RAA primer probe for detecting Nipah viruses as well as detection method
A Nipah virus and probe technology, which is applied in the field of primers, probes and detection of Nipah virus by RAA fluorescence method, can solve the problems of difficulty in applying large-scale screening, inconvenient operation, and high personnel requirements, and achieve convenience, speed and accuracy Identification, easy operation, and the effect of reducing detection time
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Embodiment 1
[0053] Embodiment 1 selects Nipah virus N gene conservative sequence to carry out primer, probe design
[0054] Find the corresponding full gene sequence in Genebank (www.ncbi.nlm.nih.gov), use DNASTAR software for homology analysis and Blast sequence analysis, and screen out the highly conserved sequence of the N gene of Nipah virus as follows:
[0055] CAGCCGAGCTTACGGCCTACGGATAACAGACATGAGCACCCTGGTCTCTGCAGTTATCACCATCGAGGCCCAGATCTGGATACTGATCGCTAAAGCAGTTACAGCTCCCGACACTGCCGAGGAAAGTGAAACTAGAAGATGGGCTAAATACGTCCAACAAAAGAGAGTCAATCCGTTCTTTGCTCTAACTCAGCAATGGCTAACAGAAATGAGGAATCTGCTCTCCCAGAGTCTATCAGTAAGGAAGTTCATGGTTGAGATCCTCATAGAAGTCAAGAAAGGAGGATCTGCTAAAGGCAGAGCAGTAGAAATAATCTCAGACATCGG(SEQ ID NO.1);
[0056] The highly conserved sequence obtained by screening was used as the target gene fragment for detection, and positive plasmids were synthesized, and primers and probes were designed and screened for detection.
[0057] According to the above conserved sequence of Nipah virus N gene, ...
Embodiment 2
[0089] Embodiment 2 primer probe concentration screening
[0090] (1) Primer
[0091] Upstream primer: 5'-CTAAAGCAGTTACAGTCCCGACACTGCCG-3'; SEQ ID NO.4
[0092] Downstream primer: 5'-CCGATGTCTGAGATTATTTTTACTGCTCTGCC-3'; SEQ ID NO.6
[0093] (2) Probe
[0094] The probe sequence is:
[0095] 5'-CAGAAATGAGGAATCTGCTCTCCCAGAGTCTATCAGTAAGGAAGTTCATG'; SEQ ID NO.8.
[0096] The probe is modified with a fluorescent reporter group (FAM) and a fluorescent quencher group (BHQ1);
[0097] The modified probe is: CAGAAATGAGGAATCTGCTCTCCCAGAGTC / i6FAM dT / / idSp / / iBHQ1dT / CAGTAAGGAAGTTCATG.
[0098] (3) Prepare primer probes of different concentrations, as shown in Table 3:
[0099] Table 3 Primer probe concentration list
[0100] Concentration 1 Concentration 2 Concentration 3 Concentration 4 Concentration 5 F 1μM 5μM 10μM 20μM 50μM R 1μM 5μM 10μM 20μM 50μM P 1μM 5μM 10μM 20μM 50μM
[0101] (4) Implementation method of pr...
Embodiment 3
[0114] A kind of method that RAA fluorescence method detects Nipah virus, comprises the steps:
[0115] (1) Construct a recombinant plasmid containing the target gene fragment, and obtain a plasmid extract.
[0116] (2) Turn on the constant temperature fluorescent gene detector RAA-F1620 for preheating, and set the reaction parameters. The reaction parameters are set to 39°C, and the reaction time is 12 minutes;
[0117] (3) Add 13.7 μL of water to 25 μL of reaction buffer, 2.1 μL of upstream and downstream primers and 0.6 μL of probe at a concentration of 10 μM, mix well, add to RAA fluorescent basic reaction reagent and mix to obtain a reaction master mix;
[0118] (4) Add 2.5 μL of Mg to the cap of the reaction tube + , fully mixing 4 μL of the nucleic acid extraction solution obtained in the step (1) with the reaction premix solution obtained in the step (3), and putting the obtained reaction system into a constant temperature fluorescent gene detector RAA-F1620 to detect...
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