Multiple rapid detection method of two food-borne pathogenic bacteria and detection primer groups as well as kit

A rapid technology for detection of primers, applied in the field of food microbial molecular biology detection, can solve the problems of indistinguishable molecular weight target product identification, amplification product analysis and identification need to be improved, achieve good specificity, optimize reaction conditions, improve detection efficiency effect

Inactive Publication Date: 2013-09-18
ZHEJIANG INST OF QUALITY INSPECTION SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, because the LAMP amplification product presents a ladder-like band in ordinary electrophoresis, multiple LAMP products cannot be identified by ordinary electrophoresis to distinguish the molecular weight of the tar...

Method used

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  • Multiple rapid detection method of two food-borne pathogenic bacteria and detection primer groups as well as kit
  • Multiple rapid detection method of two food-borne pathogenic bacteria and detection primer groups as well as kit
  • Multiple rapid detection method of two food-borne pathogenic bacteria and detection primer groups as well as kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1: The optimal Mg of Salmonella of the present invention, Staphylococcus aureus double LAMP reaction 2+ determination of concentration

[0030] The specific determination method of this embodiment is:

[0031] 1. Extraction of sample DNA

[0032] 1.1 Inoculate the standard strains of Salmonella and Staphylococcus aureus in nutrient broth respectively, and incubate at 36°C±1°C for 18 hours.

[0033] 1.2 Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract the bacterial DNA in the above culture products respectively.

[0034] 2. LAMP reaction

[0035] 2.1 The primer sets for the detection of Salmonella and Staphylococcus aureus were artificially synthesized respectively. Wherein, the sequence of the upstream primer of the outer primer in the primer set that is used for Salmonella detection is as shown in SEQ No.1, the sequence of the downstream primer of the outer primer is as shown in SEQ No.2, and the sequence of the upstream primer of the inner...

Embodiment 2

[0047] Embodiment 2: the determination of the optimal reaction temperature of Salmonella of the present invention, Staphylococcus aureus double LAMP reaction

[0048] The specific determination steps of this embodiment are as follows:

[0049] 1. Extraction of sample DNA

[0050] 1.1 Inoculate the standard strains of Salmonella and Staphylococcus aureus in nutrient broth respectively, and incubate at 36°C±1°C for 18 hours.

[0051] 1.2 Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract the bacterial DNA in the above cultured products respectively.

[0052] 2. LAMP reaction

[0053] 2.1 Artificially synthesize primer sets for the detection of Salmonella and Staphylococcus aureus respectively, and the primers and sequences of each primer set are consistent with those described in Section 2.1 of Example 1.

[0054] 2.2 LAMP reaction system

[0055] 2.2.1 LAMP reaction system of Salmonella

[0056] Six groups of Salmonella LAMP reaction systems were established ...

Embodiment 3

[0065] Example 3: The detection sensitivity of the double LAMP detection method of the present invention to Salmonella.

[0066] The concrete detection method of the present embodiment is:

[0067] 1. Extraction of sample DNA

[0068] 1.1 Inoculate the standard strain of Salmonella in nutrient broth and incubate at 36°C±1°C for 18 hours.

[0069] 1.2 Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract the bacterial DNA in the above cultured products.

[0070] 2. LAMP reaction

[0071] 2.1 The primer sets for Salmonella and Staphylococcus aureus were artificially synthesized respectively, and the primers and sequences of each primer set were consistent with those described in Section 2.1 of Example 1.

[0072] 2.2 Reaction system of LAMP

[0073] Set up 9 reaction tubes, the total volume of the reaction system in each reaction tube is 25 μL, and the composition of the reaction system in each reaction tube includes: the upstream of the outer primers for the dete...

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Abstract

The invention discloses a multiple rapid detection method of two food-borne pathogenic bacteria and detection primer groups as well as a kit, wherein the sequences of the upstream and downstream primers of an outer primer and the upstream and downstream primers of an inner primer in a quick detection primer group for salmonella are as shown in SEQ No.1, SEQ No.2, SEQ No.3 and SEQ No.4, respectively; and the sequences of the upstream and downstream primers of an outer primer and the upstream and downstream primers of an inner primer in a quick detection primer group for staphylococcus aureus, and the sequence of a loop primer for improving amplification efficiency are as shown in SEQ No.5, SEQ No.6, SEQ No.7, SEQ No.8 and SEQ No.9. The multiple rapid detection method provided by the invention realizes rapid detection of the DNAs of salmonella and staphylococcus aureus in the same system and under the same reaction conditions by using multiple LAMP detection technology in combination with a melting curve analysis method.

Description

technical field [0001] The invention belongs to the field of molecular biology detection of food microorganisms, and relates to a multiple rapid detection method for Salmonella and Staphylococcus aureus, a detection primer set and a detection kit. Background technique [0002] Food safety is a major concern of all countries in the world, and food-borne pathogens are one of the main reasons affecting food safety. According to the current national food safety supervision system, both Salmonella and Staphylococcus aureus are food-borne pathogens that are of great concern. Routine detection adopts traditional culture methods, and each pathogenic bacteria needs to be tested separately with an independent detection method. The workload is heavy, the detection cycle is long, and it is limited by various factors such as the professionalism and experience of the testing personnel, which is prone to misjudgment. With the gradual application of molecular biology methods in the detecti...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12Q1/10C12R1/42C12R1/445
Inventor 姜侃汪新金燕飞陈小珍
Owner ZHEJIANG INST OF QUALITY INSPECTION SCI
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