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Multi-suspension chip for detecting Cryptosporidium and Giardia lamblia and preparation thereof

A technology for Giardia lamblia and Cryptosporidium, applied in biochemical equipment and methods, microbe measurement/inspection, resistance to vector-borne diseases, etc., can solve the problem of lack of sensitivity, high false positive rate of PCR, and problems Problems such as floating chips, to achieve the effects of increased sensitivity, easy promotion, and low cost

Inactive Publication Date: 2009-05-27
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is a high probability of false positives in PCR, and its sensitivity is also lacking.
[0006] At present, there is no report on the suspension chip for the detection of Cryptosporidium and Giardia lamblia

Method used

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  • Multi-suspension chip for detecting Cryptosporidium and Giardia lamblia and preparation thereof
  • Multi-suspension chip for detecting Cryptosporidium and Giardia lamblia and preparation thereof
  • Multi-suspension chip for detecting Cryptosporidium and Giardia lamblia and preparation thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Primer design and synthesis

[0070] 1. Materials: Molecular biology software Primer Premier 5.0, DNAMAN, oligo 6.0 and NCBI network resources.

[0071] 2. Method results:

[0072] 1. Sequence acquisition: Through genome-wide analysis of Cryptosporidium and Giardia lamblia, the corresponding species-specific detection gene was selected as the target sequence, and the gene sequence was obtained from the GenBank public database. The accession numbers are respectively : XM_626719, X64340, X85958.

[0073] 2. Design primers: search for the sequences of all effective species of Cryptosporidium in the NCBI database, analyze and compare the sequences with DNAMAN software, find out the conserved region of the gene, use Primer Premier 5.0 software to design genus-specific primers, and use oligo 6.0 to perform Analytical verification. Cryptosporidium species-specific and Giardia species-specific primers are designed directly from the specific diagnostic sequence. The design me...

Embodiment 2

[0077] Probe Design and Synthesis

[0078] 1. Materials: Molecular biology software Primer Premier 5.0, oligo 6.0 and NCBI network resources.

[0079] 2. Method results:

[0080] 1) Sequence acquisition: designing probes in the non-primer region of the amplified fragment;

[0081] 2) Design probes: use Primer Premier 5.0 software to design probes, select the Hybridization Probes command according to the length of the probes recommended by Luminex Company at a length of 18bp-22bp, and select the Anti-sense or sense chain according to the formation of Dimer between primers and probes On design probe, parameter is with embodiment one;

[0082] 3) Probe selection: Properly adjust the probes output by the software manually, increase or decrease a few bases, then perform online Blast comparison in GenBank, select probes with high specificity, and perform NH at the 5' end 2 -(CH 2 ) 12 Modification, the selected probe sequence is ProH, ProC, ProG;

[0083] 4) Probe synthesis: D...

Embodiment 3

[0085] Preparation method of suspension chip

[0086] 1. Materials: synthesized probes, microspheres with spectral addresses, brown light-proof EP tubes, 2-(N-morpholino)ethanesulfonic acid, 1-ethyl-3-3-dimethylaminopropyl Carbodiimide-based, high-speed centrifuge.

[0087] 2. Method results:

[0088] 1. Dilute the amino-modified probe to 1 mM (1 nanomole / μl) with pure water;

[0089] 2. Shake the stored microspheres for 20 sec;

[0090] 3. Transfer 200 μl microspheres to a brown EP tube;

[0091] 4. Collect microspheres, centrifuge at 8000g for 1-2min;

[0092] 5. Discard the supernatant, add 50 μl 0.1M MES (2-[N-Morpholino]ethanesulfonic acid, 2-(N-Morpholino)ethanesulfonic acid) solution pH 4.5, shake for 20 sec;

[0093] 6. Add 2 μl of 1mM probe to the suspended microspheres and shake for 20 sec;

[0094] 7. Prepare fresh 10mg / ml EDC (1-ethyl-3-[3dimethylaminopropyl]carbodiimidehydrochloride, 1-ethyl-3-3-dimethylaminopropyl carbodiimide) with dH2O;

[0095] 8. Add 2...

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Abstract

The invention provides a multiplex suspension chip for the detection of cryptosporidium species and Giardia lambila Stiles. The chip comprises diagnosis primer and specific oligonucleotide probes coupled with microspheres, wherein the sequence of the oligonucleotide probes is a sequence in specific genes of cryptosporidium species or genus specific or giardia lamblia species. The chip is combined with a multiplex PCR technique and a liquid-phase hybridization technique, can effectively eliminate false positivity, guarantees accurate specificity, improves sensibility by 10 to 1,000 times than common PCR technique, can simultaneously identify and detect two types of aquagenic protozoa, such as cryptosporidium species, C.parvum, Giardia lambila Stiles and the like, and has the characteristics of high specificity, high sensibility, speediness, low cost, popularization easiness and the like.

Description

technical field [0001] The invention relates to a molecular biology detection chip, in particular to a multiple suspension chip for detecting Cryptosporidium species and Giardia lamblia, and also relates to a preparation method of the chip, which belongs to the technical field of biological detection. Background technique [0002] Cryptosporidium spp. includes: Cryptosporidium parvum (C.parvum), Cryptosporidium andersoni (C.andersoni), Cryptosporidium bayeiyi (C.baileyi), Cryptosporidium turkey (C. meleagridis), Cryptosporidium hominis (C.hominis) and Porcine Cryptosporidium (Porcine Cryptosporidium), are a wide range of zoonotic parasitic protozoan diseases that can cause mammals, especially ruminants and humans severe diarrhea. [0003] Giardia lambila Stiles (Giardia lambila Stiles, 1915) is a zoonotic protozoan distributed worldwide. It can cause Giardia lamblia disease in humans or animals. The main symptom is diarrhea. [0004] Cryptosporidium and Giardia lamblia bel...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCY02A50/30
Inventor 李建华张楠李巍张西臣宫鹏涛李淑红
Owner JILIN UNIV
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