CN104928379A - Kit and method for detecting mycobacterium tuberculosis complex in paraffin section tissue - Google Patents
Kit and method for detecting mycobacterium tuberculosis complex in paraffin section tissue Download PDFInfo
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Abstract
The invention discloses a kit for detecting a mycobacterium tuberculosis complex in paraffin section tissues, which comprises a primer pair shown by SEQ ID NO. 1-2 and/or SEQ ID NO. 3-4 and a probe shown by SEQ ID NO.5 and/or SEQ ID NO.6, and also discloses the primer pair shown by SEQ ID NO. 1-2 and SEQ ID NO. 3-4 and the probe shown by SEQ ID NO.5 and SEQ ID NO. 6. The kit can accurately detect whether the mycobacterium tuberculosis complex exists in the paraffin section tissue, has high sensitivity, has the minimum detection limit of MTC of 10fg, is 2.5 percent of the prior method, and has good specificity, rapid detection and good clinical application prospect.
Description
Technical field
The present invention relates to a kind of test kit and the method that detect M tuberculosis complex in paraffin section tissue.
Background technology
Tuberculosis is mainly by the chronic Amphixenosis of a class based on respiratory system infection caused in conjunction with mycobacterium complex body (M.tuberculosis complex, MTC).MTC is primarily of the highly similar but host of nucleic acid composition, phenotype and the obvious different population composition of difference such as pathogenic: mycobacterium tuberculosis M.tuberculosis, mycobacterium africanum M.africanum, kennedy mycobacterium M.canettii, Mycobacterium bovis M.bovis, mycobacterium microti M.microti, sea dog mycobacterium M.pinnipedii and sheep mycobacterium M.caprae.In China, annual tuberculosis new cases are approximately 1,000,000, have accounted for greatly 1/10th (http://www.chinacdc.cn/) of global new cases.Therefore, special, fast and efficiently detect MTC not only can as soon as possible to patient diagnose the angle simultaneously controlled from prevailing disease to be conducive to controlling and stop tuberculosis (national legal Category B notifiable disease) crowd accurately popular.
MTC almost can infect any tissue and the organ of the mankind, and wherein modal is lung.Respiratory tract cough, expectoration, spitting of blood, pectoralgia, in various degree uncomfortable in chest or expiratory dyspnea and be admitted to hospital and seek medical advice is there is in patient after normally having closer tuberculosis contact history.By the MTC checkout and diagnosis pulmonary tuberculosis in iconography and sputum or ascites pleural fluid.Its hetero-organization beyond MTC infection lung or organ comprise: lymphoglandula, digestive tube, joint and synovial membrane, (propping up) tracheae, kidney, pleura and the wall of the chest, nose (pharynx) portion, abdominal cavity, bone etc., itself and host tissue react and occur granuloma tubercle, surgical site infections censorship pathological diagnosis.
Routine pathology diagnosis in, tuberculosis be made a definite diagnosis, except observe with granuloma be feature Histological change except, also need to find direct pathogenic bacteria-MTC.Pathology Deparment of current China carries out find the method for pathogenic bacteria the most widely for neat-antiacid special dye of Nissl, but together-Nissl is antiacid has its outstanding several large problem such as: positive rate is generally on the low side, and International reporting is generally lower than 10%; In addition, acid-fast stain can not distinguish MTC and other non-tuberculous mycobacteria (NTM) from form, causes mistaken diagnosis.
One comparatively easy, method accurately when PCR detects, but the tuberculosis clinical detection reagent box of current all PCR-based technology is all for fresh sample (body fluid, blood etc.), and MTC only remains in local organization after infecting, as in granuloma tubercle, when directly getting fresh body fluid, blood or organize, many times can not cause false negative result containing MTC because of in the tissue chosen.
After tissue being made paraffin section tissue, effectively can determine that there is granuloma tubercle at which position, therefore whether contain MTC by detecting organizing of granuloma tubercle, can determine whether accurately to infect MTC, reducing false negative result.But paraffin organization has the feature of himself, in such as its tissue, DNA is not all complete DNA is fragmentation DNA, its size is generally less than 500bp, in addition in paraffin organization, MTC is all mixed in host cell, is difficult to enrichment, and the DNA overwhelming majority extracted is host DNA, often content is very low for the DNA of MTC to be checked, is therefore difficult to effective detection by the PCR method of the fresh sample of conventional sense.
The patent No. be US7,332,597B application discloses a kind of method detecting MTC in paraffin section tissue, but the sensitivity of the method is low, the lowest detection of MTC is limited to 400fg.
Summary of the invention
In order to solve the problem, the invention provides test kit and the method for M tuberculosis complex in a kind of detection paraffin section tissue newly.
The invention provides the primer pair shown in SEQ ID NO.1 ~ 2 Yu SEQ ID NO.3 ~ 4.
The invention provides the probe shown in SEQ ID NO.5 and SEQ ID NO.6.
Present invention also offers the purposes in the reagent of SEQ ID NO.1 ~ 2 and/or the primer pair shown in SEQ ID NO.3 ~ 4 and SEQ ID NO.5 and/or the M tuberculosis complex in preparation detection paraffin section tissue of the probe shown in SEQ ID NO.6.
Present invention also offers a kind of test kit detecting M tuberculosis complex in paraffin section tissue, it comprises SEQ ID NO.1 ~ 2 and/or the primer pair shown in SEQ ID NO.3 ~ 4 and SEQ IDNO.5 and/or the probe shown in SEQ ID NO.6.
Present invention also offers a kind of method detecting M tuberculosis complex in paraffin section tissue, it comprises the steps:
(1) extract sample DNA: get paraffin section tissue to be checked, extract DNA wherein;
(2) gene amplification: increase with the DNA that test kit according to claim 3 is treated in sample basis;
(3) result detects: detect DNA cloning result.
Test kit provided by the invention can accurately detect in paraffin section tissue whether have M tuberculosis complex, and highly sensitive, and the lowest detection of MTC is limited to 10fg, and specificity is good, and detect fast, potential applicability in clinical practice is good.
Obviously, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
The embodiment of form by the following examples, is described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.All technology realized based on foregoing of the present invention all belong to scope of the present invention.
Accompanying drawing explanation
Fig. 1 is that Chinese food medicine identification research institute provides people's tuberculosis reference culture H37Rv after formalin anchor stone Lasaxing Oilfield, the present invention and commercialization fresh sample tuberculosis detection kit (import) contrast experiment under different concns.P represents weak positive reference, N is negative reference, 1 bacterial strain H37RvDNA concentration to be checked is under 10fg condition, by the result that the inventive method and test kit detect, 2 represent that bacterial strain H37Rv DNA concentration to be checked is under 10fg condition, the result detected by commercialization fresh sample tuberculosis detection kit (import);
Fig. 2 is that Chinese food medicine identification research institute provides people's tuberculosis reference culture H37Rv after formalin anchor stone Lasaxing Oilfield, the present invention and commercialization fresh sample tuberculosis detection kit (import) contrast experiment under different concns.P represents weak positive reference, N is negative reference, 1 bacterial strain H37RvDNA concentration to be checked is under 100fg condition, by the result that the inventive method and test kit detect, 2 represent that bacterial strain H37Rv DNA concentration to be checked is under 100fg condition, the result detected by commercialization fresh sample tuberculosis detection kit (import);
Fig. 3 is that Chinese food medicine identification research institute provides people's tuberculosis reference culture H37Rv after formalin anchor stone Lasaxing Oilfield, the present invention and commercialization fresh sample tuberculosis detection kit (import) contrast experiment under different concns.P represents weak positive reference, N is negative reference, 1 bacterial strain H37RvDNA concentration to be checked is under 1000fg condition, by the result that the inventive method and test kit detect, 2 represent that bacterial strain H37Rv DNA concentration to be checked is under 1000fg condition, the result detected by commercialization fresh sample tuberculosis detection kit (import).
Embodiment
Experiment reagent:
PCR reaction solution (article No. R007A), dUTP, MgCl
2, UNG enzyme, Taq enzyme, all purchased from Dalian Takara company; Paraffin organization extracts test kit, purchased from Qiagen company.
Embodiment 1 detection kit of the present invention and detection method
One, the composition of test kit of the present invention
Pcr amplification reagent (50 person-portion):
Primer 1: upstream primer (F) is: 5'-TGG CCA TGC TCT TGA TGC-3'(SEQID NO.1); Downstream primer (R) is: 5'-CAA ACA AAA CCC AAA CAC TCC C-3'(SEQ ID NO.2);
Taqman fluorescence probe 1 primer sequence of FAM mark is 5'-CAGGATATTTCTAAATACCTTTGGCTCCCT-3'(SEQ ID NO.5);
Primer 2: upstream primer (F) is: 5'-GACCTACTACGACCACATCAA-3'(SEQID NO.3); Downstream primer (R) is: 5'-TCAGGGTTAGCCACACTTT-3'(SEQ IDNO.4);
Taqman fluorescence probe 2 primer sequence of FAM mark is: 5'-ATGGCGAACTCAAGGAGCACATCA-3 (SEQ ID NO.6).
Two, test kit of the present invention is adopted to detect
1, sample DNA extracts: can use Qiagen company, Tian Gen company, Promega company etc.
Paraffin organization extracts test kit, and this example, for Qiagen company, extracts DNA:
Scalpel is utilized to take out the useless paraffin of organizational boundary;
Paraffin-embedded tissue is cut into 4 μm of thick thin slices;
Rapid sterilizing pincet is got in 2-6 pieces of loading DNase/RNase Free EP pipes and (is often opened and spread area 500 (maximum) mm out
2, i.e. the square size of length of side 1.6cm;
Add 800 μ l dimethylbenzene, vibrator shakes 10s, centrifugal 3 minutes of maximum revolution;
Add 800 μ l dimethylbenzene, vibrator shakes 10s, centrifugal 3 minutes of maximum revolution;
Abandon dimethylbenzene, add 800 μ l dehydrated alcohols, centrifugal 3 minutes of maximum revolution;
Abandon dehydrated alcohol, volatilize sample;
Add 180 μ l ALT buffer and 20 μ lproteinase K, 56 DEG C more than one hour, until limpid;
90 DEG C one hour;
Centrifugal 6000g 1min, gets supernatant; (this step prevents precipitate occlusion pillar)
Add 200 μ lAL, mixing, adds 200 μ l ethanol, then mixes;
Simple centrifugal clean tube wall;
Whole mixed solution is joined DNA separator column, closes lid, centrifugal 6000g 1min, the collection tube renewed;
Add 500 μ lAW1, the centrifugal 1min of 6000g; Change collection tube;
Add 500 μ lAW2, the centrifugal 1min of 6000g, changes collection tube;
Centrifugal 3min at full speed, dry pillar;
Change a clean 1.5ml collection tube, prepare to collect DNA; Add 20-30 μ lATE, keep 1min with dissolving DNA in room temperature.Centrifugal 1min collects DNA at full speed.
2, multiplexed PCR amplification
PCR primer and probe:
Primer 1: upstream primer is: 5'-TGG CCA TGC TCT TGA TGC-3'(SEQ IDNO.1); Downstream primer is: 5'-CAA ACA AAA CCC AAA CAC TCC C-3'(SEQ IDNO.2);
Taqman fluorescence probe 1 primer sequence of FAM mark is 5'-CAGGATATTTCTAAATACCTTTGGCTCCCT-3'(SEQ ID NO.5);
Primer 2: upstream primer is: 5'-GACCTACTACGACCACATCAA-3'(SEQ IDNO.3); Downstream primer is: 5'-TCAGGGTTAGCCACACTTT-3'(SEQ ID NO.4);
Taqman fluorescence probe 2 primer sequence of FAM mark is: 5'-ATGGCGAACTCAAGGAGCACATCA-3 (SEQ ID NO.6).
Multiplex PCR system (PCR system and enzyme are purchased from Dalian Takara company) is prepared:
| Working concentration | ||
| DNA | 10μl | |
| Primer 1+Probe 1 | 2μl | Upstream and downstream primer concentration is 500nM, and concentration and probe concentration is 150nM |
| Primer 2+Probe 2 | 2μl | Upstream and downstream primer concentration is 500nM, and concentration and probe concentration is 150nM |
| 10Xbuffer | 5μl | |
| dUTP PLUS dNTP | 4μl | dATP 0.2mM、dCTP 0.2mM、dGTP 0.2mM、dUTP 0.6mM |
| 25mM MgCl 2 | 3.5μl | 3.25mM |
| Taq | 0.3μl | 1.5U |
| UNG | 0.05μl | 0.1U |
| H2O | 23.2μl | |
| total | 50μl |
It is that to be 500nM, IS6110 specificity taqman probe face concentration be 150nM, Tris-HCl (pH 8.3) 10mM, KCl 50mM, MgCl to 150nM, IS6110 upstream and downstream primer working concentration that DNA 10 μ l, 16S rRNA sequence upstream and downstream primer working concentration are 500nM, 16S rRNA specificity taqman probe face concentration
23.25mM, dATP 0.2mM, dCTP 0.2mM, dGTP 0.2mM, dUTP 0.6mM, Taq enzyme 1.5U, UNG 0.1U, distilled water 23.2 μ l; Total reaction system is 50 μ l.
PCR instrument cycling program:
| Step | Temperature | Time |
| 1 | 37℃ | 5 minutes |
| 2 | 94℃ | 1 minute |
| 3 | 94℃ | 5 seconds |
| 4 | 60℃ | 30 seconds |
| 5 | Return 3 steps, totally 15 times | |
| 6 | 94℃ | 5 seconds |
| 7 | 60℃ | 30 seconds |
| 8 | Acquired signal | Acquired signal |
| 9 | Return 6 steps, totally 25 times | |
| END | END |
3, result interpretation:
According to amplification curve and CT value sentence read result thereof: it is positive that CT value is less than 23 interpretations; CT value needs to repeat experiment then interpretation between 23-25; Do not have amplification curve or CT value to be greater than 25, interpretation is negative.
Below by the mode of experimental example, beneficial effect of the present invention is described:
The sensitivity of experimental example 1 detection kit of the present invention and specificity
One, experimental technique
1, sensitivity technique
Chinese food medicine identification research institute provides people's tuberculosis reference culture H37Rv (reference culture H37Rv, M.tuberculosis), through formalin anchor stone Lasaxing Oilfield (50% alcohol 1 hour; 70% alcohol 1 hour; 80% alcohol 1 hour; 90% alcohol 1 hour; 95% alcohol 1 hour; Raw spirit 1 hour; Raw spirit/xylene mixture 1 hour; Dimethylbenzene transparent 4; Dimethylbenzene/mineral wax mixture spends the night; Paraffin 1 hour) after, detect by the test kit of the embodiment of the present invention 1, method under different concns, with commercialization fresh sample tuberculosis detection kit (German Qiagen company article No.: tbCARE) in contrast.
2, specific detection
The non-tuberculous mycobacteria that negative reference product provide for Chinese food medicine identification research institute and Bacteria infecting respiratory are formed.Its concrete bacterial classification comprises: mycobacterium avium, soil mycobacterium, Amur mycobacterium, mycobacterium kansasii, Asia mycobacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, tortoise purulence mycobacterium, mycobacterium fortuitum, Mycobacterium phlei, Nocardia brasiliensis, Beijing corynebacterium, streptococcus pneumoniae, legionella pneumophilia, Bordetella pertussis amount to 15 kinds.Above reference material is also " mycobacterium tuberculosis PCR detection kit National reference ".
Two, experimental result
1, sensitivity
As shown in Figures 1 to 3:
Curve P represents positive reference, and curve N is negative with reference to (positive reference: reference culture H37Rv; Negative reference: the feminine gender reference in " mycobacterium tuberculosis PCR detection kit National reference "), illustrate and normally work.
Curve 1 detects the detected result of bacterial strain H37Rv DNA concentration to be checked at 10fg, 100fg, 1000fg by the inventive method, result display CT value is less than 23, be judged to be the positive, illustrate that the inventive method is when bacterial strain H37Rv DNA concentration to be checked is 10fg, 100fg, 1000fg, all can effectively detect;
Curve 2 is the detected results with the fresh sample reagent box of commercialization, in detection bacterial strain H37RvDNA concentration to be checked at 10fg, the CT value of its amplification curve, much larger than 25, is judged to be feminine gender, and the fresh sample reagent box of explanation commercialization accurately cannot detect the H37Rv that DNA concentration is 10fg.
Experimental result illustrates, the inventive method and test kit effectively can detect mycobacterium tuberculosis, and highly sensitive, lowest detectable limit is low to moderate 10fg, lower than detection kit (German Qiagen company article No.: a tbCARE) order of magnitude.
2, specificity
Through qualification, use detecting mycobacterium avium, soil mycobacterium, Amur mycobacterium, mycobacterium kansasii, Asia mycobacterium, Mycobacterium scrofulaceum, mycobacterium gordonae, tortoise purulence mycobacterium, mycobacterium fortuitum, Mycobacterium phlei, Nocardia brasiliensis, Beijing corynebacterium, streptococcus pneumoniae, legionella pneumophilia, Bordetella pertussis of this test kit, detected result is all negative.
Experimental result illustrates, the inventive method and test kit is special has detected M tuberculosis complex, and can not detect other bacteriums, and specificity is good.
To sum up, test kit of the present invention can accurately detect in paraffin section tissue whether have M tuberculosis complex, and highly sensitive, and the lowest detection of MTC is limited to 10fg, and be now methodical 2.5%, specificity is good, and detect fast, potential applicability in clinical practice is good.
Claims (5)
- Primer pair shown in 1.SEQ ID NO.1 ~ 2 Yu SEQ ID NO.3 ~ 4.
- Probe shown in 2.SEQ ID NO.5 and SEQ ID NO.6.
- Purposes in the reagent of 3.SEQ ID NO.1 ~ 2 and/or the primer pair shown in SEQ ID NO.3 ~ 4 and SEQ ID NO.5 and/or the M tuberculosis complex in preparation detection paraffin section tissue of the probe shown in SEQ ID NO.6.
- 4. detect a test kit for M tuberculosis complex in paraffin section tissue, it is characterized in that: it comprises SEQ ID NO.1 ~ 2 and/or the primer pair shown in SEQ ID NO.3 ~ 4 and SEQ ID NO.5 and/or the probe shown in SEQ ID NO.6.
- 5. detect a method for M tuberculosis complex in paraffin section tissue, it is characterized in that: it comprises the steps:(1) extract sample DNA: get paraffin section tissue to be checked, extract DNA wherein;(2) gene amplification: increase with the DNA that test kit according to claim 4 is treated in sample basis;(3) result detects: detect DNA cloning result.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107284A (en) * | 2021-12-03 | 2022-03-01 | 辽宁康惠生物科技有限公司 | Paraffin section metagenome extraction kit and extraction method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050287534A1 (en) * | 2004-06-28 | 2005-12-29 | Lele Subodh M | Primers and probe to identify mycobacterium tuberculosis complex |
| CN102533959A (en) * | 2010-12-30 | 2012-07-04 | 华中农业大学 | Multiplex polymerase chain reaction (PCR) kit for identifying mycobacterium tuberculosis |
| CN103038348A (en) * | 2010-05-27 | 2013-04-10 | 蔚山大学校产学协力团 | Method for detecting mycobacterium tubericulosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction |
-
2015
- 2015-06-12 CN CN201510323866.0A patent/CN104928379B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050287534A1 (en) * | 2004-06-28 | 2005-12-29 | Lele Subodh M | Primers and probe to identify mycobacterium tuberculosis complex |
| CN103038348A (en) * | 2010-05-27 | 2013-04-10 | 蔚山大学校产学协力团 | Method for detecting mycobacterium tubericulosis and nontuberculous mycobacteria by using dual real-time polymerase chain reaction |
| CN102533959A (en) * | 2010-12-30 | 2012-07-04 | 华中农业大学 | Multiplex polymerase chain reaction (PCR) kit for identifying mycobacterium tuberculosis |
Non-Patent Citations (3)
| Title |
|---|
| FRANCESCO BROCCOLO ET AL: "Rapid Diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR Assays", 《JOURNAL OF CLINICAL MICROBIOLOGY》 * |
| LINYONG SUN ET AL: "Rapid and accurate identification fo mycobacterium tuberculosis complex by simultaneous detection of 16S rRNA and IS6110 sequence in FFPE samples", 《INT J CLIN EXP PATHOL》 * |
| 李武秀等: "石蜡包埋组织实时荧光定量聚合酶链反应检测在结核病诊断中的价值", 《中国防痨杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114107284A (en) * | 2021-12-03 | 2022-03-01 | 辽宁康惠生物科技有限公司 | Paraffin section metagenome extraction kit and extraction method |
| CN114107284B (en) * | 2021-12-03 | 2023-09-05 | 辽宁康惠生物科技有限公司 | Paraffin slice metagenome extraction kit and extraction method |
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