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Detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and kit

A technology for Legionella pneumophila and Legionella pneumophila, which is applied in microorganism-based methods, biochemical equipment and methods, and determination/inspection of microorganisms, etc., can solve problems such as inability to judge results and inconspicuous melting curve characteristics of Legionella pneumophila. , to achieve stable and reliable results, easy and fast operation, and avoid misdiagnosis.

Active Publication Date: 2011-05-25
SHANGHAI XINGYAO MED TECH DEV CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Stolhaug et al. (Appl Environ Microbiol, 2006(72): 6394-6398) used FRET probe fluorescent PCR technology to detect the 16S rRNA gene of Legionella, and distinguished Legionella pneumophila from other Legionella by melting curve analysis of the amplified product ; But it can be analyzed, due to the variation of the gene sequence of different serotype strains, the melting curve of Legionella pneumophila may not be able to judge the result due to the inconspicuous characteristics

Method used

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  • Detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and kit

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Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1: the preparation of Legionella nucleic acid detection kit (PCR-fluorescent probe method)

[0052] (1) Primer probe synthesis

[0053] Design the primer probe according to the target sequence SEQ ID No.1, and entrust a commercial sequence synthesis company (Shanghai Sangon Bioengineering Technology Service Co., Ltd.) to synthesize:

[0054] The upstream primer is 5'-TgTgAAATTCCTgggCTTAAC-3', as shown in SEQ ID No.2;

[0055] The downstream primer is 5'-TTCgTgCCTCAgTgTCAg-3', as shown in SEQ ID No.3;

[0056] The Legionella fluorescent probe is 5'-CAggTAgCCgCCTTCgCC-3', the 5' end is labeled with FAM (6-carboxyfluorescein), and the 3' end is labeled with BHQ (Black Hole Quencher)), as shown in SEQ ID No.4.

[0057] The fluorescent probe for Legionella pneumophila is 5'-TgggACggTCAgATAATACTggTT-3' (5' end-labeled HEX (6-carboxyhexachlorofluorescein) or JOE (6-carboxy-4', 5'-dichloro-2', 7 '-dimethoxyfluorescein), VIC fluorophore, 3' end labeled BHQ), as sho...

Embodiment 2

[0077] Embodiment 2: the application of Legionella nucleic acid detection kit (PCR-fluorescent probe method)

[0078] (1) Sample processing

[0079] Take the collected water sample, filter it through a filter membrane (pore size 0.22-0.45 μm), put it in a centrifuge tube after elution, centrifuge at 13,000 rpm for 6 minutes, and carefully discard the supernatant (in order to prevent the precipitation from being sucked, 5 μl residual liquid). Add 50 μl of nucleic acid extraction solution to the pellet, vortex to mix, keep at 100°C for 10 minutes, centrifuge at 13,000 rpm for 6 minutes, and take the supernatant for fluorescent PCR amplification detection. The negative and positive controls of the kit are processed in the same way as the sample eluent.

[0080] (2) Amplification detection

[0081] According to the method in the summary of the invention, each component of the unsealed kit was freeze-thawed and oscillated, and then the internal reference was added to the PCR buf...

Embodiment 3

[0084]Embodiment 3: the application of Legionella nucleic acid detection kit (PCR-fluorescent probe method)

[0085] (1) Sample processing

[0086] Take clinically collected throat swabs, sputum, and bronchoalveolar lavage fluid (the throat swabs are first rinsed with 1ml of normal saline, and the sputum is first mixed and liquefied with an equal volume of 4% NaOH), and the throat swab rinse fluid, liquefied phlegm The solution and bronchoalveolar lavage solution were placed in centrifuge tubes respectively, the sample solution was centrifuged at 13,000 rpm for 6 minutes, and the supernatant was carefully discarded (5-10 μl of the residual solution can be retained to prevent the precipitation from being sucked away). Add 50 μl of nucleic acid extraction solution to the pellet, vortex to mix, keep at 100°C for 10 minutes, centrifuge at 13,000 rpm for 6 minutes, and take the supernatant for fluorescent PCR amplification detection. The negative and positive controls of the kit a...

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Abstract

The invention discloses a detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and a kit. The method detects legionella and legionella pneumophilia through single tube amplification of multi-wavelength fluorescent polymerase chain reaction (PCR) technology of a TaqMan probe by adopting a legionella target sequence SEQ ID No.1 design-based primer probe, and distinguishes the non-pneumophilia legionella and the legionella pneumophilia according to the result; and meanwhile, an artificially constructed internal reference system for avoiding false negative detection results is also adopted in the kit. The kit comprises nucleic acid extract, PCR buffer solution, a fluorescent probe, Taq enzyme, an internal reference, and negative and positive contrasts; and two steps comprising sample treatment and amplification detection are used in the kit. The kit is simple and convenient to operate, has high sensitivity, can avoid false positive generated by nucleic acid pollution of PCR products, can solve the problem of false negative caused by a PCR inhibitor in the sample, and can be widely applied to disease control and quick detection of the legionella and the legionella pneumophilia in clinic.

Description

technical field [0001] The invention belongs to a detection method and a kit for distinguishing non-pneumophilic and pneumophilic legionella by using fluorescent PCR. Background technique [0002] Legionella (Legionella) is a class of Gram-negative bacteria, 34 species and 53 serotypes have been found so far. Legionella often hides in air-conditioning coolers (towers), hot water pipes, shower nozzles, etc., spreads through the air, and inhales the human body through the respiratory tract in the form of aerosols, causing Legionnaires’ disease in humans. Patients will have high fever, chills, cough, Chest tightness and other symptoms similar to upper respiratory diseases can cause death in severe cases. Now it is generally reported that its mortality rate is 5-30%. Because Legionella exists widely in places where human beings live, once the disease breaks out, it will cause considerable harm; it is reported that the positive rate of Legionella in cooling towers is relatively ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64C12R1/01
Inventor 吴大治李志远夏懿
Owner SHANGHAI XINGYAO MED TECH DEV CO LTD
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