RT-LAMP (reverse transcription loop-mediated isothermal amplification) nucleic acid detection primers and kit of Hantaan viruses
A technology of RT-LAMP and detection kit, which is applied in the application field of biological detection technology, can solve the problems of easy pollution of the environment, inconvenient to carry, increase costs, etc., and achieve the effects of high sensitivity, easy reaction results, and low cost of use
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Embodiment 1
[0041] Example 1 Design and synthesis of LAMP primers
[0042] According to the sequence of S segment of Hantavirus 76-118 gene published by GenBank (GenBank: M14627.1), the relatively conserved region was analyzed by biological software BLAST, and the LAMP online design software PrimerExplorer V4 (http: / / primerexplorer.jp / e / ) 6 primers were designed for relatively conserved sequence regions, including two inner primers FIP and BIP, two outer primers F3 and B3, and two loop primers LF and LB, which respectively bind to 8 binding regions in the target sequence For matching, the primers were evaluated using Oligo 6 software and BLAST. See Table 1 for details.
Embodiment 2
[0043] Example 2 Tissue sample total RNA extraction
[0044] The total RNA of tissue samples was extracted by Trizol method and micro-extraction; the specific operation is as follows:
[0045] A. Take 100 μl of blood or 100 μg of tissue samples (ground and homogenized) in a 1.5ml centrifuge tube, add 500 μl of Trizol solution, vortex and mix for 1 min, then place at room temperature for 5-10 min, and place the above microcentrifuge tube at 12000 rpm at 4 °C Centrifuge for 15 minutes;
[0046] B. Take 500 μl of the supernatant and put it into a new 1.5ml centrifuge tube, and add 500 μl of water-saturated phenol:chloroform (1:1), shake vigorously by hand for 15 seconds, and then place it at room temperature for 10 minutes;
[0047] C. Centrifuge at 12000rpm for 10min at 4°C, then take 500μl of the supernatant and add it to a new 1.5ml centrifuge tube, add 500μl of isopropanol, mix it upside down, place it at -20°C for 1h, and then put it at 12,000rpm , centrifuge at 4°C for 10 m...
Embodiment 3
[0049] Example 3 RT-LAMP detection kit and detection method for detecting Hantavirus
[0050] The RT-LAMP detection kit that detects Hantavirus includes: a group of RT-LAMP primers for detecting Hantavirus, specifically, this group of primers includes the forward outer primer (F3 ), the reverse outer primer (B3) shown in the sequence listing SEQ ID No.2, the forward inner primer (FIP) shown in the sequence listing SEQ ID No.3, the reverse primer (B3) shown in the sequence listing SEQ ID No.4 Internal primer (BIP), the forward loop primer (LF) shown in the sequence table SEQ ID No.5, the reverse loop primer (LB) shown in the sequence table SEQ ID No.6; the primers are all entrusted to Shanghai Shenggong Synthesized by Biotech Ltd.
[0051] Further, the above-mentioned RT-LAMP detection kit for detecting Hantavirus also includes 10×ThermoPol buffer solution, dNTPs, MnCl 2 , MgSO 4 Strand displacement DNA polymerase (purchased from New England Biolabs, U.S.); AMV reverse trans...
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