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Kit and method for detecting angiostrongylus cantonensis in pomacea canaliculata

A technology of Angiostrongylus and apple snails, which is applied in the field of bioengineering, can solve problems such as false negatives and PCR reaction failures, and achieve the effects of strong specificity, good repeatability, and avoiding false negatives

Inactive Publication Date: 2010-09-08
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when detecting molluscs, the PCR reaction fails due to the presence of a large number of PCR inhibitors in the molluscs, and false negatives occur.

Method used

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  • Kit and method for detecting angiostrongylus cantonensis in pomacea canaliculata
  • Kit and method for detecting angiostrongylus cantonensis in pomacea canaliculata
  • Kit and method for detecting angiostrongylus cantonensis in pomacea canaliculata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Carrying out DNA extraction of apple snails with small tubes using a kit

[0050] 1) After pulverizing the lung sac tissue of apple snails, take about 50 mg, add 600 μl solution A (CTAB lysate (2% CTAB, 20mmol / L EDTA, 0.1mol / L Tris-Cl, 1.4mol / LNaCl)), 9μl B solution (mercaptoethanol), 6 μl solution C (proteinase K (20mg / mL)), digest overnight at 60°C;

[0051] 2) Add 600 μl D solution (phenol: chloroform: isoamyl alcohol (25:24:1)), mix thoroughly, and centrifuge at 10,000 rpm for 10 min;

[0052] 3) Take the supernatant, add an equal volume of solution D, mix well, and centrifuge at 10,000rpm for 10min;

[0053] 4) repeat step 3) step;

[0054] 5) Take the supernatant, add 2 times the volume of absolute ethanol, and centrifuge at 11,000rpm for 10min;

[0055] 6) Discard the supernatant, take the precipitate, add 500 μl of 75% ethanol to wash, and centrifuge at 11,000 rpm for 5 min;

[0056] 7) Discard the supernatant, precipitate naturally or dry in an ov...

Embodiment 2

[0058] Embodiment 2 adopts kit to carry out the detection of existing DNA sample

[0059] 1) Preparation of PCR reaction system: 5.9 μl of double steamed sterilized water, 7.5 μl of solution F (2×Master PCRMix), 0.6 μl of solution G (10 μmol / L primer, containing primer 1 and primer 2), Step 8 of Example 1 1μl of the existing DNA sample obtained after the completion of ), thoroughly mixed and briefly centrifuged at 500rpm for 5s;

[0060] 2) The PCR reaction system is as follows:

[0061]

[0062] 3) Take 6 μl of the PCR product and perform 1.5% agarose gel electrophoresis, observe the bands, and judge the infection of apple snails.

Embodiment 3

[0063] Example 3 Using a kit to extract and detect DNA from apple snails

[0064] 1) After pulverizing the lung sac tissue of apple snails, take about 100mg, add 700μl solution A (CTAB lysate (2% CTAB, 20mmol / L EDTA, 0.1mol / L Tris-Cl, 1.4mol / LNaCl)), 10μl B solution (mercaptoethanol), 7 μl solution C (proteinase K (20mg / mL)), digest overnight at 60°C;

[0065] 2) Add 700 μl D solution (phenol: chloroform: isoamyl alcohol (25:24:1)), mix thoroughly, and centrifuge at 16,000 rpm for 10 min;

[0066] 3) Take the supernatant, add an equal volume of solution D, mix well, and centrifuge at 16,000rpm for 10min;

[0067] 4) repeat step 3) step;

[0068] 5) Take the supernatant, add 3 times the volume of absolute ethanol, and centrifuge at 13,000rpm for 10min;

[0069] 6) Discard the supernatant, take the precipitate, add 500 μl of 75% ethanol to wash, and centrifuge at 13,000 rpm for 5 min;

[0070] 7) Discard the supernatant, precipitate naturally or dry in an oven;

[0071] 8) ...

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Abstract

The invention discloses a kit for detecting angiostrongylus cantonensis in pomacea canaliculata. The kit comprises (A) CTAB lysis buffer ( containing 2% CTAB, 20 m mol / L EDTA, 0.1mol / L Tris-Cl, 1.4mol / L NaCl); (B) mercaptoethanol; (C) protease K (20mg / mL); (D) phenol, chloroform and isoamyl alcohol (the ratio of the three componets is 25:24:1); (E) Tris- EDTA buffer (10 m mol / L Tris-Cl, 1m mol / L EDTA, pH 8.0); (F) 2*Master PCR Mix; and (G) a primer (10 mu mol / L). Furthermore, the invention also discloses a method for detecting angiostrongylus cantonensis in the pomacea canaliculata. The invention has the advantages that the kit has high sensitivity, strong specificity and good repetitiveness, and the detection method is simple and practical, provides convenience for carrying out detection on pomacea canaliculata molecules for the primary level and provides effective means for detection of winkles after the angiostrongylus cantonensis disease breaks out.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a detection kit, specifically a PCR technology-based detection kit for Angiostrongylus cantonensis in the snail, and a corresponding detection method. Background technique [0002] Angiostrongylus cantonensis (Angiostrongylus cantonensis) was discovered and named by Chinese scholar Chen Xintao in the lungs of Rattus rattus and Rattus norvegicus in Guangzhou City, Guangdong Province in 1933, belonging to the order Strongylata , Metastronylidae (Metastronylidae), Angiostrongylus (Angiostrongylus). Mainly distributed in the Pacific Ocean, some islands in the Indian Ocean and Southeast Asian countries. The development process of Angiostrongylus cantonensis is divided into larval and adult stages, and larvae are divided into stage I, stage II, stage III, stage IV and stage V larvae. Adult Angiostrongylus cantonensis parasitizes in the pulmonary artery of mice. The female worm lays eggs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 危芙蓉张仪刘和香周晓农吕山胡玲
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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