Rapid HPS (haemophilus parasuis) isothermal amplification detection primers, kit and detecting method
An isothermal amplification and kit technology, applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc., to achieve high specificity, wide application, and obvious and reliable results.
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Embodiment 1
[0041] Example 1, HPS loop-mediated isothermal amplification technology rapid detection kit includes LAMP amplification reaction solution, Calcein chromogen tube, positive control tube, negative control tube and sterilized deionized water tube, wherein:
[0042] The LAMP amplification reaction solution tube is composed of the following reaction solution: 1.0 μL upstream of the 50 mmol / L HPS internal primer whose sequence is SEQ ID NO.1; 1.0 μL downstream of the 50 mmol / L HPS internal primer whose sequence is SEQ ID NO.2 ; The sequence is 1.0 μL upstream of the 5mmol / L HPS outer primer of SEQ ID NO.3; the sequence is 1.0 μL downstream of the 5mmol / L HPS outer primer of SEQ ID NO.5; the sequence is 30mmol / L of SEQ ID NO.5 1.0 μL upstream of the HPS loop primer; 1.0 μL downstream of the 30 mmol / L HPS loop primer whose sequence is SEQ ID NO.6; 12.5 μL of 10×LAMP buffer; 0.8 μL of 5U / μL Bst DNA polymerase; sterile deionized 3.7 μL of water; a total of 23 μL is the amount for a sing...
Embodiment 2
[0048] Embodiment 2, the design and screening of primer
[0049] The HPS isothermal amplification primer set was designed based on the HPS 16S rRNA gene reference sequence published by GenBank, and multiple alignments were performed with Clustal W to analyze the conserved regions of the sequence. LAMP primer design software Primer Explorer V4.0 was used to design specific primers, respectively marked as: SEQ ID NO.1...SEQ ID NO.6. In the primer set, the upstream FIP of the internal primer: SEQ ID NO.1; the downstream BIP of the internal primer: SEQ ID NO.2; the upstream F3 of the external primer: SEQ ID NO.3; the downstream B3 of the external primer: SEQ ID NO.4; the loop primer Upstream LF: SEQ ID NO.5; Loop primer downstream LB: The concentration of SEQ ID NO.6 is respectively 50mmol / L, 50mmol / L, 5mmol / L, 5mmol / L, 30mmol / L, 30mmol / L; the volume ratio is 1:1:1:1:1:1. Meanwhile, during PCR amplification, PCR upstream primer: SEQ ID NO.3; PCR downstream primer: SEQ ID NO.4; t...
Embodiment 3
[0052] Embodiment 3, the preparation of positive control substance
[0053] Use the nucleic acid kit to extract the DNA of the standard positive HPS culture, electrophoresis the extracted nucleic acid, use PCR upstream primer SEQ ID NO.3 and PCR downstream primer SEQ ID NO.4 to amplify, and use the gel recovery kit to recover the amplified Bands. According to the ratio of 1:10 and the pMD-19T vector for ligation reaction, ligated at 16°C for 2 hours, transformed into JM109 bacteria, after resistance selection and PCR identification positive, re-sequencing verification, using a spectrophotometer to measure the concentration of nucleic acid to make it Concentration was controlled at 80-100ng / μL, and divided into 50μL tubes.
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