TaqMan probe for real-time fluorescence PCR (Polymerase Chain Reaction) detection of angiostrongylus cantonensis infected snails

A technology of angiostrongylus and probe, which is applied in the direction of fluorescence/phosphorescence, measurement/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as long detection period, emergency treatment of unfavorable disease-causing emergencies, and unsatisfactory

Inactive Publication Date: 2010-11-03
周卫川 +2
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Problems solved by technology

The traditional detection of Angiostrongylus cantonensis still remains at the stage of manual separation, microscopic examination, expert identification, etc., but the shortcomings of long detection cycle and extremely low sensitivity cannot be widely used. The applicant has successfully used the conventional PCR detecti...

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  • TaqMan probe for real-time fluorescence PCR (Polymerase Chain Reaction) detection of angiostrongylus cantonensis infected snails
  • TaqMan probe for real-time fluorescence PCR (Polymerase Chain Reaction) detection of angiostrongylus cantonensis infected snails
  • TaqMan probe for real-time fluorescence PCR (Polymerase Chain Reaction) detection of angiostrongylus cantonensis infected snails

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Embodiment Construction

[0012] 5.1 Preparation of materials

[0013] Collected from the main epidemic foci, the collection site is mainly the ditch vegetable field next to the garbage dump in the suburbs where rats are often infested.

[0014] 5.2 Establishment of real-time fluorescent PCR method

[0015] 5.2.1 Design and synthesis of primers and TaqMan probes

[0016] TaqMan probes and primers were designed simultaneously with primer design software, and their specificity was tested by NCBI Blast. Synthesized by Shanghai Sangon Biotechnology Service Co., Ltd. The primers and probes are shown in Table 1, and the length of the amplified fragment is 115bp. The 5' end of the TaqMan probe is labeled with a fluorescent reporter group of 6-carboxyfluorescein (6-FAM), and the 3' end of which is labeled with a fluorescent quencher group of tetramethylrhodamine (TAMRA).

[0017] Primers and probes for Table 1AC

[0018]

[0019] 5.2.2 Extraction of intermediate host snail DNA

[0020] 5.2.2.1 Isocyan...

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Abstract

The invention discloses a TaqMan probe for real-time fluorescence PCR (Polymerase Chain Reaction) detection of angiostrongylus cantonensis infected snails on the basis of a modern molecular biological technology, which is a detection method applied to snails infected with angiostrongylus cantonensis. The angiostrongylus cantonensis is food-borne zoonosis and is infected mainly through eating infected snails by mistake. The invention aims to provide a rapid and accurate method for detecting the snails infected with the angiostrongylus cantonensis. The TaqMan probe is technically characterized in that (1) a primer(upstream) having the sequence of 5'-TGA TGC TTG TGC GGT TTT C-3', a primer- (downstream) having the sequence of 5'-CCGAAG GCA AAG CAC ACA C-3' and a probe having the sequence of5'-CGC TAT ACA CGC ACA TTT GCC GG-3' are provided; (2) the total volume of a reaction system is 20 mu L, wherein the reaction system comprises 10 muL of 2*Taq PCR MasterMix, 1 muL of Taqman probe, 1 muL of the upstream primer, 1 muL of the downstream primer, 1 mu L of template and the balance of ddH2O; and (3) in a reaction process, the pre-degeneration time is 3min at 94 DEG C, 10s at 94 DEG C and 30s at 60 DEG C, and the circulation is carried out for 40 times. The invention provides a new technological measure for food quarantine as well as investigation and monitoring of epidemic situations and has far reaching importance on preventing and controlling the occurrence of the angiostrongylus cantonensis diseases.

Description

1 technical field [0001] The invention relates to a novel molecular biology detection method, in particular to establishing a PCR detection method for snails infected with Angiostrongylus cantonensis (Angiostrongylus cantonensis, AC). 2 background technology [0002] Angiostrongylus cantonensis is a new food-borne zoonotic disease. Frequent outbreaks of the disease have been caused by eating shellfish such as apple snails, escargots, ring snails, and brown cloud agate snails by mistake. So far there is no report on the detection of Angiostrongylus cantonensis from intermediate hosts. The traditional detection of Angiostrongylus cantonensis still remains at the stage of manual separation, microscopic examination, expert identification, etc., but the shortcomings of long detection cycle and extremely low sensitivity cannot be widely used. The applicant has successfully used the conventional PCR detection method to detect snails. cantonensis, but the detection cycle is long a...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 周卫川魏纪玲佘书生邵碧英王寿昆陈文炳陈寿玲
Owner 周卫川
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