Rapid detection method for enterococcus, detection primer group and detection kit

A technology for detecting primers and enterococci, which is applied in the field of microbial detection to achieve the effects of accurate detection, shortened detection cycle and high yield

Inactive Publication Date: 2013-03-27
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 enterococci

Method used

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  • Rapid detection method for enterococcus, detection primer group and detection kit
  • Rapid detection method for enterococcus, detection primer group and detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The PCR detection of embodiment 1 enterococcus

[0028] The specific detection steps of this embodiment are:

[0029] 1. Extraction of sample DNA

[0030] 1.1 Inoculate the standard strain of Enterococcus in 10 mL of nutrient broth medium, and culture at 36°C±1°C for 24 hours.

[0031] 1.2 Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract bacterial genomic DNA from the cultured products.

[0032] 2. PCR reaction

[0033] 2.1 The upstream and downstream primers of the external primers of Enterococcus, 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.

[0034] 2.2 PCR reaction system

[0035] The PCR reaction system is: DNA polymerase 0.05U / μL, dATP, dTTP, dGTP, dCTP each 0.2mM, MgSO 4 2mM, 10% (volume) 10×PCR buffer, 0.5μM upstream primer F3 and downstream primer B3, 1μL template DNA.

[0036] 2.3 PCR reaction conditions

[0037] Using a PCR machin...

Embodiment 2

[0042] Example 2 Enterococcus LAMP rapid detection of Mg 2+ concentration test

[0043] The specific detection steps of this embodiment are:

[0044] 1. Extraction of sample DNA

[0045] 1.1 Inoculate the standard strain of Enterococcus in 10 mL of nutrient broth medium, and culture at 36°C±1°C for 24 hours.

[0046] 1.2 Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract bacterial genomic DNA from the cultured products.

[0047] 2. LAMP rapid detection

[0048] 2.1 artificially synthesized Enterococcus 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 nucleotide sequence shown in SEQ No.2; 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 has the nucleotide sequence shown in SEQ No.4.

[0049] 2.2 LAMP reaction system

[0050] Application of different Mg ...

Embodiment 3

[0055] Example 3 Enterococcus LAMP Rapid Detection Sensitivity Test

[0056] The specific detection steps of this embodiment are:

[0057] 1. Extraction of sample DNA

[0058] 1.1 Inoculate the standard strain of Enterococcus in 10 mL of nutrient broth medium, and culture at 36°C±1°C for 24 hours.

[0059] 1.2 Use the QIAGEN Bacterial Genomic DNA Extraction Kit to extract bacterial genomic DNA from the cultured products.

[0060] 2. LAMP rapid detection

[0061] 2.1 artificially synthesized Enterococcus LAMP detection primer set, wherein, the upstream primer F3 of the outer primer has the nucleotide sequence shown in SEQ No.1, and the downstream primer B3 of the outer primer has the nucleotide sequence shown in SEQ No.2 Sequence, the upstream primer FIP of the inner primer has the nucleotide sequence shown in SEQ No.3, and the downstream primer BIP of the inner primer has the nucleotide sequence shown in SEQ No.4.

[0062] 2.2 LAMP reaction system

[0063] Set up 9 reacti...

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Abstract

The invention discloses a rapid detection method for enterococcus, a detection primer group and a detection kit According to loop-mediated isothermal amplification (LAMP) technology, the primer group is obtained by analysis, design and artificial synthesis of the highly conservative part in 16sRNA gene sequence of enterococcus. Containing nucleotide sequences shown in SEQ No.1-4, the primer group shows high specificity to enterococcus. The rapid detection method for enterococcus is realized by performing a LAMP reaction to the enterococcus DNA in a sample with the detection primer group. Through identification of reaction products, whether enterococcus exists in the sample can be determined. In the invention, on the basis of the above detection method a rapid detection kit for enterococcus is also designed for rapid, simple, accurate and efficient detection and identification of enterococcus.

Description

technical field [0001] The invention belongs to the field of microorganism detection, and relates to a rapid detection method for enterococci, a detection primer set and a detection kit. Background technique [0002] Enterococcus (Enterococcus) belongs to the Streptococcus family and is part of the normal intestinal flora of humans and animals. It has strong tolerance to cold and heat, and is usually found in the mixed hyphae isolated from abdominal and pelvic infections. In the past, enterococci were considered to be harmless commensal bacteria to humans, but recent studies and clinical cases have confirmed the pathogenicity of enterococci. Among aerobic Gram-positive cocci, it is second only to Staphylococcus an important nosocomial infection pathogen; enterococci can also cause nosocomial infections. Enterococcus can not only cause urinary tract infection, skin and soft tissue infection, but also life-threatening abdominal infection, sepsis, endocarditis and meningitis. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 姜侃张东雷金燕飞陈小珍
Owner 浙江省质量技术监督检测研究院
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