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Method for detecting giardia by pyrosequencing technology

A Giardia and pyrosequencing technology, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, fluorescence/phosphorescence, etc., can solve the problems of low recovery rate, small amount of DNA, and large subjectivity, and achieve The effect of small sample volume, easy operation and low cost

Active Publication Date: 2014-07-30
INSPECTION & QUARANTINE TECH CENT SHANDONG ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
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Problems solved by technology

However, when density detection is used, the recovery rate is low, and microscopic analysis depends on the experience and professional background knowledge of personnel, which is highly subjective; when PCR technology is used for confirmation, if the specificity of the primers is not strong, false detection may occur. Positive; when using the fluorescent hybridization method, due to the small amount of DNA, the method is not sensitive enough

Method used

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  • Method for detecting giardia by pyrosequencing technology
  • Method for detecting giardia by pyrosequencing technology
  • Method for detecting giardia by pyrosequencing technology

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0024] Example 1: Detection of Giardia in fecal samples from 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 nucleotide sequence of Giardia tim, which does not contain unknown base component sequences. Only one strain was selected, and the tim gene nucleotide sequence of the selected sample was edited with Editseq and MegAlign software in the DNAStar7. Nucleotide sequences are compared for homology to find the specific nucleotide sequence (target detection sequence) that characterizes the insect.

[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 scores is used for experiments. According to the homology analysis results of DNASTAR, the more conservative sequence regions in the sample genes are selected, and the design Amp...

Embodiment 2

[0044] Embodiment 2: Detect whether Giardia 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 (source water 10L, filtered water 50L), after filtering through a 1μm pore size filter membrane, wash the container with PBS+0.1%Tween80, ethanol, ultrapure water in sequence, dissolve the filter membrane with 40mL 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 6 mL of PBS+0.1% Tween80 to the concentrated precipitate, shake it with a shaker for 2 minutes, mix it evenly, and divide the solution evenly into four 15 mL centrifuge tubes, and add 3 mL of centrifugal medium Percoll-sucrose to each centrifuge tube, Centrifuge at 20°C and 3000r...

Embodiment 3

[0055] Embodiment 3: Detect whether different protozoan samples contain Giardia gene

[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 Giardia strains

[0060] The Giardia strain used in this experiment was purchased from BTF, Australia. 2.2 Control DNA samples

[0061] DNA samples of Schistosoma japonicum, Toxoplasma gondii, Cryptosporidium parvum and Entamoeba histolytica were provided by Shanghai Veterinary Research Institute.

[0062] 2.3 Isolation and purification of Giardia cysts and DNA preparation

[0063] Filter the sample containing Giardia cysts through a 100-mesh fine copper mesh, centrifuge the filtrate (2000r / min, 10min), discard the supernatant, add water to the precipitate to 2mL-3mL; take another centrifuge tube, add 0.85mol / L sucrose 5 mL; add the above encapsulation solution to the upper layer of sucrose, centrifuge (2000r / min, 5min), slowly absorb 2 mL...

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Abstract

The invention discloses a method for detecting giardia by a pyrosequencing technology, belonging to the technical field of detection of food-borne parasites. The method comprises the following steps of: designing a specificity primer and a pyrosequencing primer according to triose phosphate isomerase (tim) of the giardia; then extracting desoxyribonucleic acid (DNA) of a sample to be detected, and performing polymerase chain reaction (PCR) amplification by the specificity primer with a tim gene; detecting an amplified product by agarose gel electrophoresis, if an amplified section is 165bp in length, preparing a pyrosequencing single-chain template, and performing pyrosequencing reaction; and finally, judging whether the sample contains the giardia according to a PCR amplification result and a pyrosequencing result. The method is suitable for quick detection and confirmation for the giardia and can be widely applied to water quality monitoring in an environment, oocyst detection in a human body and confirmation of protozoons of the type in import and export trades.

Description

Technical field: [0001] The invention belongs to the technical field of detection of food-borne parasites, and relates to a method for detecting giardia using pyrosequencing (Pyrosequencing) technology. Background technique: [0002] Giardia lamblia is a multi-flagellated parasitic protozoan with 2 nuclei, which can parasitize in the intestines of humans and various vertebrates, and then cause Giardiasis, which is mainly diarrhea. One of the common parasites of intestinal infection, which is widespread worldwide. Due to its long survival time in the water supply system and high tolerance to chlorine and other disinfectants, polluted water poses a great threat to water supply safety. Studies have shown that the pathogenic dose of Giardia is 10 to 100 live cysts. After the human body is infected with Giardia, the main symptoms are abdominal pain, diarrhea, abdominal distension, vomiting, fever, and anorexia. Pus and blood. Diarrhea can cause malnutrition such as anemia in ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04G01N21/64
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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