ELISA kit for detecting salmonella antibody

A Salmonella and kit technology, which is applied in the field of ELISA rapid detection kits for rapid detection of Salmonella, can solve problems such as unsatisfactory results, and achieve the effects of reducing the occurrence rate of false positives, wide application range, high specificity and repeatability

Inactive Publication Date: 2017-01-04
NANJING AGRICULTURAL UNIVERSITY +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the existing Salmonella antibody detection methods are not very satisfactory in terms of effect, and it is nece

Method used

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  • ELISA kit for detecting salmonella antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Prokaryotic expression of target gene

[0050] 1.1 Amplification of the target gene pagC

[0051] According to the pagC gene sequence of Salmonella typhimurium LT2 strain (GenBank: AE006468.1), the 1-69bp signal peptide was removed, and a pair of cloning primers PagC1 / PagC2 were designed for the pagC gene. The cleavage sites and protective bases of Dicer EcoR I and Xho I, primers were designed with Primer Premier 5.0 software and synthesized by Beijing Dingguo Changsheng Biological Company. The primer sequences are as follows:

[0052] P1: 5'-CCAGAATTCGATACTAACGCCTTTTCC-3';

[0053] P2: 5'-CAACTCGAGTCAGAAACGGTATCCAAC-3'.

[0054] Using the genome of Salmonella typhimurium LT2 strain as a template, it was pre-denatured at 94°C for 5 minutes; denatured at 94°C for 30 s, annealed at 51°C for 30 s, and extended at 72°C for 30 s. It is the target gene band of 489bp.

[0055] 1.2 Construction of prokaryotic expression plasmids

[0056] After the PCR product wa...

Embodiment 2

[0063] Embodiment 2: Establishment of the optimal condition of ELISA reaction and establishment of diagnostic method

[0064] 2.1 Determination of the optimal coating concentration of antigen and the optimal dilution of serum

[0065] Using the square array titration method, the solution in the dialysis bag collected at the end of 1.4 was diluted by 2 times with the coating solution (0.05mol / L carbonate buffer solution of pH 9.6), and the final concentrations were 0.5, 1, 2, 4, 8, 16 μg / mL, add 100 μL to each well, repeat two rows for each dilution, place at 4°C for overnight coating; then wash 5 times with washing solution (PBST solution with pH 7.4), 3 min each time; each well Add 200 μL of blocking solution (5% skimmed milk powder) and block at 37°C for 2 hours; wash 5 times with washing solution for 3 minutes each time; use blocking solution for Salmonella negative serum and positive serum at 1:50, 1:100, and 1:200, respectively , 1:400, 1:800, 1:1 600-fold dilution, 100 ...

Embodiment 3

[0080] Embodiment 3: the applicability test of indirect ELISA diagnostic kit

[0081] The positive sera of the four most common serotypes of Salmonella in the genus of Salmonella were selected, namely, Salmonella pullorum positive serum, Salmonella choleraesuis positive serum, Salmonella typhimurium positive serum, and Salmonella enteritidis positive serum, and were detected by ELISA with established methods , to confirm whether the method is broadly applicable within the Salmonella genus.

[0082] The results showed that the positive serum results of 4 different serotypes of Salmonella were all judged as positive, which indicated that the method was widely applicable to the Salmonella genus.

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Abstract

The invention discloses an ELISA kit for detecting a salmonella antibody. The kit comprises a solid-phase carrier coated with recombinant protein PagC, an enzyme-labeled antibody, salmonella negative serum and positive serum. The ELISA kit and method for detecting the salmonella antibody can be widely applied to salmonella, and application range is wide. The kit has high specificity and repeatability, the influence of temperature on reaction plates is small, and stability is high. Compared with an ELISA method with polysaccharide antigen as detection antigen, the method has the advantages that the occurrence rate of false positivity can be reduced to the maximum, and interference of other bacterial antigens in enterobacteriaceae on detection is avoided. Compared with a slide agglutination antigen detection method mostly adopted clinically, the method has the advantages that sensitivity and accuracy are higher, and detection throughput is increased greatly.

Description

[0001] 1. Technical field [0002] The present invention relates to the field of veterinary medicine. Specifically, the present invention relates to an ELISA rapid detection kit for rapid detection of Salmonella (Salmonella), and a detection method using the kit. [0003] 2. Background technology [0004] Salmonella (Salmonella) is an important type of intestinal pathogenic bacteria, which can cause diarrhea, enteritis, sepsis and abortion in animals, and can also infect humans. It is the most important pathogen that causes food poisoning in humans. Hygiene means a lot. Salmonella includes 2 species, Salmonella enterica and Salmonella Bongor, and 7 subspecies and more than 2,500 serotypes. Among them, 292 serotypes have been reported in my country, most of which are pathogenic. The wide variety of serotypes brings great difficulty to the diagnosis of salmonellosis. [0005] ELISA detection method has high sensitivity and strong specificity, and is one of the hotspots of sero...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569
CPCG01N33/68G01N33/56911G01N2333/255G01N2800/065
Inventor 范红结马喆方一臻周红
Owner NANJING AGRICULTURAL UNIVERSITY
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