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Inert carrier indirect agglutination test detection system and its application

A technology of agglutination test and detection system, which is applied in the fields of biomedicine and immunodiagnosis, can solve the problems of low antibody detection rate and possible positive reaction of Enterobacteriaceae, and achieve clear and easy-to-judgment results and rapid positive reactions

Active Publication Date: 2021-01-05
YANGZHOU UNIV
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Problems solved by technology

[0006] Lipopolysaccharide was extracted from pullorum and Salmonella gallinarum typhi as antigen, and an enzyme-linked immunosorbent assay (ELISA) for the detection of pullorum pullorum and Salmonella gallinarum typhi has also been developed. This technology can be used in laboratory scale serum samples. Infection antibody detection, to a certain extent, overcomes the above-mentioned low sensitivity of the classic plate agglutination reaction, including the low detection rate of antibodies in infected chicks with subclinical infection and relatively weak immune response ability
It has also been reported that purified flagellin, outer membrane protein, and pili protein are used as ELISA detection antigens to detect Salmonella Enteritidis infection in chickens, but like other serological tests, ELISA technology is positive to a certain extent for other Salmonella. Possibly positive for Enterobacteriaceae, especially Escherichia coli

Method used

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  • Inert carrier indirect agglutination test detection system and its application
  • Inert carrier indirect agglutination test detection system and its application
  • Inert carrier indirect agglutination test detection system and its application

Examples

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Embodiment 1

[0048] The Salmonella S9 used in the present invention has been preserved in the China General Microorganism Culture Collection Management Center (CGMCC), the preservation address is Beijing, China, the preservation number is CGMCC No.17340, the preservation date is March 18, 2019, and the classification is named Salmonella (Salmonella sp.), strain code S9. Example 1 Identification and verification of Salmonella pullorum P antigenic factor

[0049] Search the uploaded P antigen factor coding gene P in Salmonella pullorum pullorum (Table 1) through the GenBank database on NCBI, and compare the downloaded p gene sequences with DNAMAN Windows version software, and the results show that p in different Salmonella pullorum pullorum strains The gene sequence is highly conserved ( figure 1 ). In fact, it is also highly conserved with the p gene sequences of multiple strains of Salmonella pullorum isolates isolated and identified by our laboratory and determined by DNA sequence.

[...

Embodiment 2

[0054] Example 2 Construction of inert carrier indirect agglutination test detection system S9-P

[0055] According to the full-length fragment of the p gene of Salmonella pullorum in NCBI, the amplification primers were designed with Olige7 software, and NheI and BamHI restriction sites and protective bases were added to the 5' ends of the upstream and downstream primers respectively. The upstream and downstream primers were respectively:

[0056] p-UP: 5′-ATG AAA CGT TCA CTT ATT GCT GCT-3′

[0057] p-LO: 5'-TTAA TCA GTT AAT ACC GTC ATC GTC AG-3';

[0058] Prepare CVCC526 Salmonella pullorum template by boiling method, p-PCR system: 5×pfu DNA polymerase buffer 10 μL, dNTP 5 μL, upstream primer 2 μL, downstream primer 2 μL, template 2 μL, pfu high-fidelity enzyme (2.5 units / uL) 2 μL , 27 μL of deionized water. PCR reaction conditions: 94°C for 5min, 94°C for 1min, 52°C for 1min, 72°C for 5min, 30 cycles, 72°C for 10min. After the above PCR reaction was completed, 2.4 μL of ...

Embodiment 3

[0069] Verification of Example 3 Inert carrier detection system bacterial strain expressing P factor

[0070] Single colonies of strain S9 and recombinant strain S9-P were inoculated on LB and ampicillin-resistant LB agar medium respectively, cultured at 37°C for 24 hours, and single colonies were picked and inoculated in LB and ampicillin-resistant LB liquid medium respectively, blindly After two passages, draw a small amount of bacterial liquid and inoculate them in LB and ampicillin-resistant LB liquid medium respectively. After static culture at 37°C for 48 hours, centrifuge at 10,000 rpm for 2 minutes, resuspend the pellet with sterilized PBS, absorb a small amount of supernatant, and suspend On the copper grid, and negatively stained with phosphotungstic acid for 5 minutes, PhilipsTecnai 12 transmission electron microscope TEM observation and shooting results showed that the surface of S9 seems to have no P antigen factor components, while the surface of the inert carrier...

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Abstract

The invention discloses an inert carrier indirect agglutination test detection system and its application. The indirect agglutination test detection system comprises an inert carrier bacterium S9 and a complex S9-P expressing and carrying P antigen factors on its surface. The inert carrier indirect agglutination test detection system only carries the P antigen factor, has a single component, and is specifically targeted. It will produce a positive particle agglutination reaction visible to the naked eye under certain concentration conditions with the whole blood or serum of chickens infected with pullorum and Salmonella gallinarum typhi. However, non-specific cross-agglutination reactions did not occur in chicken-derived serum and whole blood with different backgrounds of non-pulmonary pullorum and Salmonella gallinarum infection. The S9‑P is based on the glass plate agglutination reaction operation platform, which is easy to operate, sensitive and fast, the agglutination reaction particles are visible to the naked eye, and the results are clear and easy to judge. The test and result judgment can be completed within two minutes. The S9‑P is suitable for chickens in chicken flocks The target-specific detection of pullorum and Salmonella gallinarum typhi infection has a good application prospect in the field monitoring and diagnosis of chicken flocks.

Description

technical field [0001] The invention belongs to the technical field of biomedicine and immunodiagnosis, and in particular relates to an inert carrier indirect agglutination test detection system. The indirect agglutination test detection system comprises a new inert carrier indirect agglutination test pullorum and / or Salmonella gallinarum typhi detection system S9-P established by expressing an inert carrier Salmonella S9 and carrying a single antigenic factor P. Background technique [0002] Pullorum is a bacterial infectious disease of poultry such as chickens and turkeys caused by Salmonella pullorum (Salmonella pullorum). It mainly harms chickens within three weeks of age and has a high mortality rate. Adult chickens have no obvious symptoms after infection, but they can be passed through egg sources. Vertical transmission to offspring chicks is an important disease that endangers the chicken industry in my country. Salmonella gallinarum (Salmonella gallinarum) infection ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569C12N15/31C12N15/74C07K14/255
CPCG01N33/56916C07K14/255C12N15/74G01N2333/255G01N33/554
Inventor 朱国强杨斌羊扬孟霞夏芃芃段强德朱晓芳
Owner YANGZHOU UNIV
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