Haemophilus parasuis nested PCR amplification primer combination, kit and application
A technology of Haemophilus suis and amplification primers, which is applied in the field of Haemophilus parasuis nested PCR amplification primer combinations, can solve the problems of cumbersome methods, easy contamination of fluorescent quantitative PCR, and limited sensitivity, and achieve good detection sensitivity and accuracy Accuracy, good detection application value, high amplification sensitivity effect
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Embodiment 1
[0050] This embodiment provides an amplification primer combination for Haemophilus parasuis, which includes a first primer pair and a second primer pair. The primer names of the first primer pair are respectively P1:HPS-F and P2:HPS-R, and the sequences are shown in Table 1.
[0051] The primer names of the second primer pair are P5:HPS-F and P6:HPS-R respectively, and the sequences are shown in Table 1.
[0052] Use the NCBI website to download the 16S rRNA gene sequence of HPS (accession number: FJ667982), and use the Primer 5.0 primer design software to design a primer pair for HPS. P5 and P6 are primers designed in the amplified fragments of P1 and P2, and then pass Screening was used for nested PCR amplification. The primer sequences and the size of the target fragment are shown in Table 1. The primer sequences were synthesized by Shanghai Sangong.
[0053] Table 1 Primer sequences for nested PCR amplification of Haemophilus parasuis
[0054]
experiment example 1
[0058] This experiment explores the best common PCR amplification conditions. Specifically, the combination of amplification primers provided in Example 1 was used for ordinary PCR amplification.
[0059] (1) Bacterial recovery
[0060] Select LB medium, add 50mL / L calf serum and 40mg / kg NAD, then use sterilized cotton swabs to inoculate and mark Haemophilus parasuis preserved in the laboratory, place it in a 37°C incubator for 24 hours, and then put it away. Store in refrigerator at 4°C for later use.
[0061] (2) Ordinary PCR amplification
[0062] According to the instructions of Tiangen DP302 Bacterial Genomic DNA Extraction Kit, the extracted Haemophilus parasuis genome was stored at 4°C for later use. Use the P1 and P2 primers in Table 1 for ordinary PCR amplification. The amplification reaction system is: 12.5 μL of 2×HiFiTaq high-fidelity premixed PCR reaction, 1.0 μL of P1 and P2 primers (25 pmol / L), and the DNA template is 2ul, finally with ddH 2 O to 25 μL. PC...
experiment example 2
[0065] This experiment explores the optimal nested PCR amplification conditions and nested PCR amplification primer pairs. Specifically, nested PCR amplification was carried out by using the second primer pair provided in Example 1 and the nested primers provided in Comparative Example, respectively.
[0066] The product amplified by ordinary PCR in Experimental Example 1 was selected as the template for the second amplification, and the P3 and P4 primer pairs and the P5 and P6 primer pairs were respectively selected for the second amplification screening (nested PCR). The amplification reaction system is: 12.5 μL of 2×HiFiTaq high-fidelity premixed PCR reaction, 1.0 μL of P3, P4 primer pair and P5, P6 primer pair upstream and downstream primers (25 pmol / L), amplified by the first common PCR step The product is template 4ul, and finally use ddH 2 O to 25 μL. PCR reaction program: 95°C for 5min; 95°C for 45s, annealing temperature (54.5, 55.5°C, 56.5°C, 57.5°C, 58.5°C, respec...
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