Method for detecting cryptosporidium by using pyrosequencing technology

A technology of pyrosequencing and cryptosporidium, which is applied in microorganism-based methods, microbial determination/inspection, biochemical equipment and methods, etc. Small volume and easy operation

Active Publication Date: 2014-04-09
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this classic method has complex detection procedures, long cycle, time-consuming and laborious, expensive detection reagents, microscope inspection must be performed by experienced professionals, and the results are highly subjective
Very unsuitable for confirming use in various grassroots

Method used

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  • Method for detecting cryptosporidium by using pyrosequencing technology
  • Method for detecting cryptosporidium by using pyrosequencing technology
  • Method for detecting cryptosporidium by using pyrosequencing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Detecting whether Cryptosporidium is contained in fecal samples of infected persons

[0025] 1. Design specific primer sequences

[0026] Log in to the National Center for Bioinformatics (NCBI) of the United States through the Internet to query and retrieve the published 18SrRNA nucleotide sequence of Cryptosporidium, which does not contain unknown base component sequences. Select only one strain, use the Editseq and MegAlign software in the DNAStar7.1 software package to edit the 18S rRNA gene nucleotide sequence of the selected sample, compare them one by one, and use the default parameters of the Clustal W Method (software) to align the selected 18S The rRNA nucleotide sequence is compared for homology, and the specific nucleotide sequence (target detection sequence) that characterizes the insect is found.

[0027]The design of PCR amplification primers and sequencing primers can be carried out by Assay Design SW software, and a set of primers with higher...

Embodiment 2

[0044] Embodiment 2: Detect whether cryptosporidium is contained in certain waters

[0045] 1. Design specific primer sequences

[0046] With embodiment 1.

[0047] 2. Extract the DNA of the sample to be tested:

[0048] Collect a certain volume of water samples (10L of source water, 50L of filtered water), filter through a 1μm pore size filter membrane, wash the container with PBS+0.1%Tween80, ethanol, and ultrapure water in sequence, dissolve the filter membrane with 40mL of acetone, and dissolve the filter membrane at 3000r / After centrifuging at 2 min to remove the supernatant, add 40 mL of acetone solution again and centrifuge to remove the supernatant to obtain the corresponding precipitate.

[0049] Add 6mL of PBS+0.1%Tween80 to the concentrated precipitate, shake it with a shaker for 2min, mix evenly and divide the solution into four 15mL centrifuge tubes, add 3mL of centrifuge medium Percoll-sucrose to each centrifuge tube, Centrifuge at 20°C and 3000r / min for 10mi...

Embodiment 3

[0055] Example 3: Detecting whether Cryptosporidium genes are contained in different protozoan samples

[0056] 1. Design specific primer sequences

[0057] With embodiment 1.

[0058] 2. Extract the DNA of the sample to be tested

[0059] 2.1 Source of Cryptosporidium strains

[0060] The Cryptosporidium strains used in this experiment were purchased from BTF Company in Australia, and mice were used to preserve the species.

[0061] 2.2 Control DNA samples

[0062] DNA samples of Schistosoma japonicum, Toxoplasma gondii and Entamoeba histolytica were provided by Shanghai Veterinary Research Institute. Cryptosporidium (Giardia lamblia) samples were prepared in our laboratory

[0063] 2.3 Isolation and purification of Cryptosporidium and DNA preparation

[0064] Filter the sample containing Cryptosporidium cysts through a 100-mesh fine copper mesh, centrifuge the filtrate (3000r / min, 10min), discard the supernatant, add normal saline to the precipitate to 2mL; take anothe...

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Abstract

The invention discloses a method for detecting cryptosporidium by using a pyrosequencing technology. The method comprises the following steps of: firstly, designing specific primers and pyrosequencing primers according to cryptosporidium 18S rRNA (Ribosomal Robonucleic Acid); extracting desoxyribonucleic acid (DNA) of a sample to be tested; performing polymerase chain reaction (PCR) proliferation by using the specific primers of 18SrRNA genes; detecting a proliferated product by using agarose gel electrophoresis; preparing a pyrosequencing single-chain template if the proliferation segment length of the primer is 201bp; carrying out pyrosequencing reaction; and finally, determining whether the sample contains giardia according to a PCR proliferation result and a pyrosequencing result. The method is suitable for rapidly detecting and confirming the cryptosporidium, can be widely applied to water quality monitoring and control in an environment, oocyst detection of excrement of a human body and confirmation of protozoon in import and export trades, and is convenient to operate and small in required sample amounts.

Description

Technical field: [0001] The invention belongs to the technical field of detection of food-borne parasites, and relates to a method for detecting cryptosporidium by adopting pyrosequencing (Pyrosequencing) technology. Background technique: [0002] Cryptosporidium is a tiny coccidia parasite. Widely exist in a variety of vertebrates, the main one that parasitizes humans and most mammals is C.parvum, and the disease caused by Cryptosporidium is called cryptosporidiosis. Diarrhea is the main clinical manifestation of zoonotic protozoan disease. It is distributed worldwide, and its incidence rate is similar to Campylobacter jejuni, Salmonella, Shigella, and pathogenic Escherichia coli, and it ranks first or second in parasitic diarrhea. [0003] In 1984, two consecutive large-scale diarrhea occurred among about 6,000 residents in San Antonio, Texas, and a total of 2,006 people were infected by Cryptosporidium, with an incidence rate of about 34%. It was the first confirmed cas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/90
Inventor 孙涛邓明俊肖西志王群郑小龙凌宗帅
Owner INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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