Primer probe sets and methods for detecting dengue, chikungunya and measles viruses
A technology for dengue virus and measles virus, applied in the field of primer probe sets for the detection of dengue virus, chikungunya virus and measles virus, can solve the problems of ignoring early clinical symptoms, difficult to distinguish and identify diseases, etc., to reduce labor costs and time cost effect
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0057] Embodiment 1 primer, probe synthesis
[0058] The primer probe set of the present disclosure selects specific detection genes or conserved sequences, and it is necessary to ensure that the primer probe segments can fully cover dengue virus, chikungunya virus and measles virus respectively. Moreover, the problem of co-amplification of primer probes of different target genes in a reaction system should be fully considered during the design process. Avoid hairpin structures, primer-dimers, etc., and because RPA requires a primer length of 30-38nt and a probe length of 45nt, such a sequence is likely to produce a large number of primer-dimers under constant temperature conditions, which will affect the experimental results Therefore, higher requirements are put forward for the design of primers to ensure the probability of simultaneous amplification of different primers and probes in the later stage. Finally, a set of specific primer probe sequences provided by the present...
Embodiment 2
[0067] Embodiment 2 specificity verification:
[0068] Yellow fever virus, West Nile virus, forest encephalitis virus (Senzhang strain, Mudanjiang strain), Japanese encephalitis virus, and Marburg virus rubella virus transcribed in vitro were selected as specificity evaluation samples. The nucleic acid concentration of each sample was 0.001ng / μL, and the nucleic acid of each sample was mixed as a specific detection template, and RPA amplification was performed using the primer-probe combination in Table 1 and the final concentration in Table 2.
[0069] Table 2 Final concentration of dengue virus, chikungunya virus and measles virus primers and probes
[0070] Target Primer Final Concentration Probe Final Concentration dengue virus 0.4μM 0.1μM chikungunya virus 0.4μM 0.1μM measles virus 0.4μM 0.1μM
[0071] RPA reaction system configuration: total system 50 μL, add 29.5 μL of rehydration buffer, 2.5 μL (280 mmol / L) of magnesium acet...
Embodiment 3
[0075] Embodiment 3 minimum detection limit verification:
[0076] Use restriction endonucleases SpeI and PvuII to digest the standard plasmids recombined with gene sequences of dengue fever virus, chikungunya virus and measles virus, linearize them and carry out in vitro transcription; the initial concentration is 10 4 Copy / μL Transcript products were mixed in equal proportions and serially diluted as template I, diluted to 10 3 copies / μL, 10 2 copies / μL, 10 copies / μL, 2 copies / μL and 1 copy / μL as templates II, III, IV, V, VI. System preparation and RPA reaction conditions are the same as those in Example 2 for specificity evaluation. In the reaction system, the concentration of the virus was 5×10 4 copy / system, 5×10 3 copy / system, 5×10 2 copies / system, 50 copies / system, 10 copies / μL and 5 copies / system.
[0077]Judgment of RPA reaction results: blank control, IAC and negative control are established, otherwise the experimental results will be considered invalid; if the...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More