CN103525902A - Rapid identification method for legionella pneumophila - Google Patents

Rapid identification method for legionella pneumophila Download PDF

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CN103525902A
CN103525902A CN201210346366.5A CN201210346366A CN103525902A CN 103525902 A CN103525902 A CN 103525902A CN 201210346366 A CN201210346366 A CN 201210346366A CN 103525902 A CN103525902 A CN 103525902A
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legionella pneumophilia
legionella
rapid identification
probe
legionella pneumophila
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朱庆义
胡朝晖
赵利伟
戈立秀
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Gold Territory Nanning Co Ltd Of Medical Test Institute
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Abstract

The invention provides a rapid identification method for legionella pneumophila. The rapid identification method comprises the following steps: adopting a pair of legionella pneumophila specific primers to amplify a region on a 16S rRNA (ribosomal Robonucleic Acid) gene; designing a pair of hybridization probes in an amplified segment aiming at specific sequences of the legionella pneumophila specific primers; anchoring a probe 3'-end marked FAM fluorescent gene and quenching a probe 5'-end marked BHQ1 (Black Hole Quencher 1); carrying out phosphorylation on the 3' end; analyzing the legionella pneumophila in a sample by real-time fluorescence PCR (Polymerase Chain Reaction) melting curve analysis. The detection method mainly comprises the following three steps: extracting a sample DNA (Deoxyribose Nucleic Acid), carrying out real-time fluorescence PCR and analyzing a melting curve. The method is simple and rapid to operate, is intuitive in result, is high in specificity and good in stability, and can be widely applied to rapid identification of the legionella pneumophila in clinical and environmental water samples.

Description

Legionella pneumophilia rapid identification method
Technical field
The invention belongs to medical field, specifically a kind of method of utilizing fluorescent PCR melting curve to identify legionella pneumophilia.
Background technology
Legionella is a kind of facultative born of the same parents' endophyte, is the important pathogen body that causes mankind's légionaires' disease.In recent years, légionaires' disease has eruption and prevalence during all over the world, and China has been listed in one of 14 kinds of emerging infectious diseases.According to external, in institute, unknown cause pneumonia has 10% to be to be caused by legionella.Current known legionella has 52 kinds, more than 70 serotype.But 80% above infection of legionella is to be caused by legionella pneumophilia clinically.Therefore, for légionaires' disease, diagnosis has great value in the evaluation of legionella pneumophilia.
The detection authentication method of legionella mainly contains separation and Culture, Serum Antibody Detection, urinary soluble antigen detection at present, in addition, also have molecule parting detection technique, mainly comprise traditional polymerase chain reaction (PCR) electrophoresis and probe hybridization detection method, amplification length polymorphism analysis (AFLP), restriction fragment length polymorphism analysis (RFCP), Random amplified polymorphic DNA (RAPD), restricted enzyme cutting analysis, DNA sequencing analysis etc.But the above equal Shortcomings of detection method, as direct separation and Culture, required time reaches 3～10 days, and positive rate is low, and substratum configuration is complicated, expensive; Urine Detection of antigen only has specificity to legionella pneumophilia serotype 1 type; Although molecule parting technology has improved susceptibility and specificity that legionella detects to a great extent, but still has shortcoming separately, as traditional PCR detects complex operation, length consuming time, easily pollutes; Hybridization probe labeling technique based on FRET (fluorescence resonance energy transfer) principle is expensive, complicated operation; DNA sequencing is species of legionella authentication method the most accurately, but consuming time longer, analyzes difficulty, can not adapt to quick requirement clinically, and other molecular detecting methods all exist various deficiencies.
Summary of the invention
The present invention is based on FRET (fluorescence resonance energy transfer) principle, according to legionella pneumophilia 16S rRNA gene conserved sequence design primer and probe, choose a kind of quench fluorescence group and replace original hybridization probe fluorescent marker, set up novel real-time fluorescence PCR melting curve method legionella pneumophilia is identified.
The present invention is achieved in that a kind of legionella pneumophilia rapid identification method, based on FRET (fluorescence resonance energy transfer) principle, chooses a kind of quench fluorescence group, sets up novel real-time fluorescence PCR melting curve method legionella pneumophilia is identified.
Further, the step of described authentication method following (1) is listed to (4),
(1) real-time fluorescence PCR reaction adopts in setting up pair of primers and a pair of probe:
Upstream primer: 5 '-AGGGTTGATAGGTTAAGAGC-3 ',
Downstream primer: 5 '-CCAACAGCTAGTTGACATCG-3 ',
Grappling probe: 5 '-CCCATACTCGAGTCAACC (FAM)-3 ',
Quenching probe: 5 '-(BHQ1) TATTATCTGACCGTCCCAG (ph)-3 ';
(2) extract DNA: get sputum sample 1ml and add equivalent 0.1% dithiothreitol (DTT) phlegm Digestive system processing, at 37 ℃, place 30min, centrifugation, the extracting genome DNA liquid 60 μ l that add autogamy in precipitating species, fully mix, 55 ℃ of water-bath 30min, 100 ℃ of water-bath 10min, supernatant liquor is genomic dna template;
(3) the above genomic dna of obtaining is template, carries out real-time fluorescence PCR response procedures: first 95 ℃ of denaturation 3min, then 95 ℃ of sex change 10s, and 57 ℃ of annealing 20s, 72 ℃ are extended 30s, carry out 50 circulations;
(4) detected result is judged, melting curve analysis program is: 95 ℃ of 1min; 40 ℃ of 2min; With the speed of 1 ℃/5s, be warming up to 85 ℃ from 40 ℃, the Tm value of legionella pneumophilia is 57 ℃, and other non-legionella pneumophilia and non-legionella are all without the Tm value of 57 ℃.
Described PCR reaction system is as follows:
Figure BDA0000215432422
The present invention is the rapid identification method of a kind of legionella pneumophilia, it adopts the region on a pair of legionella special primer amplification 16S rRNA gene, in this amplified fragments (386bp), for legionella pneumophilia distinguished sequence, design a pair of hybridization probe, grappling probe 3' end flag F AM fluorophor; Quenching probe 5' end mark BHQ1,3' holds phosphorylation; By real-time fluorescence PCR melting curve quencher group, replace original hybridization probe fluorescent marker to analyze to detect the legionella pneumophilia in sample.
The present invention is according to primer probe and the technical scheme of above-mentioned design, and by the new detection method of legionella pneumophilia of optimizing reaction system and amplification condition foundation, its principal feature is as follows:
(1) high specific: the present invention is by carrying out genotype tests to 16 strain legionella pneumophilias (comprising 15 serotypes), the non-legionella pneumophilia of 35 strain and other non-legionella of 12 strains, the method can accurately identify legionella pneumophilia, illustrates that the method is the legionella pneumophilia detection method that a species specificity is very high;
(2) hypersensitivity: the melting curve analysis method that the present invention sets up, has improved the susceptibility detecting greatly.By restricted dilution method, find that the method lowest detection limit reaches 100fg/ μ l;
(3) high stability: according to the probe of legionella pneumophilia specific target sequences Design, mark fluorescent group and quenching of fluorescence group detect legionella pneumophilia, and result is reliable and stable;
(4) detection method stopped pipe operation, easy to be quick;
(5) sensing range is wide: as a kind of legionella pneumophilia rapid detection means, Technical Design of the present invention is reasonable, can be widely used in the Rapid identification of legionella pneumophilia in clinical and ambient water sample.
The present invention can be for clinical sputum sample, also can be for environmental water sample this and other clinical samples as the legionella pneumophilia Rapid identification of bronchial perfusate etc., but be not limited in this.
Embodiment
Real-time fluorescence PCR melting curve method rapid detection legionella pneumophilia method of the present invention is a complete integral body.Below in conjunction with specific embodiment, further set forth the present invention.
Real-time fluorescence PCR melting curve method rapid detection legionella pneumophilia method of the present invention, mainly comprises the steps:
1, the preparation of main agents required for the present invention
Test the primer is synthetic by Shanghai Ying Weijie base trade Co., Ltd, and probe is synthetic by Ji Ke bio tech ltd, Xiamen, and 10 * PCR Master Mix, warm start Taq enzyme are purchased from Ji Ke bio tech ltd, Xiamen.
(1) real-time fluorescence PCR probe design: analyze by the 16S rRNA sequence alignment to legionella pneumophilia, non-legionella pneumophilia and other non-legionella, find legionella pneumophilia high conservative and have fine homology sequence, according to the principle of hybridization probe fluorescent PCR design, design a pair of legionella pneumophilia specific probe, grappling probe 3' end flag F AM, quenching probe 5' end mark quencher group B HQ1,3' holds phosphorylation.
Concrete sequence is as follows: grappling probe 5 '-CCCATACTCGAGTCAACC (FAM)-3 ', quenching probe 5'-(BHQ1) TATTATCTGACCGTCCCAG (ph)-3'.
(2) real-time fluorescence PCR primer design: compare by the 16S rRNA gene sequencing to legionella pneumophilia and non-legionella pneumophilia, the a pair of conservative primer of design alternative, the conserved sequence of one section of 386bp in legionella 16S rRNA gene is used for increasing, upstream primer 5 '-AGGGTTGATAGGTTAAGAGC-3 ', downstream primer 5 '-CCAACAGCTAGTTGACATCG-3 '.
2, extract the genomic dna (take clinical sputum specimen as example, extract by this laboratory self-control extracting solution) of sample to be checked
Get sputum sample 1ml and add the processing of equivalent 0.1% dithiothreitol (DTT) (DTT) phlegm Digestive system, place 30min for 37 ℃, centrifugation, in precipitating species, add the CTEN extracting genome DNA liquid 60 μ l(of autogamy as shown in table 1), fully mix, 55 ℃ of water-bath 30min, 100 ℃ of water-bath 10min, supernatant liquor is genomic dna template.
Table 1 CTEN extracting genome DNA liquid formula (mM in table is every liter of mmole)
Composition Concentration
Chelex-100 solution 5%
Trishydroxymethyl ammonia methane hydrochloride salt (Tris-HCL) [PH 8.3] 10 mM
Disodium ethylene diamine tetraacetate (EDTA-Na 2） 0.1 mM
Sodium azide (NaN 3） 0.1%
Proteinase K (Protease-k) 100μg/ml
Water H 2O ------
3, real-time fluorescence PCR reaction
The genomic dna of more than obtaining is template, carries out real-time fluorescence PCR reaction, and concrete reaction system is as table 2:
Table 2 real-time fluorescence PCR reaction system
Composition Concentration
10×PCR Master Mix 15 μl
Upstream primer (2 pmol/ μ l) 2.0 μl
Downstream primer (40 pmol/ μ l) 1.0 μl
Grappling probe (10 pmol/ μ l) 1.0 μl
Quenching probe (10 pmol/ μ l) 0.5 μl
Warm start Taq enzyme 0.2 μl
Distilled water ddH2O 0.3 μl
Genomic dna (template) 5.0μl
Cumulative volume 25 μl
Response procedures: first 95 ℃ of denaturation 3min; 95 ℃ of sex change 10s again, 57 ℃ of annealing 20s, 72 ℃ are extended 30s, carry out 50 circulations;
4, melting curve analysis result is judged
After above reaction system and response procedures complete, carry out melting curve analysis, routine analyzer is: 95 ℃ of 1min; 40 ℃ of 2min; With the speed of 1 ℃/5s, from 40 ℃, be warming up to 85 ℃.The Tm value of legionella pneumophilia is 57 ℃, and other non-legionella pneumophilia and non-legionella are all without the Tm value of 57 ℃.Therefore, as there is the Tm value of 57 ℃, can think that this detection sample is legionella pneumophilia; Otherwise, think that legionella pneumophilia is negative.
Figure IDA00002154324400011

Claims (4)

1. a legionella pneumophilia rapid identification method, is characterized in that, based on FRET (fluorescence resonance energy transfer) principle, chooses a kind of quench fluorescence group, sets up novel real-time fluorescence PCR melting curve method legionella pneumophilia is identified.
2. the legionella pneumophilia rapid identification method described in claims 1, is characterized in that, the step of described authentication method is listed as follows,
(1) real-time fluorescence PCR reaction adopts in setting up pair of primers and a pair of probe:
Upstream primer: 5 '-AGGGTTGATAGGTTAAGAGC-3 ',
Downstream primer: 5 '-CCAACAGCTAGTTGACATCG-3 ',
Grappling probe: 5 '-CCCATACTCGAGTCAACC (FAM)-3 ',
Quenching probe: 5 '-(BHQ1) TATTATCTGACCGTCCCAG (ph)-3 ';
(2) extract DNA: get sputum sample 1ml and add equivalent 0.1% dithiothreitol (DTT) phlegm Digestive system processing, at 37 ℃, place 30min, centrifugation, the extracting genome DNA liquid 60 μ l that add autogamy in precipitating species, fully mix, 55 ℃ of water-bath 30min, 100 ℃ of water-bath 10min, supernatant liquor is genomic dna template;
(3) the above genomic dna of obtaining is template, carries out real-time fluorescence PCR response procedures: first 95 ℃ of denaturation 3 min, then 95 ℃ of sex change 10s, and 57 ℃ of annealing 20s, 72 ℃ are extended 30s, carry out 50 circulations;
(4) detected result is judged, melting curve analysis program is: 95 ℃ of 1min; 40 ℃ of 2min; With the speed of 1 ℃/5s, be warming up to 85 ℃ from 40 ℃, the Tm value of legionella pneumophilia is 57 ℃, and other non-legionella pneumophilia and non-legionella are all without the Tm value of 57 ℃.
3. legionella pneumophilia rapid identification method as claimed in claim 2, is characterized in that described PCR reaction system is as follows:
Figure FDA0000215432411
Figure FDA0000215432412
。
4. legionella pneumophilia rapid identification method as claimed in claim 2, is characterized in that described extracting genome DNA liquid is:
Figure FDA0000215432413
。
CN201210346366.5A 2012-09-18 2012-09-18 Rapid identification method for legionella pneumophila Pending CN103525902A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755379A (en) * 2016-12-13 2017-05-31 海南医学院 4 kinds of Aspergillus are synchronously quantified and the dimer of Genotyping is mutated fluorescent primer quantifying PCR method
CN114410847A (en) * 2022-03-03 2022-04-29 广东省人民医院 Legionella pneumophila and dengue fever nucleic acid quantitative combined detection reagent

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101597647A (en) * 2009-07-09 2009-12-09 广州金域医学检验中心有限公司 Species of legionella quick detection kit and detection method thereof
CN102226160A (en) * 2011-05-13 2011-10-26 广州金域医学检验中心有限公司 Sputasol for isolated culture of Legionnella

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101597647A (en) * 2009-07-09 2009-12-09 广州金域医学检验中心有限公司 Species of legionella quick detection kit and detection method thereof
CN102226160A (en) * 2011-05-13 2011-10-26 广州金域医学检验中心有限公司 Sputasol for isolated culture of Legionnella

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
XIAO-YONG ZHAN ET AL.: "Two-step scheme for rapid identification and differentiation of Legionella pneumophila and non-Legionella pneumophila species", 《JOURNAL OF CLINICAL MICROBIOLOGY》, vol. 48, no. 2, 9 December 2009 (2009-12-09), pages 433 - 439 *
戈立秀等: "实时荧光PCR熔解曲线法快速鉴定嗜肺军团菌", 《临床减压杂志》, vol. 30, no. 3, 31 March 2012 (2012-03-31) *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106755379A (en) * 2016-12-13 2017-05-31 海南医学院 4 kinds of Aspergillus are synchronously quantified and the dimer of Genotyping is mutated fluorescent primer quantifying PCR method
CN106755379B (en) * 2016-12-13 2021-06-04 海南医学院 Dimer mutation fluorescent primer quantitative PCR method for synchronously quantifying and genotyping 4 aspergillus
CN114410847A (en) * 2022-03-03 2022-04-29 广东省人民医院 Legionella pneumophila and dengue fever nucleic acid quantitative combined detection reagent

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Application publication date: 20140122