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PCR detection kit for rapidly identifying specific serotype salmonella

A detection kit and Salmonella technology, applied in the field of biotechnology detection, can solve the problems of pathogenic bacteria contamination, Salmonella injury, unsuitable routine detection, etc., and achieve good repeatability

Active Publication Date: 2016-09-14
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Traditional detection methods, namely, non-selective and selective enrichment, biochemical characteristics and serological identification are laborious and time-consuming, and it takes 4-7 days to complete. Other methods, such as antibody detection, are fast, but their high false positives make them Not suitable for routine testing
In addition, factors such as low levels of pathogenic bacteria contamination, "injury" of Salmonella after food processing and interference from other food ingredients have limited the detection of Salmonella

Method used

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  • PCR detection kit for rapidly identifying specific serotype salmonella
  • PCR detection kit for rapidly identifying specific serotype salmonella
  • PCR detection kit for rapidly identifying specific serotype salmonella

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Bioinformatics method to identify the distribution of tcpS gene

[0036] In NCBI, use the Blastn online comparison software (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to search for the tcpS gene in the genome-wide database. The search results show that the tcpS gene only exists in Salmonella enteritidis, chicken Pullorum / Typhi Salmonella and Salmonella Dublin, and the nucleotide sequence similarity is 100%, while other Salmonella serotypes and other species do not have this gene ( figure 1).

Embodiment 2

[0037] The preparation of embodiment 2 kit

[0038] Primer design and synthesis: using the tcpS gene as a template, design and analyze primers, and select the best pair of detection primers according to the genomic DNA sequence. The nucleotide sequences are shown in Table 1 below:

[0039] Table 1

[0040]

[0041] The above-mentioned primer pairs can be packaged individually, or can be made into a PCR detection solution. In the PCR detection solution, the amount of the above-mentioned primer pairs can be the conventional amount known to those skilled in the art.

[0042] That is to say, the kit of the present invention may contain the aforementioned independently packaged primer pair, or may contain a configured PCR detection solution containing the primer pair.

[0043] Further, the kit can also contain sterile water (ddH 2 O), dNTP, PCR buffer, rTaq enzyme, sample genomic DNA extraction reagent, etc.

Embodiment 3

[0044] Example 3 The kit identifies that the tcpS gene only exists in specific serotype Salmonella

[0045] Using the primers in the kit described in Example 2, using the genomes of different serotypes of Salmonella and other bacteria as templates, the distribution characteristics of the tcpS gene in different bacteria were identified by PCR.

[0046] The PCR reaction system is (25μL): ddH 2 O 16.25 μL, dNTP 2 μL, 10×PCR buffer 2.5 μL, tcpS-F 1 μL, tcpS-R 1 μL, template 2 μL, rTaq enzyme 0.25 μL.

[0047] The PCR program was 94°C for 5min; 94°C for 45s, 59°C for 45s, 72°C for 1min, 30 cycles; 72°C for 10min.

[0048] The PCR products were subjected to 1% agarose gel electrophoresis, and the PCR electrophoresis results showed that only in the swimming lanes with enteritis, pullorum / typhoid fever and Salmonella Dublin genome as the template, there were target bands ( figure 2 , image 3 ), the sequencing results after gel recovery showed that the nucleotide similarity of all...

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Abstract

The invention belongs to the field of biological technology detection, and specifically relates to a PCR detection kit for rapidly identifying specific serotype salmonella. The kit comprises a tcpS gene detection primer. The tcpS gene detection primer comprises a forward primer, whose nucleotide sequence is represented by SEQ ID No.1, and a reverse primer, whose nucleotide sequence is represented by SEQ ID No.2. The provided kit can rapidly identify enteritis serotype salmonella, pullorum disease / typhoid serotype salmonella, and Dublin serotype salmonella, can be used to assist the conventional serological typing of salmonella, and is capable of monitoring three specific serotype salmonella simply and rapidly in lab diagnosis, and the repeatability is good.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a PCR detection kit for rapidly identifying specific serotype Salmonella. Background technique [0002] Salmonellosis is one of the important zoonotic diseases in public health. The pathogen Salmonella belongs to Enterobacteriaceae. Eggs, livestock and meat products are the main media. It can not only cause a variety of livestock and poultry diseases, but also cause Systemic sepsis and enteritis can also be used as pathogens of foodborne diseases, causing human gastroenteritis and food poisoning. In my country, about 70% to 80% of bacterial food poisoning is caused by Salmonella. At present, according to the Kauffman-White (K-W) serotyping method, according to the difference of Salmonella bacterial antigens and flagellar antigens, there are more than 2,500 serotypes of Salmonella, and 292 different serotypes have been reported in China. Belongs to 35 O groups. [0003] T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/10C12R1/42
CPCC12Q1/689C12Q2600/156C12Q1/10Y02A50/30C12R2001/42C12N1/205
Inventor 焦新安潘志明熊丹宋丽焦扬孙林陈祥耿士忠黄金林殷月兰
Owner YANGZHOU UNIV
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