Loop mediated isothermal amplification detection primer group, detection method and kit for enterococcus faecalis

A ring-mediated isothermal and detection kit technology, applied in the biological field, can solve the problems of unsatisfactory rapid detection, cumbersome detection methods, low sensitivity, etc., achieve rapid detection and ensure the quality and safety of drinking water, short detection time and high sensitivity Effect

Active Publication Date: 2013-04-03
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After the water sample is filtered, transfer the filter membrane to KF medium for 48 hours. If there are red or pink colonies on the filter membrane, perform pure medium and further biochemical identification. The detection method is cumbersome. The entire detection process takes about 3-5 days and the sensitivity is high. low, and cannot meet the needs of on-site rapid detection, especially the rapid detection of large quantities of water samples

Method used

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  • Loop mediated isothermal amplification detection primer group, detection method and kit for enterococcus faecalis
  • Loop mediated isothermal amplification detection primer group, detection method and kit for enterococcus faecalis
  • Loop mediated isothermal amplification detection primer group, detection method and kit for enterococcus faecalis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Detection of Enterococcus faecalis in source water

[0029] 1. Water sample filtration

[0030] Take 250 mL of water sample and filter it through a 0.45 μm filter membrane by aseptic operation, transfer the filter membrane to 30 ml of brain heart infusion broth (BHI) for cultivation, and incubate at 36°C±1°C for 10 hours to obtain bacterial cultures.

[0031] 2. Bacterial DNA extraction

[0032]Take 1 mL of bacterial culture and centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50μL TE to fully suspend and mix well, bathe in 100℃ water for 10min, ice bath for 3min, centrifuge at 12000r / min for 5min, take the supernatant for later use, to obtain bacterial DNA.

[0033] 3. LAMP amplification

[0034] The loop-mediated isothermal amplification system has a total volume of 25 μL, including 2.5 μL 10× Thermopol reaction buffer, 0.4 μL 10 mmol / L dNTPs, 0.5 μL 10 μmol / L upstream outer primer F3, 0.5 μL 10 μmol / L downstream oute...

Embodiment 2

[0043] Example 2: Detection of Enterococcus faecalis in bottled water

[0044] Take a 250ml bottled water sample and carry out LAMP detection according to the method of Example 1, except that the reaction temperature of LAMP is 65° C. and incubate for 45 minutes, and the others are the same as in Example 1.

[0045] The result is: the color of the reaction tube turns orange (the negative control (genomic DNA of Escherichia coli 8099) is orange, the positive control (the genomic DNA of positive strain Enterococcus faecalis ATCC29212) is green), indicating that the sample is not contaminated by Enterococcus faecalis. This is consistent with the detection results of traditional biochemical methods, which proves that the data of this method is reliable.

Embodiment 3

[0047] 1. Screening of target genes by LAMP method

[0048] The present invention designs LAMP primers based on three target genes of Enterococcus faecalis, mtlF, EF0027 and groES. As shown in Table 1, these LAMP primers are used to target Enterococcus faecalis (CMCC32223, ATCC29212) and Enterococcus faecium (E.Faecium) ATCC19434 For loop-mediated isothermal amplification, the specific steps are as follows:

[0049] Table 1: LAMP Primers

[0050]

[0051] Enterococcus faecalis (CMCC32223), Enterococcus faecalis (ATCC29212) and Enterococcus faecium (E.Faecium) ATCC19434 were cultured in tryptone soybean broth TSB at 37°C for 24 hours to obtain bacterial cultures, and the bacteria were extracted according to the method in Example 1 The bacterial DNA of Enterococcus faecalis (CMCC32223), Enterococcus faecalis (ATCC29212) and Enterococcus faecium (E.Faecium) ATCC19434 were obtained respectively.

[0052] LAMP amplification: The loop-mediated isothermal amplification (LAMP) re...

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Abstract

The invention discloses a loop mediated isothermal amplification detection primer group, a detection method and a kit for enterococcus faecalis. The detection primer group is as follows: an upstream outer primer is F3:5'-GCCATTCCTCATGGAACAG-3'; a downstream outer primer is B 3:5'-CCACGTTATCCATATCACTACA-3'; an upstream inner primer is FIP:5'-CGGTGCCAAAATTAACACCCTTTAGTGAAAAAATCAGGAATCTGTG-3'; a downstream inner primer is BIP:5'-TGCTACCGTATTATTTGGGATTGCAAAGTGCAATTTGTTGGACTA-3'. The detection method has the advantages of high flexibility, strong specificity, high accuracy, short detection time. From sample treatment to report only takes 12 hours, a PCR (polymerase chain reaction) and an electrophoresis apparatus do not need, the operation process is simple, compared with other PCR technology, the detection method has higher specificity, is applicable to self-detection of basic level detection organizations and drinking water processors.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a loop-mediated isothermal amplification detection primer set, detection method and kit for Enterococcus faecalis. Background technique: [0002] Enterococcus faecalis, also known as Streptococcus faecalis, is a Gram-positive, catalase-negative coccus, a representative species of the Enterococcus genus, and an opportunistic pathogenic bacterium that commonly exists in water environments. It is resistant to multilateral adverse environments such as chlorine treatment, municipal sewage treatment, high salinity and is heat resistant, so it can survive in water for a long time. Enterococcus faecalis is an important indicator of fecal pollution in water bodies, and it can more objectively reflect the hygienic status of water quality. In various fresh water and seawater, the diseases caused by Enterococcus faecalis have exceeded that of coliform bacteria. For this reason, the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11C12R1/01
Inventor 徐晓可吴清平张菊梅张淑红郭伟鹏周锦祯程健恒
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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