I-F: TTCTTTGATTGGRATGTTGTGC;
[0036]...
Embodiment 2
[0046] Embodiment 2: The nested RT-PCR detection kit of porcine blue ear virus
[0047] A detection kit for identifying porcine PRRS virus and distinguishing three subtypes such as classic strains, highly pathogenic variant strains or NADC-30, said kit comprising: two pairs of detection primers and amplification reagents , positive and negative controls. Among them, two pairs of primers are outer primers PRRSV-O-F, PRRSV-O-R and inner primers PRRSV-I-F, PRRSV-I-R, the negative control is RNase-free water, and the positive control is recombinant positive plasmid pMD18-T-NSP2. The construction method of the positive plasmid is as follows:
[0048] Step 1: Cloning of NSP2 gene
[0049] Genomic RNA of porcine blue ear virus (PRRSV) was extracted, and using the genomic RNA as a template, a part of the conserved sequence of the NSP gene was amplified by nested RT-PCR outer primers PRRSV-O-F and PRRSV-O-R. PCR reaction system (25 μl): 12.5 μl 2×1 Step Buffer, 1 μl PrimeScript 1Ste...
Embodiment 3
[0056] Embodiment 3: Feasibility analysis of nested RT-PCR primers
[0057] 1. Specificity test
[0058] Extract the genomes of porcine blue ear virus (PRRSV), porcine epidemic diarrhea virus (PEDV), porcine tricoronavirus (PDCoV), Escherichia coli and Staphylococcus aureus as templates, and two pairs of primers for nested RT-PCR PRRSV-O-F, PRRSV-O-R and PRRSV-I-F, PRRSV-I-R were tested for specificity, and the specificity of the two pairs of primers was tested. The result is as figure 1 , as shown in 2, both pairs of primers have good specificity and high amplification efficiency.
[0059] 2. Sensitivity test
[0060] The positive plasmid obtained in Example 2 was diluted 10 times to a single-digit copy number, and each dilution plasmid was selected as a template for the first PCR amplification with primers PRRSV-O-F and PRRSV-O-R, and the PCR product was amplified with 1 % agarose gel detection. The result is as image 3 As shown, the results of the first PCR amplifica...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More