A kind of multiple dpo-pcr primer combination and method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma
A DPO-PCR, primer combination technology, applied in microorganism-based methods, biochemical equipment and methods, recombinant DNA technology, etc., can solve the problems of complex system, low stability, limited use, etc., to achieve high throughput, Good specificity and improved detection efficiency
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Embodiment 1
[0028] Example 1. Multiplex DPO-PCR method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma
[0029] 1. Design of Multiplex DPO-PCR Primer Combinations
[0030] According to the conserved regions of Trichinella spiralis ATP6 target gene, Toxoplasma gondii P22 target gene and Schistosoma japonicum 28S rRNA, the following multiplex DPO-PCR primer combinations for detecting the conserved regions of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum were respectively designed (I in the primer sequence xanthine):
[0031] Trichina-F: 5'-TCTCCCTACTCAGATACAACTGAATIIIIIIACAGCCAA-3' (SEQ ID NO: 1)
[0032] Trichina-R: 5'-GGATTTATGTGTTTTTTTGTGTGTGTTIIIIITTCTGGTC-3' (SEQ ID NO: 2)
[0033] Toxo-F: 5'-CGGCGCAACGAAGACTGTTGIIIIICCCTCCAGT-3' (SEQ ID NO: 3)
[0034] Toxo-R: 5'-ACCTGCTTCGGCAACGCACTTIIIIIIAGAGAACC-3' (SEQ ID NO: 4)
[0035] Schistosome-F: 5'-TGAGATACCACAAAAGGTGTTGGIIIIICCAGACAGC-3' (SEQ ID NO: 5)
[0036] Schistosome-R: 5'-GGGCTGCGATCTAC...
Embodiment 2
[0052] Example 2. Sensitivity detection of multiple DPO-PCR primer combinations
[0053] 1. Extraction of DNA
[0054] According to the instructions of the kit, the gene DNA of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum was extracted. The obtained genomic DNA was mixed and diluted 10 times, and the genomes of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum were prepared at concentrations of 18ng / μL, 1.8ng / μL, 0.18ng / μL, 0.018ng / μL and 0.0018ng / μL, respectively. DNA.
[0055] 2. Multiplex DPO-PCR amplification
[0056] Adopt the method for detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma in the step 2 of embodiment 1, carry out multiple DPO-PCR amplification with the genomic DNA of following 5 groups respectively as template, obtain multiple DPO-PCR amplification products respectively:
[0057] Group 1: Genomic DNA (1 μL) of 18 ng / μL Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum was used as templat...
Embodiment 3
[0065] Example 3. Detection of Annealing Temperature Sensitivity of Multiplex DPO-PCR Primer Combinations
[0066] 1. Extraction of DNA
[0067] Genomic DNA of Trichinella spiralis, Toxoplasma gondii and Schistosoma japonicum was extracted according to the kit instructions.
[0068] 2. Multiplex DPO-PCR amplification
[0069] Using the Trichinella spiralis, Toxoplasma gondii and Schistosoma genomic DNA obtained in step 1 as a template, using conventional primers (as shown in Table 1) or the method for simultaneously detecting Trichinella spiralis, Toxoplasma gondii and Schistosoma in step 2 of Example 1, using different The annealing temperatures (48.3°C, 49.5°C, 51.4°C, 53.7°C, 56.4°C, 59.1°C, 61.7°C, 64.1°C, 66.1°C, 67.4°C, 68°C) were used for PCR amplification, and the rest of the reaction conditions were unchanged, respectively Obtain the PCR amplification product.
[0070] Table 1. Sequences of conventional primers for amplifying 28S rRNA of Schistosoma japonicum, ATP6...
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