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Fast detection method for clostridium perfringens, detection primer group and detection kit

A technology of Clostridium perfringens and detection primers, which is applied in the field of microbial detection to achieve accurate detection, reduce detection links, and shorten the detection cycle

Inactive Publication Date: 2011-07-27
浙江省质量技术监督检测研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are no relevant reports on the application of LAMP in the detection of Clostridium perfringens

Method used

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  • Fast detection method for clostridium perfringens, detection primer group and detection kit
  • Fast detection method for clostridium perfringens, detection primer group and detection kit
  • Fast detection method for clostridium perfringens, detection primer group and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Clostridium perfringens PCR detection

[0027] In this embodiment, irrigation water is used as a detection sample, and the specific detection steps are:

[0028] 1. Extraction of sample DNA

[0029] 1.1. Take 1mL of the water sample to be tested and put it into 9mL blister liquid culture medium, and apply the Mart II anaerobic system to incubate for 24 hours at 36°C±1°C after sealing.

[0030] 1.2. Use bacterial genome DNA extraction kit to extract bacterial DNA from cultured products.

[0031] 2. PCR reaction

[0032] 2.1. The upstream and downstream primers of the artificially synthesized Clostridium perfringens outer primers, wherein the upstream primer F3 has the nucleotide sequence shown in SEQ No.1, and the downstream primer B3 has the nucleotide sequence shown in SEQ No.2 acid sequence.

[0033] 2.2. PCR reaction system

[0034] The PCR reaction system is: DNA polymerase 0.05U / μL, dATP, dTTP, dGTP, dCTP each 0.2mM, MgSO 4 2 mM, 10% (volume) 10×DN...

Embodiment 2

[0041] Example 2 Clostridium perfringens LAMP rapid detection of Mg 2+ concentration test

[0042]In this embodiment, irrigation water is used as a detection sample, and the specific detection steps are:

[0043] 1. Extraction of sample DNA

[0044] 1.1. Take 1mL of the water sample to be tested and put it into 9mL blister liquid culture medium, and apply MartⅡ anaerobic system after sealing for 24 hours at 36°C±1°C.

[0045] 1.2. Use bacterial genome DNA extraction kit to extract bacterial DNA from cultured products.

[0046] 2. LAMP rapid detection

[0047] 2.1. Artificially synthesized Clostridium perfringens LAMP detection primer set, the upstream primer F3 of the outer primer has the nucleotide sequence shown in SEQ No.1; the downstream primer B3 of the outer primer has the sequence shown in SEQ No.2 The nucleotide sequence shown; the upstream primer FIP of the inner primer has the nucleotide sequence shown in SEQ No.3; the downstream primer BIP of the inner primer ha...

Embodiment 3

[0057] Example 3 Clostridium perfringens LAMP rapid detection sensitivity test

[0058] In this embodiment, irrigation water is used as a detection sample, and the specific detection steps are:

[0059] 1. Extraction of sample DNA

[0060] 1.1. Take 1mL of the water sample to be tested and put it into 9mL blister liquid culture medium, and apply the Mart II anaerobic system to incubate for 24 hours at 36°C±1°C after sealing.

[0061] 1.2. Use bacterial genome DNA extraction kit to extract bacterial DNA from cultured products.

[0062] 2. LAMP rapid detection

[0063] 2.1. Artificially synthesized Clostridium perfringens LAMP detection primer set, the upstream primer F3 of the outer primer has the nucleotide sequence shown in SEQ No.1; the downstream primer B3 of the outer primer has the sequence shown in SEQ No.2 The nucleotide sequence shown; the upstream primer FIP of the inner primer has the nucleotide sequence shown in SEQ No.3; the downstream primer BIP of the inner pr...

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Abstract

The invention discloses a detection method for clostridium perfringens, a primer group and a detection kit for same. The primer group is based on the Loop-Mediated Isothermal Amplification (LAMP), and is obtained through analysis, design and artificial synthesis on the special alpha toxin (C. perfringens alpha toxin, CPa) gene sequence of the clostridium perfringens, the contained nucleotide sequence is shown in SEQ No.1-4, and the primer group has considerably high specificity to the clostridium perfringens. The fast detection method for the clostridium perfringens carries out LAMP reaction on the DNA of the clostridium perfringens in a sample by utilizing the detection primer group, and whether the clostridium perfringens is contained in the sample or not is judged by identifying reaction products. The invention also designs the fast detection kit for the clostridium perfringens according to the detection method, so that fast, simple, accurate and efficient clostridium perfringens detection and identification can be carried out on the sample.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and relates to a rapid detection method of Clostridium perfringens, a detection primer set and a detection kit. Background technique [0002] Clostridium perfringens (Clostridium perfringens) is an important pathogen that can cause human food poisoning, antibiotic-associated diarrhea and animal diarrhea. The bacterium is a Gram-positive spore-forming obligate anaerobic bacillus, which widely exists in soil, water sources and human and animal intestines in nature. Studies have found that the bacterium can produce at least 15 kinds of toxins. At present, according to the ability of four important pathogenic toxins (α, β, ε, iotoxin) produced by Clostridium perfringens, it is divided into A, β, ε, and ι toxin. B, C, D and E five types, all types of Clostridium perfringens produce α-toxin, so α-toxin is considered to be the most basic and important pathogenic factor. Clostridium perfringens t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 姜侃张东雷陈小珍鲁燕骅金燕飞
Owner 浙江省质量技术监督检测研究院
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