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Real-time fluorescence PCR (polymerase chain reaction) primer and probe for identifying culex tritaeniorhynchus and application of real-time fluorescence PCR primer and probe

A real-time fluorescence and probe technology, applied in the field of insect identification, achieves high sensitivity, solves the problem of mosquito species identification, and is easy to operate

Inactive Publication Date: 2016-02-17
FUJIAN INT TRAVEL HEALTH CARE CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the fluorescent PCR identification method for Culex tritaeniorhynchus

Method used

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  • Real-time fluorescence PCR (polymerase chain reaction) primer and probe for identifying culex tritaeniorhynchus and application of real-time fluorescence PCR primer and probe
  • Real-time fluorescence PCR (polymerase chain reaction) primer and probe for identifying culex tritaeniorhynchus and application of real-time fluorescence PCR primer and probe
  • Real-time fluorescence PCR (polymerase chain reaction) primer and probe for identifying culex tritaeniorhynchus and application of real-time fluorescence PCR primer and probe

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Primer Design:

[0041] According to the gene sequence of Culex tritaeniorhynchus COI (cytochromeoxidaseI) in GenBank (gene accession numbers AB690857, AB738091, HM638220, HQ398885, JQ728031, KC970299, KF407833, KM502254), the primers Culex-F and Culex-R, and the specific probe Tritaeniorhynchus were designed -P.

[0042] Fluorescence PCR upstream primer Culex-F: 5'-TTCAAGTAGTTTAGTAGAAAATGGAGC-3' (as shown in SEQ ID NO.1)

[0043] Fluorescent PCR downstream primer Culex-R: 5'-TGTAAAGAAAAAATAGCTAAATCAACTG-3' (as shown in SEQ ID NO.2)

[0044] Fluorescent PCR probe Tritaeniorhynchus-P: FAM-5'-ACAGTTTTATCCACCTCT-3'-MGB (as shown in SEQ ID NO.3)

Embodiment 2

[0045] Example 2 mosquito DNA extraction:

[0046] Mosquito DNA was extracted using commercial InsectDNAKit (OMEGA). The extracted DNA was tested directly on the machine or stored at -20°C.

Embodiment 3

[0047] The preparation of embodiment 3 standard plasmids

[0048] Referring to the literature DNAprimersforamplificationofmitochondrialcytochromecoxidasesubunitIfromdiversemetazoaninvertebrates (FolmerO, BlackM, HoehW, etal.MolMarBiolBiotechnol.1994,3(5):294-299) synthesis and amplification of COI gene upper and lower primers LCO1490 and HCO2198.

[0049] Culex tritaeniorhynchus DNA was extracted according to the instructions of InsectDNAKit (OMEGA).

[0050] The PCR reaction was performed using the commercialized ExTaq from Treasure Bio (Dalian) Co., Ltd. Add 5 μL of 10×Buffer, 4 μL of 2.5 mmol / LdNTP, 1 μL of 20 μmol / L upstream and downstream primers LCO1490 and HCO2198, 0.25 μL of ExTaq, 5 μL of DNA template, and supplemented with dHO in the PCR reaction tube. 2 0 to 50 μL. The reaction program was: pre-denaturation at 94°C for 3 minutes; 94°C for 30s, 45°C for 30s, 72°C for 1min, 5 cycles; 94°C for 30s, 51°C for 1mins, 72°C for 1min, 35 cycles; 72°C for 10min. PCR produc...

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Abstract

The invention discloses a real-time fluorescence PCR (polymerase chain reaction) primer and probe for identifying culex tritaeniorhynchus and an application of the real-time fluorescence PCR primer and probe. The primer comprises an upstream primer Culex-F and a downstream primer Culex-R, wherein the sequence of the upstream primer Culex-F is shown in SEQ ID NO.1, and the sequence of the downstream primer Culex-R is shown in SEQ ID NO.2; the probe is Tritaeniorhynchus-P, and the sequence of the MGB probe Tritaeniorhynchus-P is shown in SEQ ID NO.3. The real-time fluorescence PCR detection method established through the primers and the probe can rapidly, accurately and specifically identify culex tritaeniorhynchus from mosquito species of a seaside culex subgroup; in case of need, DNA can be extracted from a single mosquito leg for identification, stages of a life history of mosquitoes can be identified, and an incomplete specimen can be identified. As for actual work, a medium specialist is not required to be on the scene to perform medium identification work, an ordinary experimenter can perform the work, and better popularization and application value is achieved.

Description

technical field [0001] The invention belongs to the technical field of insect identification. More specifically, it relates to a real-time fluorescent PCR primer and probe for identification of Culex tritaeniorhynchus subgroup of Culex littorale and their application. Background technique [0002] Culextritaeniorhynchus (Culextritaeniorhynchus) belongs to Diptera Culexidae Culex subgenus Culex subgenus Culex seashore Culex group seaside Culex subgroup, sucking human and animal blood, is the main vector of Japanese encephalitis. Main distribution: In China, there is no record except Xinjiang and Tibet, and it is distributed all over the country; abroad, it is distributed in Pakistan, India, Bangladesh, Sri Lanka, Myanmar, Thailand, Cambodia, Vietnam, Malaysia, Singapore, Indonesia, Philippines, Japan, North Korea, the original Soviet Union, Middle East, East Africa, etc. [0003] The subgroup of Culex littoralis includes: Culex littoral, Culex tritaeniorhynchus, Culex albof...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2563/107C12Q2561/113
Inventor 王宇平高博张建庆方义亮
Owner FUJIAN INT TRAVEL HEALTH CARE CENT
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