Oligonucleotide primers for detecting arbo encephalitis viruses and detection method thereof
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A technology of encephalitis virus, detection method, applied in the direction of microorganism-based methods, biochemical equipment and methods, resistance to vector-borne diseases, etc., capable of solving problems such as harm
Inactive Publication Date: 2012-04-04
CHONGQING UNIV +1
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At the same time, human consumption of food contaminated by these viruses can also cause fatal hazards
However, in the existing reports, there is no technology that can simultaneously detect and screen the above 12 arboviruses simultaneously
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Embodiment 1
[0074] Embodiment 1, sample RNA extraction
[0075] Collect mosquitoes and other blood-sucking insects, add 800 μL TriPure Isolation Reagent (Roche, USA), grind to a transparent emulsion, add 200 μL chloroform, shake for 60 s, then centrifuge at 12000 r / min at 4 °C for 15 min at high speed, take 500 μL supernatant, and Add an equal volume of isopropanol and let stand at room temperature for 30 min, centrifuge for 15 min as before, discard the supernatant, and wash once with 70% ethanol. The RNA pellet was dissolved in 20 μL RNAse-free water.
Embodiment 2
[0076] Example 2, reverse transcription amplification (RT) reaction
[0077] Thaw the RT reaction reagent and sample RNA on ice, and prepare the reaction solution according to the system shown in the table below.
[0078] Table 1. Preparation of RT reaction solution
[0079]
[0080] RT reaction conditions: 50°C, 30 min, 85°C, 5 min.
[0082] Melt the PCR reagents and cDNA at room temperature, and prepare the reaction solution according to the system shown in the table below.
[0083] Table 2. Preparation of PCR reaction solution
[0084]
[0085] PCR reaction conditions: pre-denaturation at 95°C for 5 min; 5 cycles of pre-amplification: 94°C for 30 s; 49°C for 35 s; 72°C for 60 s; the second stage of 35 cycles: 94°C for 40 s and 60 ℃, 40 s, 70 ℃, 3 min; 72 ℃, 10 min.
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Abstract
The invention relates to oligonucleotide primers for detecting arbo encephalitis viruses, which comprise 12 pairs of virus specific primers, 1 pair of universal amplification primers and 1 pair of contrast primers inside a mosquito. In the detection step, firstly, an object to be detected is required to carry out an RT (Reverse Transcriptase) amplification reaction and a PCR (Polymerase Chain Reaction) amplification reaction, and a signal to be detected is amplified. The amplification product is detected by using an electrophoresis system, and the rapid detection and the synchronous identification of 12 kinds of viruses are realized by analyzing the segment length of the amplification product. The primers can be used for rapidly realizing detection and identification about whether the unknown sample carries the 12 kinds of arbo encephalitis viruses. The detection has high sensitivity and strong specificity. The primers provided by the invention can be applied to the efficient detection and virus identification of mosquitoes and other blood sucking insects under different environments such as departure and entry ports, baffle fields, livestock houses, rainforests, swamps and the like.
Description
technical field [0001] The invention belongs to the field of virus detection by molecular biological methods, in particular a method for detecting 12 kinds of encephalitis viruses. Background technique [0002] Eastern Equine Encephalomyelitis Virus, Western Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Virus, West Nile Virus, Highland J Virus, A Coruña Virus, Sindbis Virus, Japanese Encephalitis Virus, St. Louis Encephalitis Viruses, Ockelbo, Chikungunya, and Dengue III are encephalitis viruses transmitted by mosquitoes (or other wild animals). Traditional detection techniques and methods are mainly for serological or molecular biological detection of a small number of such viruses, generally no more than 5. The detection throughput is small, and the detection time takes a long time. The present invention is mainly based on adding a pair of external primers to both ends of the virus-specific primers, and on the basis of specific amplification, the ex...
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