Loop-mediated isothermal amplification detection primer pair of Pseudomonas aeruginosa, detection method and detection kit

A Pseudomonas aeruginosa, ring-mediated isothermal technology, applied in biochemical equipment and methods, microbial determination/inspection and other directions, can solve the problems of inability to meet rapid detection and low sensitivity, and achieve short detection time and high sensitivity. , the effect of simple operation process

Active Publication Date: 2011-07-27
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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Problems solved by technology

[0004] At present, in terms of detection technology for Pseudomonas aeruginosa, traditional physical and chemical detection methods are still used in domestic and foreign standards, including coating method, multi-tube MPN method, membrane filtration method, etc., which require multiple steps such as pure culture and biochemical identification , the entire detection period is 3-5 days, the sensitivity is low, and it cannot meet the needs of on-site rapid detection, especially the rapid detection of large quantities of water samples

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  • Loop-mediated isothermal amplification detection primer pair of Pseudomonas aeruginosa, detection method and detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: Detection of Pseudomonas aeruginosa in bottled water

[0037] 1. Water sample filtration

[0038]Take 250mL of a bottled water sample (1), filter it through a 0.45um filter membrane by aseptic operation, transfer the filter membrane to 100ml selective enrichment solution for cultivation, and incubate at 36°C±1°C for 20h to obtain a bacterial culture.

[0039] 2. Bacterial DNA extraction

[0040] Take 1mL of bacterial culture and centrifuge at 12000r / min for 5min, discard the supernatant, collect the bacteria, add 50μL TE solution to suspend and mix well, put in 100℃ water bath for 10min, ice bath for 3min, centrifuge at 12000r / min for 5min, take the supernatant for later use , the supernatant contains bacterial DNA, which serves as template DNA.

[0041] 3. LAMP amplification

[0042] Add 1 μL of Bst DNA polymerase (8 U / μL) and 1 μL of template DNA to a reaction tube containing 23 μL of loop-mediated isothermal amplification (LAMP) reaction solution, in...

Embodiment 2

[0047] Example 2: Detection of Pseudomonas aeruginosa in bottled water

[0048] This embodiment is basically the same as Embodiment 1, except that the water sample is 250 mL of a certain bottled water sample (2), and is detected according to the operation of Embodiment 1. After testing, the color of the reaction tube turns orange, indicating that the sample is negative for Pseudomonas aeruginosa and does not contain Pseudomonas aeruginosa.

[0049] The sample was detected by the traditional biochemical identification method (GB8538-2008), and the test result was negative, and the sample did not contain Pseudomonas aeruginosa, and the test results were consistent, which proved that the data of this method was reliable.

Embodiment 3

[0050] Embodiment 3: the detection of Pseudomonas aeruginosa in bottled water

[0051] This example is basically the same as Example 1, and the water sample is also a bottled water sample (1), except that in the LAMP amplification in step (3), the 23 μL loop-mediated isothermal amplification (LAMP) reaction solution is composed of: 2.5 μL 10× Thermopol reaction buffer, 3.5 μL 10 mmol / L dNTPs, 0.375 μL 10 μmol / L upstream outer primer F3, 0.375 μL 10 μmol / L downstream outer primer B3, 0.75 μL 40 μmol / L upstream inner primer FIP, 0.75 μL 40 μmol / L Downstream internal primer BIP, 1 μL 100mmol / LMgSO 4 , 8 μL 2.5mol / L betaine, 5.75 μL sterilized double distilled water ddH 2 O.

[0052] After detection, the color of the reaction tube turned green, indicating that the sample contained Pseudomonas aeruginosa. It is consistent with the detection result of Example 1.

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Abstract

The invention discloses a loop-mediated isothermal amplification (LAMP) detection primer pair of Pseudomonas aeruginosa, a detection method and a detection kit. Aiming at the ecfX gene of Pseudomonas aeruginosa, the invention designs and screens a set of specific detection primer pair, a detection kit containing the detection primer pair and an LAMP detection method utilizing the detection kit. The detection method is adopted to detect a sample to be detected, specially for drinking water to confirm whether the sample contains the special gene segment of Pseudomonas aeruginosa and further confirm whether the sample contains Pseudomonas aeruginosa. The detection kit and detection method disclosed by the invention have high sensitivity and specificity and short detection time, and the wholeprocess from sample processing to result reporting is only 24h; and the polymerase chain reaction (PCR) amplifier and electrophoresis meter are not adopted, the operation process is simple and the method and kit are especially suitable for the self-checking of grassroots detection institutions and drinking water processing enterprises.

Description

Technical field: [0001] The present invention relates to a rapid detection technology for pathogenic bacteria based on loop-mediated isothermal amplification (loop-mediated isothermalamplification, LAMP) technology, in particular to the specific detection of fragments of the specific gene ecfX of Pseudomonas aeruginosa (Pseudomonas aeruginosa) The primer set is a detection method and a detection kit for detecting Pseudomonas aeruginosa by using the detection primer set using the loop-mediated isothermal amplification method LAMP. Background technique: [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa), also known as Pseudomonas aeruginosa, is a solitary flagella, active movement, no spores, obligate aerobic Gram-negative bacilli, widely present in water, soil, air, and human It is one of the main sources of pollution in drinking water. Pseudomonas aeruginosa can produce a variety of endotoxins, leading to acute intestinal diseases and skin inflammation. In addition, b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 张淑红吴清平徐晓可张菊梅
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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