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Multi-PCR detection kit for poultry salmonella and non-diagnostic detection method of poultry salmonella

A detection kit and Salmonella technology, applied in the field of Salmonella enteritidis serotype, Salmonella avian multiplex PCR detection kit and its non-diagnostic detection, Salmonella gallinarum typhi and Salmonella typhimurium, can solve cumbersome optimization and limit wide application , complex design and other issues, to achieve the effects of high sensitivity, strong specificity, detection time and advantages

Active Publication Date: 2016-06-08
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because the design of multiplex PCR experiments is more complicated than that of single PCR, the mutual interference between primers needs to be considered when establishing a multiplex PCR reaction system, and the main components and reaction conditions need to be tediously optimized, thus limiting the wide application of this method. use

Method used

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  • Multi-PCR detection kit for poultry salmonella and non-diagnostic detection method of poultry salmonella
  • Multi-PCR detection kit for poultry salmonella and non-diagnostic detection method of poultry salmonella
  • Multi-PCR detection kit for poultry salmonella and non-diagnostic detection method of poultry salmonella

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Experimental program
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Effect test

Embodiment 1

[0025] The establishment of embodiment 1 poultry source Salmonella multiplex PCR detection method

[0026]Design of primers: According to the genomic DNA sequences of Salmonella enteritidis, Salmonella gallinarum and Salmonella typhimurium in the GenBank database, the specific gene fragments (SEQ ID NO.1, SEQ ID NO.1, SEQ ID NO. .4, SEQ ID NO.7), and on this basis, design and optimize specific detection primers for the three serotypes of Salmonella, as shown in the table below.

[0027]

[0028] The amplification primer sequences of Salmonella enteritidis SEQIDNO.2, SEQIDNO.3, the amplification primer sequences of Salmonella gallinarum typhimurium SEQIDNO.5, SEQIDNO.6, the amplification primer sequences of Salmonella typhimurium SEQIDNO.8, SEQIDNO. The complete SEQ sequence of .9 is shown in the Sequence Listing.

[0029] Preparation of PCR templates: Salmonella enteritidis ATCC13076, Salmonella gallinarum ATCC9184 and Salmonella typhimurium ATCC14028 were cultured in TSB ...

Embodiment 2

[0035] Embodiment 2 specificity evaluation experiment

[0036] Carry out the specificity evaluation of the multiplex PCR detection method of the present invention with the method of embodiment 1, after the test bacterial strain is carried out enrichment culture, boil the method to extract genomic DNA, carry out multiplex PCR detection according to the method described in embodiment 1, the result is as follows figure 2 As shown, only three serotype strains of Salmonella typhimurium (lanes 21, 30), Salmonella enteritidis (lanes 28, 39) and Salmonella gallinarum typhi (lanes 6, 12, 19, 36) showed specific amplification bands, There was no non-specific amplification of other serotypes of Salmonella and non-Salmonella strains. The strains and strain numbers used are shown in the table below. In the table, "+" in the test result column means positive; "-" means negative.

[0037]

Embodiment 3

[0038] Embodiment 3 Sensitivity evaluation experiment

[0039] The method of Example 1 was used to evaluate the sensitivity of the multiplex PCR detection method of the present invention. Salmonella enteritidis ATCC13076, Salmonella typhi ATCC9184 and Salmonella typhimurium ATCC14028 were cultured in TSB broth for 12 hours respectively, and the bacterial genome was extracted using a commercial bacterial genomic DNA extraction kit, and the original concentration was determined and then 10-fold serially diluted. Sensitivity evaluation tests were performed on different dilution gradients of bacterial genomic DNA. After the reaction, take 5 μl of the amplification product, add 1 μl of 6×Loadingbuffer to mix, and use 2% concentration agarose gel electrophoresis to detect. Electrophoresis results such as image 3 As shown, M is DL-500marker (500bp, 400bp, 300bp, 200bp, 150bp, 100bp and 50bp in sequence), and lanes 1-5 are Salmonella typhimurium ATCC14028 (the reaction concentratio...

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Abstract

The invention discloses a multi-PCR detection kit for poultry salmonella and a non-diagnostic detection method of the poultry salmonella. The kit comprises 10*PCR buffering liquid, 2.5 U / mu l Taq DNA polymerase, 10 Mm dNTPs, a multi-PCR detection primer group, a positive contrast material and a negative contrast material. The positive contrast material comprises salmonella enteritidis ATCC13076 genome DNA, salmonella typhimurium ATCC14028 genome DNA and salmonella gallinarum ATCC 9184 genome DNA; the negative contrast material is sterilized double distilled water. The invention further discloses a multi-PCR method for detecting the poultry salmonella by applying the kit. The method has the advantages of being rapid, simple, high in specificity and high in sensitivity, three types of salmonella can be detected and classified rapidly through a one-time PCR reaction, compared with traditional serological typing and ordinary PCR detection, great advantages are achieved on the aspect of detection time and detection cost, and the multi-PCR detection kit and the detection method are suitable for batch detection.

Description

technical field [0001] The invention belongs to the technical field of molecular biology detection, and relates to a multiplex PCR detection kit for avian-derived Salmonella and a non-diagnostic detection method thereof, in particular to the serotypes of Salmonella enteritidis, Salmonella gallinarum typhi and Salmonella typhimurium. Background technique [0002] Salmonella is an important zoonotic pathogen that mainly lives in the intestines of humans and animals. Studies have shown that food poisoning caused by Salmonella is the most common and most harmful type of bacterial food poisoning. According to the survey of the World Health Organization (WHO), there are 1.3×10 8 of human gastroenteritis is caused by Salmonella infection, among which the death toll is as high as 3 million. [0003] Poultry is considered a major reservoir of Salmonella, and human infections are often attributed to contaminated poultry products, including poultry meat and eggs. The total number of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12N15/11C12R1/42
CPCC12Q1/686C12Q1/689C12Q2600/16C12Q2537/143C12Q2545/113C12Q2565/125
Inventor 龚建森庄林林陆光武王成明沈海玉盛中伟张笛张林吉束婧婷俞燕刘加圣窦新红徐步邹剑敏
Owner JIANGSU INST OF POULTRY SCI
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