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Isospora suis oocyst detection method based on LAMP real-time fluorescence and isospora suis oocyst detection primer

A detection method and real-time fluorescence technology, applied in the biological field, can solve the problems of low sensitivity and complicated operation, and achieve the effect of strong specificity

Inactive Publication Date: 2015-08-26
LONGYAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] The purpose of the present invention is to provide a simple, convenient and fast method for detecting Isospora suis oocysts based on the LAMP real-time fluorescence method in view of the deficiencies such as cumbersome operation and low sensitivity in the current method for detecting Isospora suis and its primers

Method used

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  • Isospora suis oocyst detection method based on LAMP real-time fluorescence and isospora suis oocyst detection primer
  • Isospora suis oocyst detection method based on LAMP real-time fluorescence and isospora suis oocyst detection primer
  • Isospora suis oocyst detection method based on LAMP real-time fluorescence and isospora suis oocyst detection primer

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Embodiment 1

[0066] The method for detecting Isospora porcine oocysts based on LAMP real-time fluorescence method comprises the following steps

[0067] 1. Collection and purification of Isospora suis oocysts

[0068] Collect fresh piglet feces samples, collect oocysts with saturated saline flotation method, and use Sheather's sucrose solution gradient centrifugation method to purify the collected oocysts. The specific operation method is: take the feces containing Isospora suis oocysts Dissolve in 10 times the volume of clean water, filter with 80-mesh and 120-mesh metal screens, then centrifuge at 2000rpm for 3min, discard the supernatant, add 10 times the volume of precipitated saturated saline, centrifuge at 3000rpm for 3min, take the supernatant and add it to the 2.5% potassium dichromate solution with a concentration of 6 times the volume of the serum is the collected oocyst fluid;

[0069] Take the collected oocyst fluid for 1:1 Sheather's sucrose solution gradient centrifugation, ...

Embodiment 2

[0083] The method for detecting Isospora porcine oocysts based on LAMP real-time fluorescence method comprises the following steps

[0084] 1. Collection and purification of Isospora suis oocysts

[0085] Collect fresh piglet feces samples, collect oocysts with saturated saline flotation method, and use Sheather's sucrose solution gradient centrifugation method to purify the collected oocysts. The specific operation method is: take the feces containing Isospora suis oocysts Dissolve in 9 times the volume of clear water, filter through 80 mesh and 120 mesh sieves successively, then filter with 80 mesh and 120 mesh metal screens, then centrifuge at 2500rpm for 2min, discard the supernatant, add 9 times the volume of precipitated saturated brine, 3500rpm Centrifuge for 2 minutes, take the supernatant and add 2.5% potassium dichromate solution of 5 times the volume of the supernatant, which is the collected oocyst fluid;

[0086] Take the collected oocyst fluid for 1:1 Sheather's...

Embodiment 3

[0101] The method for detecting Isospora porcine oocysts based on LAMP real-time fluorescence method comprises the following steps

[0102] 1. Collection and purification of Isospora suis oocysts

[0103] Collect fresh piglet feces samples, collect oocysts with saturated saline flotation method, and use Sheather's sucrose solution gradient centrifugation method to purify the collected oocysts. The specific operation method is: take the feces containing Isospora suis oocysts Dissolve in 10 times the volume of clear water, filter with 80-mesh and 120-mesh metal screens, then centrifuge at 2000-2500rpm for 2-3min, discard the supernatant, add 9-10 times the volume of precipitated saturated brine, and centrifuge at 3000-3500rpm for 2 -3min, take the supernatant and add 2.5% potassium dichromate solution with a concentration of 5-6 times the volume of the supernatant, which is the collected oocyst fluid;

[0104] Take the collected oocyst fluid for 1:1 Sheather’s sucrose solution ...

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Abstract

The invention discloses an isospora suis oocyst detection method based on LAMP (loop-mediated isothermal amplification) real-time fluorescence and an isospora suis oocyst detection primer. A specific primer is designed according to isospora suis 18S rRNA, and together with a fluorescence quantitative PCR instrument, the isospora suis LAMP real-time fluorescence detection technology is built. By adopting the method, the detection limit of an isospora suis recombinant plasmid is 2.74*10<2> copy / [mu]L which is converted into the plasmid DNA concentration of 1 fg / [mu]L; the LAMP detection testing result of a specific number of extracted oocysts shows that the LAMP detection limit is DNA of 5 oocysts. Through building of the LAMP real-time fluorescence detection technology, an extremely valuable detection method is provided for molecular detection of isospora suis, and the shortcomings of a traditional microscope examination method are made up.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for detecting Isospora suis oocysts based on a LAMP real-time fluorescence method and a primer thereof, which is a method for detecting oocysts in feces by using a loop-mediated isothermal amplification technology combined with a fluorescent quantitative PCR instrument. A method for the rapid detection of Isospora suis oocysts. Background technique [0002] Pig coccidiosis is mainly caused by Isospora suis parasitic in the jejunum and ileum, and suckling piglets less than three weeks old may have clinical symptoms such as diarrhea and weight loss after infection. Sows have little effect on the transmission of porcine coccidiosis, and coccidia oocysts are rarely detected clinically. The farrowing room contaminated by Isospora suis oocysts is the main way for one generation of piglets to the next generation of piglets. Pig farm environment, clinical medication, and management...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/90
CPCC12Q1/6844C12Q1/6893C12Q2531/119C12Q2563/107C12Q2561/113
Inventor 王寿昆黄翠琴温福利岳良平
Owner LONGYAN UNIV
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