Reagent for legionella pneumophila detection according to in-vitro nested loop-mediated isothermal amplification method and legionella pneumophila detection method

A ring-mediated isothermal and Legionella pneumophila technology, which is applied in the determination/testing of microorganisms, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of low sensitivity and specificity, and achieve high specificity , the effect of easy operation

Inactive Publication Date: 2016-04-13
浙江国际旅行卫生保健中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The sensitivity and specificity of serum immunological methods are not high

Method used

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  • Reagent for legionella pneumophila detection according to in-vitro nested loop-mediated isothermal amplification method and legionella pneumophila detection method
  • Reagent for legionella pneumophila detection according to in-vitro nested loop-mediated isothermal amplification method and legionella pneumophila detection method
  • Reagent for legionella pneumophila detection according to in-vitro nested loop-mediated isothermal amplification method and legionella pneumophila detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 Detection of Legionella pneumophila.

[0029] Primer design.

[0030] According to the conserved sequence of the mip gene of Legionella pneumophila, a series of primers were designed, including outer primer F3, outer primer B3, inner primer FIP, inner primer BIP, loop primer Loop-f, loop primer Loop-r, set of primers FNP, Set of primers BNP. The primer gene sequence is as follows:

[0031] Outer primer F3: GCTGCAACCGATGCCACA;

[0032] Outer primer B3: TTGGGCCAATAGGTCCGC;

[0033] Inner primer FIP:

[0034] TTGCTGTTCGGTTAAAGCCAATTGTTGTCTTATAGCATTGGTGCCG;

[0035] Inner primer BIP:

[0036] ACTGAAAACAAAAACAAAGCCAGGCGCGTTGCTGGCTTACCAGTTT;

[0037] Loop primer Loop-f: CATGCAAGACGCTATGAGTGG;

[0038] Loop primer Loop-r: CGGGTTTAACACCATTTCCA;

[0039] Set of primers for FNP:

[0040] TTCAGCAGTACGCTTAGCCATCAAAATCAAGGCATAGATGTTAATCCG;

[0041] Set of primers for BNP:

[0042] AAGCGGATGAAAATAAAGTAAAAGGGCCATCAATCAGACGACCAGTAT.

[0043] Among the above prime...

Embodiment 2

[0058] Example 2, detection of Legionella pneumophila in environmental samples.

[0059] Use 20 copies of bacteria stored on magnetic beads separated from air-conditioned water samples, take out the magnetic beads for culture, pick colonies, and extract DNA according to the instructions of the extraction kit. The reaction system is as follows: 10×buffer 2.5 μL, primer mixture 1 μL (primer mixture The concentrations of the primers in the medium are as follows: 1 μmol / L for each of F3 and B3, 15 μmol / L for each of FIP and BIP, 5 μmol / L for each of Loop-f and Loop-r, 15 μmol / L for each of FNP and BNP), 1 μL of template DNA, 8 UBstDNA polymerase 1 μL, betaine (8mol / L) 2 μL, dNTP (10mmol / L) 2 μL, MgSO 4 (100mmol / L) 1.2μL, Eva_green 1μL, add sterile deionized H 2 Make up 25 μL of O, mix well, and use sterilized double distilled water as negative control. The reaction program on the fluorescent quantitative PCR instrument is 63°C, 55 seconds, 63°C, 5 seconds, read the fluorescence ...

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Abstract

The invention provides a reagent for legionella pneumophila detection according to an in-vitro nested loop-mediated isothermal amplification method. The reagent comprises an outer primer F3, an outer primer B3, an inner primer FIP, an inner primer BIP, a loop primer Loop-f, a loop primer Loop-r, a nested primer FNP and a nested primer BNP. The invention further provides a novel efficient in-vitro isothermal amplification based legionella pneumophila detection method adopting the reagent. Quickness and accuracy in legionella pneumophila detection are realized by adoption of the method, sample detection can be finished in 30-45 minutes approximately, LAMP (loop-mediated isothermal amplification) reaction time is shortened by about 30%-50% as compared with that of a method with loop primers added, and great practical significance to quick disease diagnosis is achieved.

Description

technical field [0001] The invention relates to a reagent and a method for detecting legionella pneumophila in vitro. Background technique [0002] Legionella pneumophila ( Legionella lapneumophila ) was first discovered in the United States in 1976. It is a Gram-negative conditional pathogen that is mainly transmitted by polluting water systems in buildings, and is prone to large-scale outbreaks and epidemics. After infection, the disease progresses rapidly and the mortality rate is high. In Western European countries, the annual incidence of Legionella is close to 100,000 cases. With the development of the economy and the widespread use of central air-conditioning, the risk of Legionella outbreaks is increasing. Many countries have listed Legionnaires' pneumonia as a legal infectious disease. The Ministry of Health of my country promulgated the "Hygienic Standards for Centralized Air Conditioning and Ventilation Systems" in 2003, the purpose of which is to cut off the s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q2531/119C12Q2545/113Y02A50/30
Inventor 吕沁风姜侃白颉李杰李洪波
Owner 浙江国际旅行卫生保健中心
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