CN105219882A - Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method - Google Patents
Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method Download PDFInfo
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Abstract
The invention discloses a kind of primer for detecting mycobacterium avium-intracellulare compound group and probe and its detection method, this primer comprises upstream primer for detecting mycobacterium avium and downstream primer, does is its nucleotide sequence respectively the SEQ in sequence table ID NO.1 and SEQ ID NO.2; And for the upstream primer that detects Mycobacterium intracellulare and downstream primer, its nucleotide sequence is respectively the SEQ in sequence table ID NO.3 and SEQ ID NO.4.Does does this Taqman probe comprise and have SEQ in sequence table ID NO.5 and SEQ ID two Taqman probes of NO.6; 5 ' end of two described probes is marked with reporter fluorescence group, and 3 ' end is marked with quenching fluorescence group.The method comprises extracts sample genomic dna to be detected, and configuration quantitative fluorescent PCR reaction system, carries out the steps such as PCR reaction.Primer provided by the invention, probe, method to can be used in clinical and scientific research mycobacterium avium-intracellulare compound group and detect, and detect simultaneously, differentiate mycobacterium avium and Mycobacterium intracellulare, highly sensitive, sampling less, fast and convenient.
Description
Technical field
The present invention relates to biology field, refer to a kind of mycobacterium avium-intracellulare compound group detection primer and probe and its detection method especially.
Background technology
Non-tuberculous mycobacteria is extensively present in natural soil, dust, water, and in fish and poultry, route of transmission mainly obtains infection from environment, such as sewage, and human-to-human transmission is seldom shown in.Comparatively mycobacterium tuberculosis is low to human pathogenic for this type of mycobacterium usual, if but there is predisposing factor, make host local or general immunity function generation obstacle, then can cause pathology.Non-tuberculous mycobacteria wherein with mycobacterium avium-intracellulare compound group (M.aviumcomplex) mycobacterium kansasii (M.Kartsasii) and mycobacterium xenopi (M.xenopi) for causing the most common pathogen of pulmonary lesion.
Mycobacterium avium disease infects by bird-Mycobacterium intracellulare complex body (Mycobacteriumaviumcomplex or Mycobacteriumavium-intracellularecomplex, MAIC) the Zoonosis sexually transmitted disease caused.MAIC can encroach on Various Tissues organ after infecting and comprise lung, marrow and lymphoglandula etc.Clinical disease mainly comprises single tubercle, tubercular bronchiectasis, tuberculiform infiltration and the diffustivity in immunocompromised patients and infiltrates Four types.
At present, the method that in laboratory, identification of mycobacterium adopts is smear method and culture method.Mycobacterium tuberculosis and non-tuberculous mycobacteria can not separate by smear method; Culture method is accurately but need to cultivate and could confirm for 2-3 time, and consuming time long.Also there are some molecular biological variety identification methods for mycobacterium avium-intracellulare compound group in the last few years, but also all need 4-6 hour, as reverse hybridized, PCR-RFLP etc., and easily there is laboratory pollution.
Summary of the invention
In view of this, the object of the invention is to propose a kind of mycobacterium avium-intracellulare compound group detection primer and probe and its detection method.
Based on the above-mentioned purpose primer for detecting mycobacterium avium-intracellulare compound group provided by the invention, comprise the upstream primer for detecting mycobacterium avium and downstream primer, its nucleotide sequence is respectively SEQIDNO.1 and SEQIDNO.2 in sequence table; And for the upstream primer that detects Mycobacterium intracellulare and downstream primer, its nucleotide sequence is respectively SEQIDNO.3 and SEQIDNO.4 in sequence table.
The primer sequence derived by above-mentioned primer also belongs to protection scope of the present invention.Described derived sequence refers at SEQIDNO.1 and/or SEQIDNO.2, and through primer sequence that the replacement of one to ten base, disappearance or interpolation obtain on the basis of SEQIDNO.3 and/or SEQIDNO.4.
Based on identical inventive concept, present invention also offers the Taqman probe for detecting mycobacterium avium-intracellulare compound group, comprising two Taqman probes with SEQ ID NO.5 and SEQIDNO.6; 5 ' end of two described probes is marked with reporter fluorescence group, and 3 ' end is marked with quenching fluorescence group.
Preferably, the 5 ' end described in the probe of SEQ ID NO.5 marks JOE, 3 ' end mark BHQ; Described 5 ' the end flag F AM with the probe of SEQ ID NO.6,3 ' end mark BHQ.
Also protection scope of the present invention is belonged to by the derived sequence of above-mentioned Taqman probe sequence.Described derived sequence is on the basis of SEQIDNO.5 and/or SEQIDNO.6, in the sequence that 5 ' end and/or the one or more base of 3 ' end interpolation of sequence obtain.
Based on identical inventive concept, present invention also offers mycobacterium avium-intracellulare compound group detection kit, comprising the primer for detecting mycobacterium avium-intracellulare compound group and Taqman probe.
Based on identical inventive concept, present invention also offers a kind of method detecting mycobacterium avium-intracellulare compound group, adopt above-mentioned primer for detecting mycobacterium avium-intracellulare compound group and the above-mentioned Taqman probe for detecting mycobacterium avium-intracellulare compound group, with sample genomic dna to be detected for template, carry out quantitative fluorescent PCR, its step is as follows:
1) sample genomic dna to be detected is extracted;
2) quantitative fluorescent PCR reaction system is configured: template 5 μ l, 10 × Taq enzyme buffer5 μ l, 2.5mMdNTP4 μ l, each 2 μ l of upstream primer and downstream primer for detecting mycobacterium avium, its concentration is 10 μMs, the each 2 μ l of upstream primer and downstream primer for detecting Mycobacterium intracellulare, concentration is 10 μMs, each 1 μ l of two Taqman probes of SEQ ID NO.5 and SEQIDNO.6, concentration is 10 μMs, Taq enzyme 2.5U, UNG enzyme 0.01 μ l, ddH
2o mends to 50 μ l;
3) PCR reaction is carried out: reaction conditions is: 50 DEG C of 2min; 94 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 40s, totally 40 circulations; Quantitative real time PCR Instrument increases, collects signal.
As can be seen from above, mycobacterium avium-intracellulare compound group provided by the invention detects by primer and probe and its detection method.Utilize mycobacterium avium and the genome difference between Mycobacterium intracellulare and other mycobacteriums, design 2 pairs of primers and 2 probes, target fragment according to amplification designs Taqman probe respectively, and different fluorescent marks is carried out to two probes, achieve detect mycobacterium avium and Mycobacterium intracellulare simultaneously in same reaction tubes, and differentiated by different fluorescence channels, can direct sentence read result on software while amplification, and without leakage of electricity swimming, avoid Aerosol Pollution.The feature such as there is sensitivity and specificity is high, can realize multiple reaction, level of automation is high, pollution-free, real-time and accurate.Have compared with traditional discrimination method highly sensitive, sample less, the advantage such as fast and convenient.
Accompanying drawing explanation
Fig. 1 uses mycobacterium avium-intracellulare compound group detection primer and probe in embodiments of the invention, with mycobacterium avium genomic dna for template, carry out fluorescent quantitative PCR graphic representation;
Fig. 2 uses mycobacterium avium-intracellulare compound group detection primer and probe in the embodiment of the present invention, with Mycobacterium intracellulare genomic dna for template, carry out fluorescent quantitative PCR graphic representation;
Fig. 3 uses mycobacterium avium-intracellulare compound group detection primer and probe in the embodiment of the present invention, with mycobacterium avium and Mycobacterium intracellulare genomic dna for template, carry out fluorescent quantitative PCR graphic representation.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly understand, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in more detail.
Embodiment 1: mycobacterium avium-intracellulare compound group detects the Design and synthesis with primer and probe
Various Mycobacterium tuberculosis genes group sequences according to providing in gene pool in Pub-Med are compared, find that various mycobacterium is variant in 16SrRNA sequence, mycobacterium is at the not conservative region of 16SrRNA, design primer and probe is carried out according to these difference, thus mycobacterium compound group in bird born of the same parents is therefrom distinguished, concrete sequence is as follows:
For the primer of mycobacterium avium, probe be:
Upstream primer AF:5 '-GGAAAGGCCTCTTCGGAGGT-3 ' (SEQ ID NO.1);
Downstream primer AR:5 '-CGCAAAAGCTTTCCACCAGA-3 ' (SEQ ID NO.2),
Probe AP sequence is: 5 '-CGGATAGGACCTCAAGACGCATGTC-3 ' (SEQ ID NO.5).
For the primer of Mycobacterium intracellulare, probe be:
Upstream primer: IF:5 '-CCCCTTCGGGGGTACTCG-3 ' (SEQ ID NO.3),
Downstream primer IR:5 '-CCGCAAAAGCTTTCCACCTAA-3 ' (SEQ ID NO.4),
Probe I P sequence is: 5 '-ACCGGATAGGACCTTTAGGCGCATG-3 ' (SEQ ID NO.6).
Wherein, two Taqman probes are respectively for mycobacterium avium and Mycobacterium intracellulare, and 5 ' the end mark JOE of the probe AP of mycobacterium avium, 3 ' end marks BHQ, 5 ' the end flag F AM of the probe I P of Mycobacterium intracellulare, 3 ' end mark BHQ.When only there is Mycobacterium intracellulare DNA in testing sample, the Mycobacterium intracellulare system in reaction system can increase, and has fluorescent signal to occur at FAM passage, and has real-time amplification curve; When only there is mycobacterium avium DNA in sample, in reaction system, mycobacterium avium system can increase, and has fluorescent signal to occur at JOE passage, and has real-time amplification curve; When to there is the DNA of two kinds of bacterium simultaneously, two individual system can work simultaneously (note: concentration ratio is at 1:10
3within), all can there is real-time amplification curve at two passages, thus realize the discriminating to both.Come to be mycobacterium avium and Mycobacterium intracellulare in judge templet by the fluorescence curve observed at the fluorescent signal of different passage.
The synthesis of all primers and probe and mark have all been come by the raw work in Shanghai.
Embodiment 2: apply primer provided by the invention and probe detects mycobacterium avium-intracellulare compound group
1. extract sample genomic dna to be detected:
1) get clinical sputum sample and originally carry out screening and number record smear results;
2) 8%NaOH solution is prepared;
3) in Biohazard Safety Equipment, in sputum, the above-mentioned sodium hydroxide solution of equal-volume is added;
4) the rear 37 DEG C of liquefaction of concuss mixing 1 hour;
5) sample of 1ml post liquefaction is got in 1.5ml sterile centrifugation tube;
6) 12000rpm, abandons supernatant after centrifugal 10min;
7) concussion mixing after adding stroke-physiological saline solution 1ml in this centrifuge tube;
8) 12000rpm, abandons supernatant after centrifugal 10min;
9) mix after adding sterilized water 105 μ l;
10) entire contents in pipe is transferred in new aseptic 1.5ml centrifuge tube;
11) cooling is taken out after 95 DEG C of heating in water bath 10min;
12) 12000rpm, centrifugal 2min;
13) proceed to freezing storing box ,-20 DEG C save backup for a long time.
2. configure quantitative fluorescent PCR reaction system:
Experiment material: mycobacterium avium, Mycobacterium intracellulare, Mycobacterium tuberculosis H37Rv, BCG strain, other mycobacterium, respiratory tract common bacteria, Taq enzyme suit (Takara), primer AF and AR, IF and IR, probe AP and IP, ddH
2o.
Laboratory apparatus: ABI7500 quantitative real time PCR Instrument.
| Sequence number | Component | Initial concentration | Quantity |
| 1 | Ultrapure water | / | Mend to 50 μ l |
| 2 | 10×buffer | / | 5μl |
| 3 | Primer AF | 10μM | 2μl |
| 4 | primer AR | 10μM | 2μl |
| 5 | primer IF | 10μM | 2μl |
| 6 | primer IR | 10μM | 2μl |
| 7 | Probe AP | 10μM | 1μl |
| 8 | Probe IP | 10μM | 1μl |
| 9 | Taq enzyme | 5U/L | 0.5μl |
| 10 | dNTP | 25mM | 4μl |
| 11 | UNG enzyme | 1U/μl | 0.01μl |
| 12 | Template | 5μl | |
| Total | 50μl |
3. carry out PCR reaction:
Be configured according to above-mentioned system, ABI7500 quantitative real time PCR Instrument increases, the reaction conditions of PCR is as follows: 50 DEG C, 2min, 94 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 40s, totally 40 circulations; Quantitative real time PCR Instrument increases, signal collection is carried out to FAM and JOE passage.
Respectively with the nucleic acid extracted after the nucleic acid extracted after the deactivation of mycobacterium avium pure growth, the deactivation of Mycobacterium intracellulare pure growth and both mixtures for template, detect.Fig. 1-3 is respectively in the embodiment of the present invention and uses mycobacterium avium-intracellulare compound group detection primer and probe, with above-mentioned genomic dna for template, carries out fluorescent quantitative PCR graphic representation.
As Figure 1-3, time in a template only containing mycobacterium avium genomic dna, only there is amplification curve at the passage of corresponding mycobacterium avium, and do not have amplification curve to occur for the passage of Mycobacterium intracellulare; Time in a template only containing Mycobacterium intracellulare genomic dna, only there is amplification curve at the passage of corresponding Mycobacterium intracellulare, and do not have amplification curve to occur for the passage of mycobacterium avium; Time in a template simultaneously containing mycobacterium avium and Mycobacterium intracellulare genomic dna, fluorescent signal can be detected at two passages.
As can be seen from above, two kinds of systems can well carry out work in same reaction tubes, do not have cross reaction.
Embodiment 3: with the cross reactivity of other mycobacteriums and respiratory pathogen
By the detection method in embodiment 2, respectively with common branches bacillus and respiratory pathogen for sample, detect.
For other common mycobacterium (concrete title is as following table), prepares corresponding DNA profiling, detected by this system, represented to reach the cycle number increased required for threshold value.Result is as follows:
Other common branches bacillus detected result of table 1
| Strain name | Detected result | Whether intersect |
| Mycobacterium abscessus | ND (detected result is negative) | No |
| Mycobacterium chelonei | ND (detected result is negative) | No |
| Mycobacterium fortuitum | ND (detected result is negative) | No |
| Golden mycobacterium | ND (detected result is negative) | No |
| Mycobacterium xenopi | ND (detected result is negative) | No |
| Mycobacterium marinum | ND (detected result is negative) | No |
| Mycobacterium phlei | ND (detected result is negative) | No |
| Mycobacterium avium-intracellulare compound group | 23.21 | ---- |
As can be seen from the results, this system can identify mycobacterium avium and Mycobacterium intracellulare (i.e. mycobacterium compound group in bird born of the same parents), does not intersect to other mycobacterium.
Respiratory tract common causative detected result is as following table:
Table 2 and common respiratory pathogen detected result
As can be seen from result, this system can identify mycobacterium avium and Mycobacterium intracellulare (i.e. mycobacterium compound group in bird born of the same parents), does not intersect to other respiratory tract common bacteria.
In sum, the present invention is according to the difference of mycobacterium avium-intracellulare compound group with other Mycobacterium tuberculosis genes group, design 2 pairs of primers, article two, Taqman probe is respectively for mycobacterium avium and Mycobacterium intracellulare, mark JOE held by the probe 5 ' of mycobacterium avium, 3 ' end mark BHQ, flag F AM held by the probe 5 ' of Mycobacterium intracellulare, 3 ' end mark BHQ.When only there is Mycobacterium intracellulare DNA in testing sample, the Mycobacterium intracellulare system in reaction system can increase, and has fluorescent signal to occur at FAM passage, and has real-time amplification curve; When only there is mycobacterium avium DNA in sample, in reaction system, mycobacterium avium system can increase, and has fluorescent signal to occur at JOE passage, and has real-time amplification curve; When to there is the DNA of two kinds of bacterium simultaneously, two individual system can work simultaneously (note: concentration ratio is at 1:10
3within), all can there is real-time amplification curve at two passages, thus realize the discriminating to both.Can direct sentence read result on software while amplification, and swim without leakage of electricity, avoid Aerosol Pollution.The feature such as there is sensitivity and specificity is high, can realize multiple reaction, level of automation is high, pollution-free, real-time and accurate.Have compared with traditional discrimination method highly sensitive, sample less, the advantage such as fast and convenient.
Those of ordinary skill in the field are to be understood that: the discussion of above any embodiment is only exemplary, and not intended to be implies that the scope of the present disclosure (comprising claim) is limited to these examples; Under thinking of the present invention, also can combine between technical characteristic in above embodiment or different embodiment, step can realize with random order, and there are other changes many of different aspect of the present invention as above, and they do not provide in details for the sake of simplicity.Therefore, within the spirit and principles in the present invention all, any omission made, amendment, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (9)
1. for detecting the primer of mycobacterium avium-intracellulare compound group, it is characterized in that, comprise the upstream primer for detecting mycobacterium avium and downstream primer, its nucleotide sequence is respectively SEQIDNO.1 and SEQIDNO.2 in sequence table; And for the upstream primer that detects Mycobacterium intracellulare and downstream primer, its nucleotide sequence is respectively SEQIDNO.3 and SEQIDNO.4 in sequence table.
2. the derived sequence of primer described in claim 1.
3. derived sequence according to claim 2, it is characterized in that: described derived sequence is at SEQIDNO.1 and/or SEQIDNO.2, and through primer sequence that the replacement of one to ten base, disappearance or interpolation obtain on the basis of SEQIDNO.3 and/or SEQIDNO.4.
4. for detecting the Taqman probe of mycobacterium avium-intracellulare compound group, it is characterized in that, comprising two Taqman probes with SEQ ID NO.5 and SEQIDNO.6; 5 ' end of two described probes is marked with reporter fluorescence group, and 3 ' end is marked with quenching fluorescence group.
5. the Taqman probe for detecting mycobacterium avium-intracellulare compound group according to claim 4, is characterized in that, described in have the probe of SEQ ID NO.5 5 ' end mark JOE, 3 ' end mark BHQ; Described 5 ' the end flag F AM with the probe of SEQ ID NO.6,3 ' end mark BHQ.
6. the derived sequence of Taqman probe according to claim 5.
7. according to claim 6, it is characterized in that, described derived sequence is on the basis of SEQIDNO.5 and/or SEQIDNO.6, in the sequence that 5 ' end and/or the one or more base of 3 ' end interpolation of sequence obtain.
8. a mycobacterium avium-intracellulare compound group detection kit, it is characterized in that, comprising primer for detecting mycobacterium avium-intracellulare compound group according to claim 1 and the Taqman probe for detecting mycobacterium avium-intracellulare compound group according to claim 4.
9. one kind is detected the method for mycobacterium avium-intracellulare compound group, it is characterized in that, adopt primer for detecting mycobacterium avium-intracellulare compound group according to claim 1 and the Taqman probe for detecting mycobacterium avium-intracellulare compound group according to claim 4, with sample genomic dna to be detected for template, carry out quantitative fluorescent PCR, its step is as follows:
1) sample genomic dna to be detected is extracted;
2) quantitative fluorescent PCR reaction system is configured: template 5 μ l, 10 × Taq enzyme buffer5 μ l, 2.5mMdNTP4 μ l, each 2 μ l of upstream primer and downstream primer for detecting mycobacterium avium, its concentration is 10 μMs, the each 2 μ l of upstream primer and downstream primer for detecting Mycobacterium intracellulare, concentration is 10 μMs, each 1 μ l of two Taqman probes of SEQ ID NO.5 and SEQIDNO.6, concentration is 10 μMs, Taq enzyme 2.5U, UNG enzyme 0.01 μ l, ddH2O mend to 50 μ l;
3) PCR reaction is carried out: reaction conditions is: 50 DEG C of 2min; 94 DEG C of 5min; 94 DEG C of 30s, 60 DEG C of 40s, totally 40 circulations; Quantitative real time PCR Instrument increases, collects signal.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107805666A (en) * | 2016-09-07 | 2018-03-16 | 江苏奇天基因生物科技有限公司 | A kind of method that mycobacterium avium-intracellulare complex is detected using isothermal amplification |
| CN108531622A (en) * | 2017-03-06 | 2018-09-14 | 广州市宝创生物技术有限公司 | The hybond membrane item and detection kit of mycobacterium tuberculosis complex and the compound group of the extracellular mycobacteria of bird- |
| CN110938702A (en) * | 2019-11-28 | 2020-03-31 | 北京市结核病胸部肿瘤研究所 | Method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof |
| CN112538539A (en) * | 2020-11-26 | 2021-03-23 | 中国医学科学院北京协和医院 | Kit and system for detecting mycobacterium avium |
| CN112538541A (en) * | 2020-11-26 | 2021-03-23 | 中国医学科学院北京协和医院 | Kit and system for detecting M.intracellulare |
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Cited By (9)
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| CN107805666A (en) * | 2016-09-07 | 2018-03-16 | 江苏奇天基因生物科技有限公司 | A kind of method that mycobacterium avium-intracellulare complex is detected using isothermal amplification |
| CN108531622A (en) * | 2017-03-06 | 2018-09-14 | 广州市宝创生物技术有限公司 | The hybond membrane item and detection kit of mycobacterium tuberculosis complex and the compound group of the extracellular mycobacteria of bird- |
| CN108531622B (en) * | 2017-03-06 | 2021-07-20 | 广州市宝创生物技术有限公司 | Hybrid membrane strip and detection kit for mycobacterium tuberculosis complex and avian-intracellular mycobacterium tuberculosis complex |
| CN110938702A (en) * | 2019-11-28 | 2020-03-31 | 北京市结核病胸部肿瘤研究所 | Method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof |
| CN110938702B (en) * | 2019-11-28 | 2022-11-01 | 北京市结核病胸部肿瘤研究所 | Method for detecting mycobacterium avium by real-time fluorescent PCR and application thereof |
| CN112538539A (en) * | 2020-11-26 | 2021-03-23 | 中国医学科学院北京协和医院 | Kit and system for detecting mycobacterium avium |
| CN112538541A (en) * | 2020-11-26 | 2021-03-23 | 中国医学科学院北京协和医院 | Kit and system for detecting M.intracellulare |
| CN112538539B (en) * | 2020-11-26 | 2022-09-23 | 中国医学科学院北京协和医院 | Kit and system for detecting mycobacterium avium |
| CN112538541B (en) * | 2020-11-26 | 2022-10-14 | 中国医学科学院北京协和医院 | Kit and system for detecting M.intracellulare |
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Application publication date: 20160106 |