CN102304575A - Method for identifying mycobacterium tuberculosis and nontuberculoaus mycobacteriosis quickly - Google Patents
Method for identifying mycobacterium tuberculosis and nontuberculoaus mycobacteriosis quickly Download PDFInfo
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Abstract
The invention discloses a method for identifying mycobacterium tuberculosis and nontuberculoaus mycobacteriosis quickly. In the principle of the method, four specific primers are designed according to the specificity of the mycobacterium tuberculosis and nontuberculoaus mycobacteriosis 16S ribonucleic acid (RNA), a deoxyribonucleic acid (DNA) template of a sample is amplified at the temperature of between 63 and 65 DEG C by using the four specific primers and DNA polymerase with strand displacement activity, the amplification efficiency can reach 109 to 1,010 copies within a short time, and color change is observed by adding SYBR Green I to judge whether the template is amplified or not. The method has the advantages of high sensitivity, high specificity and the like, is convenient and quick, has relatively high reference value on the diagnosis and clinical administration of the mycobacterium tuberculosis or the nontuberculoaus mycobacteriosis, and is suitable for popularization and application.
Description
Technical field
The present invention relates to the detection and the evaluation of pathogenic micro-organism, be specifically related to a kind of method with ring mediated isothermal gene amplification technology (LAMP technology) Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria.
Background technology
In recent years; Non-tuberculous mycobacteria infects obviously to be increased; The disease that tuberculosis and non-tuberculous mycobacteria cause clinically is difficult to difference; The white plaque that the most representative mycobacterium tuberculosis causes exactly likes non-tuberculous mycobacteria tuberculosis, but both are different to anti-tuberculosis medicaments sensibility, so correct identification of mycobacterium is most important in medical diagnosis on disease and treatment.Traditional biological assay method mainly according to bacterial growth speed, pigment produce, to drug tolerance, biochemical reaction etc.; Method is complicated, time-consuming; After obtaining the separation and Culture thing, still need 2～4 weeks can obtain the result; Therefore be necessary to set up the method for a kind of quick diagnosis mycobacterium tuberculosis and non-tuberculous mycobacteria, white plaque conclusive evidence and clinical application are had important directive significance.
The molecular biosciences method of differentiating mycobacterium tuberculosis at present has PCR; Method such as ELISA, immune colloid gold; Be worth though above the whole bag of tricks possibly provide preferably clinically, fail clinically large-scale promotion because of different reasons such as the expensive plant and instrument of needs, loaded down with trivial details operating process, sensitivity are low and use.Ring mediated isothermal gene amplification technology (Loop-Mediated Isothermal Amplification; Hereinafter to be referred as the LAMP method) be the gene amplification technology that Japanese Eiken Chemical developed before and after 2000; It has fast and convenient, operation accurately, popularize easily, safe and reliable advantage, do not have as yet at present the LAMP method is applied to Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria.
Summary of the invention
The object of the present invention is to provide one group of Auele Specific Primer that is used for Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria, and a kind of method with ring mediated isothermal gene amplification technology (LAMP technology) Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria.This authentication method is convenient and swift, cheap, is suitable for applying.
For achieving the above object, the technical scheme that the present invention adopted is following:
The present invention is according to 4 Auele Specific Primers of specificity design of mycobacterium tuberculosis and non-tuberculous mycobacteria 16S RNA; Utilize loop-mediated isothermal amplification technique; At 60～65 ℃, isothermal duplication 60 ~ 90 minutes is distinguished mycobacterium tuberculosis and non-tuberculous mycobacteria according to the color that developer shows.
Be used for the Auele Specific Primer of Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria, comprise:
Inner primer 1:5 '-TGGTCGTAGTAGGTCGATGGGGAGTCGATCTGCACACAGCT-3 ' (SEQ ID No.1);
Inner primer 2:5 '-TGCGCGATGGCGAACTCAAGGCACCGTAAACACCGTAG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-TCATCGCCGATCATCAGG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGAGTTTGGTCATCAGCCG-3 ' (SEQ ID No.4).
The method of a kind of Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria specifically comprises the steps:
(1) extraction of template DNA: extract the DNA of sample to be checked, said sample to be checked is to be accredited as sample or the isolating mycobacterium that contains mycobacterium;
(2) ring mediated isothermal gene amplification reaction: prepare reaction system at 200 μ l PCR pipe: primer mixed solution 1 μ l; Reaction solution 12.5 μ l; Archaeal dna polymerase 0.5 ~ 1 μ l, template DNA 2 ~ 5 μ l use sterilization deionized water polishing to 25 μ l; The sealing liquid that adds 20 μ l then reacts above-mentioned reaction tubes to 60 ~ 90min in 63 ~ 65 ℃;
Said primer mixed solution contains 4 above-mentioned Auele Specific Primers (SEQ ID No.1 ~ 4); Wherein, the concentration of inner primer 1 is that the concentration of 4 ~ 8 pmol/ μ l, inner primer 2 is that the concentration of 4 ~ 8 pmol/ μ l, outer primer 1 is that the concentration of 1 pmol/ μ l, outer primer 2 is 1 pmol/ μ l;
Said reaction solution contains 10mM dNTP, 10 * ThermoPol reaction buffer, 150mM MgSO
4, 5mM Betaine, four volume ratio is 7 ~ 9:5:2 ~ 4:9 ~ 11;
(3) result judges: in above-mentioned reaction tubes, add 1 ~ 2 μ l developer SYBR GREEN I, the color of observing response liquid behind the mixing then is mycobacterium tuberculosis if present green, and orange then is non-tuberculous mycobacteria.
Preferably, in the said reaction solution, 10mM dNTP:10 * ThermoPol reaction buffer: 150mM MgSO
4: 5mM Betaine (volume ratio)=8:5:3:10.
Preferably, said archaeal dna polymerase is the Bst archaeal dna polymerase, and concentration is 8U/ μ l.
Beneficial effect of the present invention is: the inventive method is convenient and swift, can differentiate mycobacterium tuberculosis and non-tuberculous mycobacteria in the short period of time, and does not need expensive plant and instrument; Highly sensitive, minimumly detect 100 bacterium/ml; Specificity is good, and the present invention combines with 6 zones of goal gene according to 4 pairs of primers of 16S rna gene sequences Design, has higher specificity.The present invention has higher reference value for mycobacterium tuberculosis or non-tuberculous mycobacteria diagnosis of infection and clinical application, is suitable for applying.
Embodiment
According to 4 Auele Specific Primers of specificity design of mycobacterium tuberculosis and non-tuberculous mycobacteria 16S RNA, sequence is distinguished as follows:
Inner primer 1:5 '-TGGTCGTAGTAGGTCGATGGGGAGTCGATCTGCACACAGCT-3 ' (SEQ ID No.1);
Inner primer 2:5 '-TGCGCGATGGCGAACTCAAGGCACCGTAAACACCGTAG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-TCATCGCCGATCATCAGG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGAGTTTGGTCATCAGCCG-3 ' (SEQ ID No.4).
Adopt above-mentioned Auele Specific Primer Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria, specifically comprise the steps:
(1) extraction of template DNA: extract the DNA of sample to be checked, said sample to be checked is to be accredited as sample or the isolating mycobacterium that contains mycobacterium;
(2) ring mediated isothermal gene amplification reaction: prepare reaction system at 200 μ l PCR pipe: primer mixed solution 1 μ l; Reaction solution 12.5 μ l; Archaeal dna polymerase 0.5 ~ 1 μ l, template DNA 2 ~ 5 μ l use sterilization deionized water polishing to 25 μ l; The sealing liquid that adds 20 μ l then reacts above-mentioned reaction tubes to 60 ~ 90min in 63 ~ 65 ℃;
Said primer mixed solution contains 4 above-mentioned Auele Specific Primers (SEQ ID No.1 ~ 4); Wherein, the concentration of inner primer 1 is that the concentration of 4 ~ 8 pmol/ μ l, inner primer 2 is that the concentration of 4 ~ 8 pmol/ μ l, outer primer 1 is that the concentration of 1 pmol/ μ l, outer primer 2 is 1 pmol/ μ l;
Said reaction solution contains 10mM dNTP, 10 * ThermoPol reaction buffer, 150mM MgSO
4, 5mM Betaine, four volume ratio is 7 ~ 9:5:2 ~ 4:9 ~ 11;
(3) result judges: in above-mentioned reaction tubes, add 1 ~ 2 μ l developer SYBR GREEN I, the color of observing response liquid behind the mixing then is mycobacterium tuberculosis if present green, and orange then is non-tuberculous mycobacteria.
Preferably, in the said reaction solution, 10mM dNTP:10 * ThermoPol reaction buffer: 150mM MgSO
4: 5mM Betaine (volume ratio)=8:5:3:10.
Preferably, said archaeal dna polymerase is the Bst archaeal dna polymerase, and concentration is 8U/ μ l.
Below in conjunction with embodiment the present invention is further described, but is not limited thereto.
Embodiment 1
The preparation of ring mediated isothermal gene amplification reaction system:
(1) reaction solution: 10mM dNTP, 10 * ThermoPol reaction buffer, 150mM MgSO
4, 5mM Betaine, four volume ratio is 8:5:3:10;
(2) primer liquid: comprise that 4pmol/ μ l inner primer 1,2, four primers of 4pmol/ μ l inner primer 2,1 pmol/ μ l outer primer, 1,1 pmol/ μ l outer primer are respectively:
Inner primer 1:5 '-TGGTCGTAGTAGGTCGATGGGGAGTCGATCTGCACACAGCT-3 ' (SEQ ID No.1);
Inner primer 2:5 '-TGCGCGATGGCGAACTCAAGGCACCGTAAACACCGTAG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-TCATCGCCGATCATCAGG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGAGTTTGGTCATCAGCCG-3 ' (SEQ ID No.4);
(3) archaeal dna polymerase: Bst archaeal dna polymerase, concentration are 8U/ μ l;
(4) developer: optical dye 1 * SYBR Green I.
Mycobacterium tuberculosis and non-tuberculous mycobacteria are identified by following method with above-mentioned reaction system.The sample to be checked of present embodiment certainly, also can be bronchial perfusate and other sample that is accredited as the mycobacterium patient, perhaps isolating mycobacterium for being accredited as mycobacterium patient's sputum.
1, template DNA extracts:
1) sputum specimen with 3ml places room temperature;
2) 4% of 2 times of sputum sample volumes of adding NaOH in the sputum sample sample;
3) every at a distance from 2min vortex vibration 15s, processing 15min draws in the centrifuge tube of 1ml adding band spiral cover then;
4) the centrifugal 15min of 12000r/min removes supernatant;
5) the NaCl 1ml of adding 0.9%, washing, the centrifugal 15min of 12000r/min removes supernatant;
6) purified water of adding 100 μ l;
7) 100 ℃ are boiled 20min, then ice bath 10min;
8) the centrifuging and taking supernatant is transferred in another centrifuge tube as template DNA.
2, ring mediated isothermal gene amplification reaction: prepare reaction system at 200 μ l PCR pipe: primer mixture 1 μ l; Reaction solution 12.5 μ l, archaeal dna polymerase 1 μ l, template DNA 2 μ l; Use sterilization deionized water polishing to 25 μ l; The sealing liquid that adds 20 μ l is then put into 63 ℃ of water-baths with above-mentioned reaction tubes, takes out reaction tubes behind the reaction 60min.
3, the result judges: in above-mentioned reaction tubes, add 1 μ l, 1 * SYBR Green I, the color of observing response liquid behind the mixing then is mycobacterium tuberculosis if present green, and orange then is non-tuberculous mycobacteria.
In the present embodiment, the PCR pipe shows green, shows that the mycobacterium that sample to be checked infects is a mycobacterium tuberculosis.
The comparison of embodiment 2 conventional PCR reactions and the inventive method sensitivity
It is 1.0 * 10 that the mycobacterium tuberculosis of getting separation and purification dilutes successively
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2, 1.0 * 10
1CFU/ mL identifies with authentication method of the present invention and conventional PCR method respectively.
1, LAMP detection method of the present invention
The preparation of ring mediated isothermal gene amplification reaction system:
(1) reaction solution: 10mM dNTP, 10 * ThermoPol reaction buffer, 150mM MgSO
4, 5mM Betaine, four volume ratio is 8:5:3:10;
(2) primer liquid: comprise that 8pmol/ μ l inner primer 1,2, four primers of 8pmol/ μ l inner primer 2,1 pmol/ μ l outer primer, 1,1 pmol/ μ l outer primer are respectively:
Inner primer 1:5 '-TGGTCGTAGTAGGTCGATGGGGAGTCGATCTGCACACAGCT-3 ' (SEQ ID No.1); Inner primer 2:5 '-TGCGCGATGGCGAACTCAAGGCACCGTAAACACCGTAG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-TCATCGCCGATCATCAGG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGAGTTTGGTCATCAGCCG-3 ' (SEQ ID No.4);
(3) archaeal dna polymerase: Bst archaeal dna polymerase, concentration are 8U/ μ l;
(4) developer: optical dye 1 * SYBR Green I.
Respectively the bacterium liquid of different concns is detected by following method with above-mentioned reaction system, does 3 repetitions:
(1) extraction of template DNA:
1) the bacterium liquid of getting 3ml places room temperature;
2) add 4% long-pending NaOH of 2 times of bacteria liquids;
3) every at a distance from 2min vortex vibration 15s, processing 15min draws in the centrifuge tube of 1ml adding band spiral cover then;
4) the centrifugal 15min of 12000r/min removes supernatant;
5) the NaCl 1ml of adding 0.9%, washing, the centrifugal 15min of 12000r/min removes supernatant;
6) purified water of adding 100 μ l;
7) 100 ℃ are boiled 20min, then ice bath 10min;
8) the centrifuging and taking supernatant is transferred in another centrifuge tube as template DNA.
(2) constant temperature gene amplification reaction: prepare reaction system at 200 μ l PCR pipe: primer mixture 1 μ l; Reaction solution 12.5 μ l, archaeal dna polymerase 0.5 μ l, template DNA 2 μ l; Use sterilization deionized water polishing to 25 μ l; The sealing liquid that adds 20 μ l is then put into 63 ℃ of water-baths with above-mentioned reaction tubes, takes out behind the reaction 65min.
(3) result judges: in above-mentioned reaction tubes, add 1 μ l, 1 * SYBR Green I, and the color of observing response liquid behind the mixing, it is yellow that no amplification PCR pipe is, and has the amplification PCR pipe to be green, and experimental result is seen table 1.
In the present embodiment, 1.0 * 10
6, 1.0 * 10
5, 1.0 * 10
4, 1.0 * 10
3, 1.0 * 10
2The PCR pipe of CFU/ mL is green, and expression has amplified reaction; 1.0 * 10
1The PCR pipe of CFU/ mL shows no amplified reaction for orange.
2, conventional PCR detects
(1) sample DNA extracts: consistent with the step in the LAMP method.
(2) PCR primer: PCR reaction primer adopts the outer primer 1 and outer primer 2 in present method reaction.
(3) PCR reaction: the PCR reaction system is 25 μ l systems, 10 * PCR Buffer (PCR reaction buffer, Promega company), 2.5 μ l; 10mM dNTPs (Promega company) 0.5 μ l, the respectively corresponding outer primer 1 of upstream and downstream primer and outer primer 2, each 0.5 μ l; Taq enzyme (5U/ μ l; Promega company) 0.5 μ l, template DNA 2 μ l mend to 25 μ l with the sterilization deionized water.Response procedures is 95 ℃ of preparatory sex change 5min, 95 ℃ of sex change 30s, and 58 ℃ of annealing 30s, 72 ℃ are extended 30s, and 72 ℃ are extended 7min.The PCR product is got 10 μ l and 2% agarose gel electrophoresis, 30min under the 100V voltage, and through gel imaging analysis appearance observations, the intended purposes band is 278bp.Experimental result is seen table 1.The sensitivity of conventional PCR method is 1.0 * 10
2, 1.0 * 10
1CFU/ mL is shown as feminine gender, does not promptly have amplification.
Annotate: "+" expression detected result is positive, and amplification is promptly arranged; "-" expression detected result is negative, does not promptly have amplification.
Can find out relatively that by two kinds of methods authentication method sensitivity of the present invention can reach 1.0 * 10
2The concentration of CFU/ mL, and the sensitivity of PCR method is 1.0 * 10
3CFU/ mL, and 1.0 * 10
2CFU/ mL or the following negative findings that is shown as; Through comparison, authentication method sensitivity of the present invention can detect the more sample of low levels apparently higher than the susceptibility of PCR method.
The experiment of embodiment 3 specificitys
With the authentication method of embodiment 1 respectively to the mycobacterium tuberculosis of separation and purification; Bird-Mycobacterium intracellulare; Mycobacterium kansasii; Mycobacterium fortuitum; Mycobacterium chelonei; Mycobacterium abscessus; Mycobacterium marinum; Mycobacterium buruli; Mycobacterium xenopi; Soviet Union adds the branch bacillus; The Ma Ermo mycobacterium; The ape and monkey mycobacterium; Mycobacterium haemophilum; The soil mycobacterium; Mycobacterium scrofulaceum; Mycobacterium phlei; Mycobacterium flavescens; Mycobacterium fortutitum; Mycobacterium gordonae is identified.Conventional PCR method with embodiment 2 is tested.
Qualification result shows: the reaction tubes that bird-Mycobacterium intracellulare, mycobacterium kansasii, mycobacterium fortuitum, Mycobacterium chelonei, mycobacterium abscessus, Mycobacterium marinum, mycobacterium buruli, mycobacterium xenopi, Soviet Union add branch bacillus, Ma Ermo mycobacterium, ape and monkey mycobacterium, mycobacterium haemophilum, soil mycobacterium, Mycobacterium scrofulaceum, Mycobacterium phlei, mycobacterium flavescens, mycobacterium fortutitum, mycobacterium gordonae is orange, does not promptly have amplification; The reaction tubes of mycobacterium tuberculosis is green, and amplification is promptly arranged.This result and conventional PCR result are consistent, demonstrate good specificity.
< 110>Guangdong Province's white plaque control center
Guangzhou enlightening Australia bio tech ltd
< 120>method of a kind of Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria
<130>
<160> 4
<170> PatentIn?version?3.5
<210> 1
<211> 41
<212> DNA
< 213>artificial primer
<400> 1
tggtcgtagt?aggtcgatgg?ggagtcgatc?tgcacacagc?t 41
<210> 2
<211> 38
<212> DNA
< 213>artificial primer
<400> 2
tgcgcgatgg?cgaactcaag?gcaccgtaaa?caccgtag 38
<210> 3
<211> 18
<212> DNA
< 213>artificial primer
<400> 3
tcatcgccga?tcatcagg 18
<210> 4
<211> 19
<212> DNA
< 213>artificial primer
<400> 4
cgagtttggt?catcagccg 19
Claims (4)
1. be used for the Auele Specific Primer of Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria, it is characterized in that, comprising:
Inner primer 1:5 '-TGGTCGTAGTAGGTCGATGGGGAGTCGATCTGCACACAGCT-3 ' (SEQ ID No.1);
Inner primer 2:5 '-TGCGCGATGGCGAACTCAAGGCACCGTAAACACCGTAG-3 ' (SEQ ID No.2);
Outer primer 1:5 '-TCATCGCCGATCATCAGG-3 ' (SEQ ID No.3);
Outer primer 2:5 '-CGAGTTTGGTCATCAGCCG-3 ' (SEQ ID No.4).
2. the method for Rapid identification mycobacterium tuberculosis and non-tuberculous mycobacteria; It is characterized in that; Adopt described 4 Auele Specific Primers of claim 1 and a kind of to have the active archaeal dna polymerase of strand displacement, add template DNA, at 60～65 ℃; Isothermal duplication 60 ~ 90 minutes specifically comprises the steps:
(1) extraction of template DNA: the DNA that extracts sample to be checked is as template DNA, and said sample to be checked is to be accredited as sample or the isolating mycobacterium that contains mycobacterium;
(2) ring mediated isothermal gene amplification reaction: prepare reaction system at 200 μ l PCR pipe: primer mixed solution 1 μ l; Reaction solution 12.5 μ l; Archaeal dna polymerase 0.5 ~ 1 μ l, template DNA 2 ~ 5 μ l use sterilization deionized water polishing to 25 μ l; The sealing liquid that adds 20 μ l then reacts above-mentioned reaction tubes to 60 ~ 90min in 63 ~ 65 ℃;
Said primer mixed solution contains described 4 Auele Specific Primers of claim 1; Wherein, the concentration of inner primer 1 is that the concentration of 4 ~ 8 pmol/ μ l, inner primer 2 is that the concentration of 4 ~ 8 pmol/ μ l, outer primer 1 is that the concentration of 1 pmol/ μ l, outer primer 2 is 1 pmol/ μ l;
Said reaction solution contains 10mM dNTP, 10 * ThermoPol reaction buffer, 150mM MgSO
4, 5mM Betaine, four volume ratio is 7 ~ 9:5:2 ~ 4:9 ~ 11;
(3) result judges: in above-mentioned reaction tubes, add 1 ~ 2 μ l developer SYBR GREEN I, the color of observing response liquid behind the mixing then is mycobacterium tuberculosis if present green, and orange then is non-tuberculous mycobacteria.
3. the method for a kind of Rapid identification mycobacterium tuberculosis according to claim 2 and non-tuberculous mycobacteria is characterized in that, in the said reaction solution, and 10mM dNTP:10 * ThermoPol reaction buffer: 150mM MgSO
4: 5mM Betaine (volume ratio)=8:5:3:10.
4. the method for a kind of Rapid identification mycobacterium tuberculosis according to claim 2 and non-tuberculous mycobacteria is characterized in that said archaeal dna polymerase is the Bst archaeal dna polymerase, and concentration is 8U/ μ l.
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| CN112501324A (en) * | 2020-11-26 | 2021-03-16 | 广州迪澳生物科技有限公司 | Primer and kit for detecting mycobacterium tuberculosis complex and nontuberculous mycobacterium complex based on loop-mediated isothermal amplification |
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