Swine-derived eperythrozoon detecion kit, method and applications

A technology of eperythrozoon and detection kit is applied in the field of porcine-derived eperythrozoon detection kits, which can solve the problems of pollution and easy occurrence of false positives, and achieve the effects of good repeatability, high sensitivity and strong specificity

Inactive Publication Date: 2014-11-19
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Pig blood samples collected clinically may be contaminated by various bacteria, and PCR amplification of 16SrRNA gene is prone to false positives

Method used

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  • Swine-derived eperythrozoon detecion kit, method and applications
  • Swine-derived eperythrozoon detecion kit, method and applications
  • Swine-derived eperythrozoon detecion kit, method and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Cloning and sequence analysis of porcine Eperythrosome G1 gene

[0037] (1) Primer design:

[0038] According to the porcine Eperythrosome G1 protein coding gene sequence published by GenBank (GenBank accession number: AM407404.1), the specific primers for amplifying the full-length coding gene of porcine Eperythrosome G1 protein were designed by using Primer Premier5.0 software. The sequence is as follows:

[0039] Upstream primer MF1: 5'-GCGGGATCCATGACAATCCACAAAGTAGC-3' (SEQ ID NO: 5);

[0040] Downstream primer MR1: 5'-CGCCTGCAGTTAAAGAGAAATGTAGTA-3' (SEQ ID NO: 6).

[0041] (2) Genomic DNA extraction from pig blood samples:

[0042] Use a whole blood genomic DNA extraction kit (resin type, purchased from Saibaisheng Company, product catalog number: CUC-100), and extract genomic DNA from pig blood samples according to the instructions of the kit: Take 500 μL of sterile anticoagulated whole blood to 1 mL In the purified resin, mix by inverting 5-6 times. ...

Embodiment 2

[0065] The preparation of embodiment 2 positive recombinant plasmid standard template

[0066] (1) Identification of positive plasmids

[0067] Cut the gel of the target band, recover and purify it with a gel recovery kit, clone the target fragment into the pMD18-T vector and transform it into Escherichia coli DH5α competent cells, culture it overnight at 37°C in a solid medium containing ampicillin, and select a single colony for Cultivate, extract the plasmid, and identify it by restriction enzyme analysis and sequencing. The obtained sequence was compared with the sequence published on GenBank to determine the positive recombinant plasmid.

[0068] (2) Determination of the concentration of positive plasmid

[0069] After dissolving the recombinant plasmid DNA with sterilized double distilled water, measure the plasmid concentration in a nucleic acid protein analyzer at 260nm and 280nm wavelengths, and calculate the copy number of plasmid DNA per microliter of liquid accor...

Embodiment 3

[0072] Example 3 Establishment of real-time fluorescent quantitative PCR detection method for porcine-derived Eperythrozoon

[0073] (1) Design of primers and probes:

[0074] A pair of specific primers and TaqMan probes were designed according to the full-length sequence of the porcine Eperythrosome G1 gene. The fluorescent reporter group labeled at the 5' end of the probe was FAM, and the quencher group labeled at the 3' end was MGB. The length of the fragment is 136bp, which is the nucleotide sequence between the 658th and 793rd bases of the sequence of SEQ ID NO:1. The sequences amplified by the primers and probes are shown in the box of the full-length sequence of Eperythrozoon porcine G1 in Example 1. The designed primers and probe sequences are as follows:

[0075] MF2: 5'-GCTGCTGCAATTGGAAGAGTA-3' (SEQ ID NO: 2);

[0076] MR2: 5'-TGATTTCTTCTGCAGAAACAGACT-3' (SEQ ID NO: 3);

[0077] Probe: 5'(FAM)-TTGATGGAATTGCACACAGA-3'(MGB) (SEQ ID NO: 4).

[0078] (2) Determinati...

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Abstract

The invention discloses a swine-derived eperythrozoon detecion kit, a method and applications. The kit comprises a TaqMan probe and a primer pair designed aimed at a swine-derived eperythrozoon G1 gene. The probe binds to the 658th-793th nucleotide sequence of the swine-derived eperythrozoon G1 gene specifically. The primer pair is used for amplification of the 658th-793th nucleotide sequence of the swine-derived eperythrozoon G1 gene. The swine-derived eperythrozoon real-time fluorescence quantification PCR detection kit based on the attachment protein G1 gene has characteristics of strong specificity, high sensitivity and good repeatability, can detect whether eperythrozoon is existed in a swine blood sample rapidly, accurately and with high throughput, can be applied at aspects of clinical diagnosis, molecular epidemiological investigation, therapeutic effect evaluation, pig farm purification of swine eperythrozoonosis and the like, and has an important meaning to early state rapid diagnosis, prevention and control and purification of swine eperythrozoonosis.

Description

technical field [0001] The invention belongs to the technical field of detecting mycoplasma infection in animal blood, and in particular relates to a swine-derived Eperythrozoon detection kit, method and application. Background technique [0002] Eperythrozoon porcine is a member of the Mycoplasma hemophilus family that cannot be cultured in vitro, and generally parasitizes on the surface and inside of pig red blood cells. Eperythrozoon infection of porcine origin is widely distributed all over the world, causing severe economic losses to the swine industry. Its acute infection can lead to severe bacteremia, acute red blood cell hemolysis, and sometimes death of young piglets, abortion of pregnant sows, etc. For chronically infected pigs, the blood bacteria content is low, and the clinical symptoms are variable, such as mild jaundice, sub-health, slow growth rate, low production capacity or susceptibility to other infectious diseases. Under strong immunization and antibiot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/35
CPCC12Q1/6851C12Q2531/113C12Q2561/101C12Q2561/113
Inventor 周鹏陈兆国曹薇米荣升黄燕
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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