DPO primer sequences for salmonella detection by using DPO-PCR method, and detection kit thereof

A DPO primer, Salmonella technology, applied in the direction of using vectors to introduce foreign genetic material, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of low sensitivity, high false positive rate, poor specificity, etc., and achieve good repeatability and stability effects

Inactive Publication Date: 2013-09-25
哈尔滨海关技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the VIDAS-CHL method, EIA method, colloidal gold test paper method and API method all have defects such as poor specificity and low sensitivity, and have not been widely used; LAMP method, as a new detection method, has greatly improved the detection method. The efficiency and the cost of detection are reduced, but the false positive rate is relatively high; as a highly sensitive, fast and simple detection method, PCR technology has been widely used in many fields, especially in the detection of pathogens. It is a gold standard for rapid detection of nucleic acid, and on this basis, DNA probe technology, real-time fluorescent PCR technology and PCR combined with denaturing high-performance liquid chromatography (DHPLC) technology have been developed, and the design of PCR primers has become a constraint on this Critical Factors for the Success of Class Detection Methods
Conventional PCR primer design not only needs to repeatedly compare the specificity of the primers, but also needs to optimize the parameters and reaction conditions of the primers, especially the annealing temperature, to prevent non-specific amplification, especially when multiplex PCR is involved. Experiments to verify the specificity of the detection method, time-consuming and laborious

Method used

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  • DPO primer sequences for salmonella detection by using DPO-PCR method, and detection kit thereof
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  • DPO primer sequences for salmonella detection by using DPO-PCR method, and detection kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Preparation of Salmonella DNA template

[0048] Extract and purify bacterial genomic DNA according to the instructions of TIANamp Bacteria DNA Kit, and add the corresponding reagents in order:

[0049] (1) Take 1.5 mL of bacterial liquid, centrifuge at 10,000 r / min for 1 min, discard the supernatant, add 200 μL of buffer GA, and suspend the bacterial cells thoroughly;

[0050] (2) Add 20 μL proteinase K (20mg / mL), and add 220 μL buffer solution GB, mix thoroughly, and act in a 70°C water bath for 10 minutes;

[0051] (3) Add 220 μL of absolute ethanol, mix thoroughly for 15 seconds, transfer the resulting solution (including flocculent precipitate) to the adsorption column CB3 after brief centrifugation, centrifuge at 12000 r / min for 30 seconds, and discard the liquid in the collection tube;

[0052] (4) Add 500 μL buffer GD (containing absolute ethanol) to the adsorption column CB3, centrifuge at 12000r / min for 30s, and discard the liquid in the collection tube;

[0...

Embodiment 2

[0057] Example 2 Design and synthesis of PCR primers

[0058] The Salmonella FimY gene was selected as the target gene, and after BLAST analysis, the following primers were designed and synthesized:

[0059] (1) Prepare PCR amplification primers for the positive control substance of Salmonella FimY gene (the size of the PCR product is 227bp):

[0060] SA-CGF: 5′-TATTTTCCGGAAAACGTACCGCAGCATTCCGC-3′

[0061] SA-CGR: 5′-AGCCATACGGATAAACTGTGTTATAGCGGAGGT-3′

[0062] (2) DPO primers for Salmonella DPO-PCR method identification (PCR product size is 237bp):

[0063] SA-DPOF: 5'-TATTTTCCGGAAAACGTACCGCAIIIIIGCATTCCGC-3',

[0064] SA-DPOR: 5'-AGCCATACGGATAAACTGTGTTATIIIIIAGCGGAGGT-3';

Embodiment 3

[0065] Example 3 Preparation of positive control substance

[0066] Using SA-CGF and SA-CGR as primers, the FimY gene of Salmonella was amplified by PCR.

[0067] Utilize target gene amplification primer SA-CGF / SA-CGR, adopt PCR method to amplify Salmonella FimY gene, the reaction system of PCR is:

[0068] 10×PCR Buffer 2.5μL dNTP (2.5mM for each) 2.0 μL Upstream primer SA-CGF (10μM) 1.0 μL Downstream primer SA-CGR (10μM) 1.0 μL Taq DNA Polymerase (5U / μL) 0.5μL DNA template 0.5μL wxya 2 o 17.5μL

[0069] The reaction conditions of PCR were: 95°C for 5min; 35 cycles of 94°C for 15s, 57.6°C for 20s, and 72°C for 30s; 72°C for 10min.

[0070] The PCR amplification result was detected by agarose gel electrophoresis, and the size of the PCR product was 227bp.

[0071] Connect the PCR product to the pMD-19-T vector, transform competent Escherichia coli JM109, and obtain positive recombinant bacteria, and prepare positi...

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Abstract

The invention relates to DPO primer sequences for salmonella detection by using a DPO-PCR method, and a diagnosis kit thereof, wherein the DPO primer sequences are that: SA-DPOF: 5'-TATTTTCCGGAAAACGTACCGCAIIIIIGCATTCCGC-3', and SA-DPOR: 5'-AGCCATACGGATAAACTGTGTTATIIIIIAGCGGAGGT-3'. The diagnosis kit contains the DPO primer pair, a positive control, a negative control, TaqDNAPolymerase and PCR reaction solutions. According to the invention, advantages of strong operability and accurate detection result are provided.

Description

Technical field [0001] The present invention is a biological test technology field, which involves a DPO primer sequence and the detection kit that uses the DPO-PCR method to detect Salmonella. Background technique [0002] Salmonella is an important germ of human and animals. It is extremely widely distributed in nature. It is mainly infected by gastrointestinal tract, causing clinical symptoms characterized by typhoid fever, parathyletes and acute gastroenteritis.Humans are infected with foods contaminated by Salmonella, which are mainly manifested in vomiting, abdominal pain and diarrhea. Common ones are mouse typhoid Salmonella, pig cholera Salmonella, and enteritis, etc., which is of great significance in public health. [0003] At present, the detection of Salmonella still mainly depends on traditional methods, that is, the first use of selective medium to increase bacteria, and then combine biochemical and serotics methods.The traditional method test results are accurate, ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12N15/63C12N15/66C12R1/42
Inventor 徐义刚刘忠梅吴岩李丹丹李苏龙
Owner 哈尔滨海关技术中心
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