RT-LAMP reagent kit for detecting yellow fever viruses, and special primer of RT-LAMP reagent kit
A technology of RT-LAMP and yellow fever virus, applied in the direction of microorganism-based methods, DNA/RNA fragments, microorganisms, etc., can solve the problems of high environmental requirements for virus isolation, failure to meet detection needs, and many influencing factors, etc., and reach a broad market Foreground, suitable for large-scale promotion and application, and the effect of high specificity detection
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Embodiment 1
[0037] Example 1: Primer Design for RT-LAMP Detection of Yellow Fever Virus
[0038] This embodiment retrieves 43 yellow fever virus genome sequences altogether from the GenBank database, carries out homology analysis by BLAST, screens, intercepts and finally determines that a section of specific conserved fragment of yellow fever virus is the target gene sequence (SEQ ID in the sequence listing NO: 1), then according to the target gene sequence, use the software Primer design V5 (http: / / primerexplorer.jp / lampv5e / index.html) to design primers for RT-LAMP detection of yellow fever virus, a total of Four sets of yellow fever virus detection primers were designed, and the nucleotide sequences of each set of primers are shown in Table 1 below.
[0039] Table 1: Nucleotide sequence information of yellow fever virus universal detection primers
[0040]
[0041] Utilize above-mentioned four groups of detection primers respectively to yellow fever virus positive plasmid pGSI-YF-NS (...
Embodiment 2
[0043] Embodiment 2: the establishment of yellow fever virus RT-LAMP detection method
[0044] In this embodiment, under the same reaction system (described in detail below), the NS2a-4 primer set of Example 1 was used at 58-65° C. The sample (yellow fever virus culture, derived from the Wuhan Institute of Virology, Chinese Academy of Sciences) was extracted and obtained) for RT-LAMP detection to determine the optimum reaction temperature, including the following steps:
[0045] 1) Preparation of reaction system: using yellow fever virus genomic RNA (final concentration>1ng / μl) as template, 25μl RT-LAMP reaction system includes: genomic RNA 2μl, 2.5μl 10×ThermoPol Reaction Buffer, 5mM betaine 4μl, 100mM MgSO 4 1.5μl, 10mM dNTP 3.5μl, Bst DNA polymerase 1μl, WarmStart RTxreverse transcriptase (New England Biolabs Inc., Massachusetts, USA, add when the template is RNA, that is, add when RT-LAMP) 0.5μl, the amount of primer added: 100μM FIP 0.8 μl of each BIP primer (final conc...
Embodiment 3
[0049] Embodiment 3: the specificity, the sensitivity of the RT-LAMP detection method of yellow fever virus
[0050] In this example, the NS2a-4 primer set obtained in Example 1 was used to detect yellow fever virus by using the yellow fever virus RT-LAMP detection method to test the specificity and sensitivity of the method.
[0051] 3.1. Specificity detection
[0052] This step uses yellow fever virus positive plasmid pGSI-YF-NS, hepatitis C virus HCV genome full-length plasmid, hepatitis B virus HBV genome full-length plasmid, human acquired immunodeficiency virus HIV genome full-length plasmid, SARS coronavirus Envelope protein S1 C-terminal deletion of 19 AA plasmids pCAGGS-SARS-S1Δ-19, Middle East respiratory syndrome virus MERS spike protein particle, coronavirus OC43 envelope protein S1 C-terminal deletion of 17 AA plasmids pCAGGS-OC43-S1Δ-17, Ebola virus Ebola envelope glycoprotein particle, type A influenza virus Influenza A virus, Neisseria meningitidis group B B C...
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