Primer and probe group for detecting enterohemorrhagic escherichia coli, kit and PCR (polymerase chain reaction) detection method

A detection kit, Escherichia coli technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., can solve the problems of not being adopted, time-consuming, laborious, etc., and achieve simple operation and high sensitivity. , detect fast and reliable results

Inactive Publication Date: 2016-07-20
珠海出入境检验检疫局检验检疫技术中心
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  • Abstract
  • Description
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Problems solved by technology

[0005] The traditional detection method of enterohemorrhagic Escherichia coli, like the detection of other pathogenic microorganisms, requires serological identification based on microbial proliferation and biochemical identification. Although it is reliable, it is time-consuming and laborious, and it takes 4 to 7 days to complete. Therefore, when it is necessary to evaluate the safety of microorganisms in food in a timely and rapid manner, it is usually not used

Method used

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  • Primer and probe group for detecting enterohemorrhagic escherichia coli, kit and PCR (polymerase chain reaction) detection method
  • Primer and probe group for detecting enterohemorrhagic escherichia coli, kit and PCR (polymerase chain reaction) detection method
  • Primer and probe group for detecting enterohemorrhagic escherichia coli, kit and PCR (polymerase chain reaction) detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0084] Such as figure 1 As shown, this embodiment provides a primer and probe set for the detection of enterohaemorrhagic Escherichia coli, for the stx1 gene, stx2 gene and eae gene in enterohaemorrhagic Escherichia coli, it is made up of the following primers and probes :

[0085] stx1 upstream primer: TGTCGCATAGTGGAACCTCAC;

[0086] Stx1 downstream primer: TCCCCTCTGTATTTGCCGAAA;

[0087] stx1 probe: FAM-CAGTCTGTGGCAAGAGCGATGT-BHQ1;

[0088] stx2 upstream primer: ATGGGTACTGTGCCTGTTACT;

[0089] stx2 downstream primer: GCCCTCGTATATCCACAGCAA;

[0090] stx2 probe: Cy5-AACAGACACCGATGTGGTCCCC-BHQ3;

[0091] eae upstream primer: TACTCAATGCAGTTCCGTT;

[0092] eae downstream primer: ATCCTGCTTCTTGTACTCC;

[0093] eae probe: HEX-TTCAATTTGCTGAGACCACGAT-BHQ1.

[0094] This embodiment also provides a detection kit for the above-mentioned primer and probe set. For the stx1 gene, stx2 gene and eae gene in enterohemorrhagic Escherichia coli, its composition is as follows:

[0095] ...

Embodiment 2

[0111] Such as Figure 2 to Figure 5 As shown, in this example, the specificity experiment is mainly carried out on the primers and probe sets provided in Example 1, specifically:

[0112] The DNA of the Escherichia coli strains listed in the table below was used to replace the template DNA to be tested, and the real-time fluorescent PCR amplification reaction in Step 2 in Example 1 was also carried out, and deionized water was used as a negative control. See the table below for details:

[0113]

[0114]

[0115] The experimental results are as follows: the amplification results of the E. coli strains in the above table are completely consistent with the corresponding genotypes in the table, and the real-time fluorescent PCR amplification curve of the positive amplification is shown in Figure 2 to Figure 5 .

[0116] Depend on Figure 2 to Figure 5 Known, above-mentioned primer and probe group have specificity to stx1 gene, stx2 gene and eae gene in enterohemorrhagi...

Embodiment 3

[0118] Such as Figure 6 to Figure 12 As shown, in this example, the sensitivity experiment is mainly carried out on the primers and probe sets provided in Example 1, specifically:

[0119] The standard DNA solution of enterohemorrhagic Escherichia coli ATCC35150 was 2 μL, and the concentration of the DNA solution was measured by a nucleic acid protein analyzer to be 30 ng / μL; the solution was diluted to 3 ng / μL, 300 pg / μL, and 30 pg / μL with deionized water in 10-fold increments. , 3pg / μL, 0.3pg / μL and 0.03pg / μL, all replace the template DNA to be tested, and implement the real-time fluorescent PCR amplification reaction of step 2 described in Example 1 in addition,

[0120] Depend on Figure 6 to Figure 12 It can be seen that Figure 6 to Figure 12 Corresponding to the amplification curves of standard DNA solutions with concentrations of 30ng / μL, 3ng / μL, 300pg / μL, 30pg / μL, 3pg / μL, 0.3pg / μL and 0.03pg / μL; therefore, the real-time fluorescent PCR can be judged The detection se...

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Abstract

The invention relates to a primer and probe group for detecting enterohemorrhagic escherichia coli, a kit and a PCR (polymerase chain reaction) detection method. Based upon PCR detection on stx1 gene, stx2 gene and eae gene in the enterohemorrhagic escherichia coli, the primer and probe group, which adopts real-time fluorescence PCR detection, is completely consistent with the genotype of an escherichia coli strain in amplification of the strain and is free from specific amplification; therefore, the primer and probe group, which adopts real-time fluorescence PCR detection, has the advantages of being excellent in specificity and real-time fluorescence PCR on on three genes in the enterohemorrhagic escherichia coli, namely the stx1 gene, the stx2 gene and the eae gene; and meanwhile, the corresponding PCR detection method has the characteristics of being strong in specificity, high in sensitivity, rapid and reliable in detection and simple and convenient to operate.

Description

【Technical field】 [0001] The invention relates to a real-time fluorescent PCR detection technology in the field of food safety, in particular to a primer and probe set, a kit and a PCR detection method for the detection of enterohaemorrhagic Escherichia coli. 【Background technique】 [0002] Escherichia coli (Escherichia coli) is a bacterium commonly found in the intestines of humans and warm-blooded animals, and most strains of Escherichia coli are harmless. However, some Escherichia coli can cause food-borne diseases. Among them, the antigen composition of E. coli is complex and can be divided into bacterial antigen (O), flagellar antigen (H) and surface antigen (K). According to the different bacterial antigens, Escherichia coli can be divided into more than 200 types, some of which are pathogenic and can cause diarrhea, collectively referred to as diarrhea-causing Escherichia coli. [0003] According to different biological characteristics, diarrhea-causing E. coli can b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/10C12N15/11
CPCC12Q1/689
Inventor 游淑珠冯家望王小玉姚丽锋蔡教英唐食明胡松楠符家忍蒋联振徐日文
Owner 珠海出入境检验检疫局检验检疫技术中心
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