CN104313169A - Triple PCR detection method for rapidly distinguishing non-LP (legionella pneumophila), LP and LP I - Google Patents
Triple PCR detection method for rapidly distinguishing non-LP (legionella pneumophila), LP and LP I Download PDFInfo
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- CN104313169A CN104313169A CN201410607389.6A CN201410607389A CN104313169A CN 104313169 A CN104313169 A CN 104313169A CN 201410607389 A CN201410607389 A CN 201410607389A CN 104313169 A CN104313169 A CN 104313169A
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Abstract
The invention relates to a triple PCR (Polymerase Chain Reaction) detection method for rapidly distinguishing non-LP (legionella pneumophila), LP and LP I, which is a legionella multiple PCR detection method and belongs to the field of microorganism molecule detection. According to the invention, primers of 16SrRNA, danJ and ORF9 genes which respectively aim at legionella, LP and LP I are utilized to optimize a dNTPs and primer concentration ratio and an annealing temperature so as to establish the triple PCR detection method for rapidly distinguishing the non-LP (legionella pneumophila), the LP and the LP I. The method disclosed by the invention is simple, convenient and rapid, is low in cost, has an accurate and reliable result and can rapidly and effectively judge dubious legionella strains cultured by separation.
Description
Technical field
Quick differentiation non-addicted to lung, addicted to lung with addicted to the triple PCR detection method of lung I type legionella, be a kind of legionella multi-PCR detection method, belong to microbial molecules detection field.
Background technology
The légionaires' disease of more than 90% is caused by legionella pneumophilia (Legionella Pneumophila, LP), and wherein 85% is LP1 type Legionnaires Pneumonia, so legionella pneumophilia and LP1 type legionella pneumophilia are the emphasis that we monitor.According to WS394-2012 " public place centralized air ventilation system hygienic practice ", must not detect legionella pneumophilia in all public places centralized air ventilation system cooling tower water and water of condensation, the standard detecting method of this specification is culture method.But traditional culture method exists blind area when judging the suspicious legionella bacterium colony be separated to, the uncertainty of biochemistry detection and SD many cross reactivities, cause some suspicious legionella bacterium colonies finally not differentiated.The present invention can make up deficiency that is biochemical and serological identification detection, fast and accurately to separation and Culture to suspicious legionella bacterium colony judge, and simply, conveniently, cost is low.
Summary of the invention
The object of the invention is to fast and accurately to separation and Culture to suspicious legionella bacterium colony judge, to overcome the shortcoming that culture-based method mesophytization and serological identification detect.
Technical scheme of the present invention: a kind of distinguish fast non-addicted to lung (non-LP), addicted to lung (LP) and the triple PCR detection method addicted to lung 1 type (LP1) legionella, step is:
1, prepare pcr template, picking suspicious single bacterium colony to be detected is in the deionized water of 0.5mL sterilizing nuclease free, and be prepared into bacteria suspension, 100 DEG C are boiled 15min, for subsequent use after cooling.
2, primer sequence:
16S rRNA gene: upstream primer 5 '-AAGATTAGCC TGCGTCCGAT-3 ', downstream primer 5 '-GTCAACTTAT CGCGTTTGCT-3 ', expection expanding fragment length is 654 bp;
DanJ gene: upstream primer 5 '-AGGTGGTTTT GGCGGATTTG G-3 ', downstream primer 5 '-TGAATTCTGA CTTGCCCCAT G-3 ', expection expanding fragment length is 285 bp;
ORF9 gene: upstream primer 5 '-CAGGATTACC GCTCATTATT G-3 ', downstream primer 5 '-GTAATTCCCA GCCATTTACC AGATC-3 ', expection expanding fragment length is 561 bp;
3, PCR reaction system: 1.6 μ L dNTP, three kinds of all each 2 μ L of upstream and downstream primer (working concentration is 2 μMs), template DNA 2 μ L, rTaq enzyme 0.08 μ L, 10 × buffer damping fluid 2 μ L, the deionized water of nuclease free complements to 20 μ L;
4, amplification condition: 95 DEG C of denaturation 5 min; 95 DEG C of 30 s, 52 DEG C of 30 s, 72 DEG C of 40 s carries out 30 circulations; 72 DEG C of whole ends extend 5 min;
5, gel electrophoresis: at 1.5% sepharose, electrophoresis in 0.5 × tbe buffer liquid, after electrophoresis, gel imaging obtains result;
6, decision method: the band obtaining 3 entries is LP1, the band of two entries is LP, and the band of 1 entry is non-LP, and the band of 0 entry is non-legionella.
Beneficial effect of the present invention: present method is easy, quick, cost is low, result accurately and reliably, can to separation and Culture to suspicious legionella bacterial strain judge fast and effectively, distinguish fast non-addicted to lung, addicted to lung with addicted to lung 1 type legionella.
Accompanying drawing explanation
Fig. 1 is 29 strain legionella PCR primer electrophoretograms.M:100bp DNA marker (the brightest band is 500 bp, and upwards, downwards often band increases successively, subtracts 100 bp); 1,18 is negative control, and 2 is non-legionella pneumophilia
l. Jordanis(ATCC33623), 3 is LP6 (ATCC33216), and 4 is LP1 (ATCC33152), 5 ~ 17,19 ~ 34 29 strain legionella for being separated to.
specific examples mode
Embodiment 1 one kinds is distinguished fast non-addicted to lung, addicted to lung and the triple PCR detection method addicted to lung 1 type legionella, step is,
1, prepare pcr template, picking suspicious single bacterium colony to be detected is in the deionized water of 0.5mL sterilizing nuclease free, and be prepared into bacteria suspension, 100 DEG C are boiled 15min, for subsequent use after cooling.
2, primer sequence:
16S rRNA gene: upstream primer 5 '-AAGATTAGCC TGCGTCCGAT-3 ', downstream primer 5 '-GTCAACTTAT CGCGTTTGCT-3 ', expection expanding fragment length is 654 bp;
DanJ gene: upstream primer 5 '-AGGTGGTTTT GGCGGATTTG G-3 ', downstream primer 5 '-TGAATTCTGA CTTGCCCCAT G-3 ', expection expanding fragment length is 285 bp;
ORF9 gene: upstream primer 5 '-CAGGATTACC GCTCATTATT G-3 ', downstream primer 5 '-GTAATTCCCA GCCATTTACC AGATC-3 ', expection expanding fragment length is 561 bp.
3, PCR reaction system: 1.6 μ L dNTP, three kinds of all each 2 μ L of upstream and downstream primer (working concentration is 2 μMs), template DNA 2 μ L, rTaq enzyme 0.08 μ L, 10 × buffer damping fluid 2 μ L, the deionized water of nuclease free complements to 20 μ L.
4, amplification condition: 95 DEG C of denaturation 5 min; 95 DEG C of 30 s, 52 DEG C of 30 s, 72 DEG C of 40 s carries out 30 circulations; 72 DEG C of whole ends extend 5 min.
5, gel electrophoresis: electrophoresis in 1.5% sepharose, 0.5 × tbe buffer liquid, after electrophoresis, gel imaging obtains result.
6, result judges: obtain the band of 3 entries as LP1, and the band of two entries is LP, and the band of 1 entry is non-LP, and the band of 0 entry is non-legionella.
Claims (1)
1. distinguish fast non-addicted to lung, addicted to lung and the triple PCR detection method addicted to lung I type legionella, it is characterized in that step is:
(1) prepare pcr template, picking suspicious single bacterium colony to be detected is in the deionized water of 0.5mL sterilizing nuclease free, and be prepared into bacteria suspension, 100 DEG C are boiled 15min, for subsequent use after cooling;
(2) primer sequence:
16S rRNA gene: upstream primer 5 '-AAGATTAGCC TGCGTCCGAT-3 ', downstream primer 5 '-GTCAACTTAT CGCGTTTGCT-3 ', expection expanding fragment length is 654 bp;
DanJ gene: upstream primer 5 '-AGGTGGTTTT GGCGGATTTG G-3 ', downstream primer 5 '-TGAATTCTGA CTTGCCCCAT G-3 ', expection expanding fragment length is 285 bp;
ORF9 gene: upstream primer 5 '-CAGGATTACC GCTCATTATT G-3 ', downstream primer 5 '-GTAATTCCCA GCCATTTACC AGATC-3 ', expection expanding fragment length is 561 bp;
(3) PCR reaction system: 1.6 μ L dNTP, concentration are all each 2 μ L of three kinds of upstream and downstream primers of 2 μMs, template DNA 2 μ L, rTaq enzyme 0.08 μ L, 10 × buffer damping fluid 2 μ L, the deionized water of nuclease free complements to 20 μ L;
(4) amplification condition: 95 DEG C of denaturation 5 min; 95 DEG C of 30 s, 52 DEG C of 30 s, 72 DEG C of 40 s carries out 30 circulations; 72 DEG C of whole ends extend 5 min;
(5) gel electrophoresis: electrophoresis in 1.5% sepharose, 0.5 × tbe buffer liquid, after electrophoresis, gel imaging obtains result;
(6) decision method: the band obtaining 3 entries is LP1, the band of two entries is LP, and the band of 1 entry is non-LP, and the band of 0 entry is non-legionella.
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| CN201410607389.6A CN104313169B (en) | 2014-11-03 | 2014-11-03 | A kind of quick differentiation is non-addicted to lung, addicted to lung and the triple PCR detection method addicted to lung I type legionella |
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| CN201410607389.6A CN104313169B (en) | 2014-11-03 | 2014-11-03 | A kind of quick differentiation is non-addicted to lung, addicted to lung and the triple PCR detection method addicted to lung I type legionella |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154538A (en) * | 2015-08-26 | 2015-12-16 | 广州金域医学检验中心有限公司 | Primer and method for rapidly detecting and parting legionella |
| CN105219847A (en) * | 2015-08-26 | 2016-01-06 | 广州金域医学检验中心有限公司 | A kind of legionella rapid detection and parting kit and detection method thereof |
| CN105255876A (en) * | 2015-11-13 | 2016-01-20 | 广州市圣鑫生物科技有限公司 | Primers and method for legionella quick detecting and parting |
| CN106701981A (en) * | 2017-02-06 | 2017-05-24 | 广州金域医学检验中心有限公司 | Rapid detection kit and method for legionella pneumophila ST1 type strain |
Citations (3)
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| CN101717815A (en) * | 2009-07-09 | 2010-06-02 | 广州金域医学检验中心有限公司 | Legionnella rapid detecting and parting method |
| CN102071247A (en) * | 2009-11-24 | 2011-05-25 | 上海复星医学科技发展有限公司 | Detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and kit |
| CN102134588A (en) * | 2010-11-24 | 2011-07-27 | 山东大学 | Legionella pneumophilia test kit and application thereof |
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2014
- 2014-11-03 CN CN201410607389.6A patent/CN104313169B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101717815A (en) * | 2009-07-09 | 2010-06-02 | 广州金域医学检验中心有限公司 | Legionnella rapid detecting and parting method |
| CN102071247A (en) * | 2009-11-24 | 2011-05-25 | 上海复星医学科技发展有限公司 | Detection method for distinguishing non-pneumophilia legionella and legionella pneumophilia and kit |
| CN102134588A (en) * | 2010-11-24 | 2011-07-27 | 山东大学 | Legionella pneumophilia test kit and application thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105154538A (en) * | 2015-08-26 | 2015-12-16 | 广州金域医学检验中心有限公司 | Primer and method for rapidly detecting and parting legionella |
| CN105219847A (en) * | 2015-08-26 | 2016-01-06 | 广州金域医学检验中心有限公司 | A kind of legionella rapid detection and parting kit and detection method thereof |
| CN105219847B (en) * | 2015-08-26 | 2017-06-13 | 广州金域医学检验中心有限公司 | A kind of Legionella quick detection and parting kit and its detection method |
| CN105154538B (en) * | 2015-08-26 | 2017-06-13 | 广州金域医学检验中心有限公司 | The primer and method of a kind of Legionella quick detection and parting |
| CN105255876A (en) * | 2015-11-13 | 2016-01-20 | 广州市圣鑫生物科技有限公司 | Primers and method for legionella quick detecting and parting |
| CN106701981A (en) * | 2017-02-06 | 2017-05-24 | 广州金域医学检验中心有限公司 | Rapid detection kit and method for legionella pneumophila ST1 type strain |
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