CN102925554B - For detecting the primer sets of mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof - Google Patents
For detecting the primer sets of mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof Download PDFInfo
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Abstract
The present invention relates to the primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof, the method utilizing this primer sets to detect the resistance to pyrazinoic acid amide property of medicine of mycobacterium tuberculosis is: extract the DNA in sample; The tuberculosis pack section containing total length pyrazinamidase gene (pncA) by fluorescent quantitative PCR detects mycobacterium tuberculosis; To mycobacterium tuberculosis positive sample, carry out the detection of detection of pyrazinamide resistance, the step of this detection is, 1. carries out a PCR reaction with quantitative fluorescent PCR product again for template and adds vivoexpression element; 2. this PCR primer is joined in Cell free expression system, express pyrazinamidase; 3. contrasted by the pyrazinoic acid amide enzymic activity of this sample and reference culture, provide detection of pyrazinamide resistance result.The present invention, by the pyrazinamidase enzymic activity of fluorescent quantitative PCR total length pncA gene in conjunction with vivoexpression, achieves the joint-detection of mycobacterium tuberculosis and detection of pyrazinamide resistance thereof in sample, without the need to microbial culture and DNA sequencer.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of based on quantitative fluorescent PCR and the cell free in vitro protein expression system method for mycobacterium tuberculosis in clinical sample and detection of pyrazinamide resistance joint-detection, can be used for detecting and confirming the mycobacterium tuberculosis in clinical sample and detection of pyrazinamide resistance thereof.
Background technology
Tuberculosis is the serious infectious diseases threatening human health, and the medicine at present for first-line treatment has pyrazinoic acid amide, vazadrine, Rifampin and ethylamin ethanol.Along with the popularization of chemotherapy, bacterium also occurs drug resistant M thereupon in a large number, greatly have impact on the treatment of tuberculosis patient.Efficient diagnosis lungy and Drug Resistance Detection have very important meaning for guiding clinical treatment tuberculosis.But these two aspects is all faced with the detection of pyrazinamide resistance detection of some important problems, particularly mycobacterium tuberculosis at present, significantly limit prevention and control ability lungy, is badly in need of solving.
In diagnosis lungy, laboratory tubercle mycobaterium mainly contains bacteriology, immunology and molecular biology three types of technology.In bacteriological detection, Sputum smears acid fast staining microscopy is that tuberculosis detects the most basic method.Advantage be simple to operate, low cost, sense cycle are short, the general same day just can go out result.But susceptibility is low, average recall rate only has 25%-35%, cannot distinguish dead bacterium, viable bacteria; Poor specificity, the equal pigmentable of various acid-fast bacilli, needs further test just can determine whether as tubercule bacillus.The separation and Culture of tubercule bacillus identifies the gold standard of mycobacterium tuberculosis at present.Because the breeding of mycobacterium tuberculosis is by the control of STPK (Ser/tHR protein kinase comprises PknA, PKnB, PKnd), is often in dormant state and stationary phase, thus poor growth, needs 2-8 week just can go out result.
Mycobacterium rapid culture system BACTEC460TB and BACTECMGIT960 afterwards, owing to using liquid nutrient medium, better solves and finishes this problem of pyrenomycetes poor growth.It has, and sense cycle is short, susceptibility is high, automatization is strong, just for separation rate advantages of higher.Positive detection time shortens to 3-14 days in 8 weeks by cellar culture, and also can be used for the Resistance detection of tubercule bacillus, greatly facilitate the development that tuberculosis bacteria learns diagnosis, this standard reference system as diagnosis of tuberculosis is united by many countries.But BACTEC460TB and BACTECMGIT960 expensive equipment, reagent dependence on import, complicated operation, hinders the popularization of the method., also there is stronger humoral immune reaction in the cell immune response of the existing T cell mediation of immune response of m tuberculosis infection.In immunology detection, utilize the enzyme of humoral response to join methods such as (ELISA), inoculate at differential diagnosis m tuberculosis infection and bacille Calmette-Guerin vaccine (BCG) and there is poor specificity etc. in nonpathogenic mycobacteria infection etc.In addition, conventional tuberculin test (tuberculinskintest, and IFN-γ release test (interferon-γ releaseassay TST), the immunological method such as IGRA), is not suitable for diagnosis of tuberculosis people in the high countries and regions of mycobacterium tuberculosis infection rate.
In recent years along with the development of Protocols in Molecular Biology, increasing novel method, as quantitative fluorescent PCR, isothermal duplication etc., for detection and the qualification of mycobacterium tuberculosis, the prevention and control for tuberculosis epidemic situation provide effective approach.But current most Molecular Detection means are all certain the Idiotype fragments only increased in M. tuberculosis genes group, focus on that realizing mycobacterium tuberculosis detects this single purpose, does not also have the report of joining together with Resistance detection etc.
The Resistance detection of mycobacterium tuberculosis, has very important effect for the clinical correctly taking drugs of guidance and effective tuberculosis that controls.Current use be used for the treatment of in clinical medicine lungy, pyrazinoic acid amide is one of line antitubercular agent.The resistance method detecting pyrazinoic acid amide at present clinically mainly can be divided into Phenotypic examination (microbial culture) and gene test two class, and based on the former.Because M. tuberculosis growth is slow, not only need to reach 1 ~ 2 month, and need to carry out in acidic culture to the Resistance detection of pyrazinoic acid amide, high to the pH value control overflow of substratum, even the result often causing same laboratory different time to measure is also inconsistent.And the method for gene test mainly measures the site mutation producing closely-related pyrazinamidase gene (pncA) with detection of pyrazinamide resistance.The method reported has the method such as gene chip, gene sequencing row.All gene testers can only judge whether resistance according to the mutational site producing dependency with resistance reported.But due to a lot of site mutations of pncA gene, capital causes the generation of detection of pyrazinamide resistance, and new mutational site is also at continuous report, therefore, the result of various gene test can only as a reference, also need traditional drug sensitive experiment to verify for new mutational site.A series of research shows, detection of pyrazinamide resistance generation and the loss of activity of the pyrazinamidase of tuberculosis pncA genetic expression have the dependency of more than 90%.Therefore also someone reports the detection of pyrazinamide resistance being measured tubercule bacillus by the pyrazinoic acid amide enzymic activity directly measured in mycobacterium tuberculosis.But because in tubercule bacillus, the content of pyrazinamidase is few, the method needs to carry out just carrying out after cultivation is amplified to tubercule bacillus equally, and time and traditional susceptibility time difference of needs are few.
Based on the problem of above mycobacterium tuberculosis pyrazinamide drug resistance, to develop before us a kind of based on the method (application number 20111004779.2 detection method of mycobacterium tuberculosis pyrazinamide drug resistance) of external Cell free expression system for Rapid Detection of Mycobacterium Tuberculosis clinical separation strain detection of pyrazinamide resistance.External protein Cell free expression system is a kind of is template with foreign DNA, utilizing the protein synthesis machine in cell extract, the protein folding factor and other related enzyme systems, realizing by adding amino acid, T7 polysaccharase and energy matter etc. the system that aleuroplast expresses fast outward.The cell pyrolysis liquid having rabbit reticulated red blood cells (rabbitreticulocyte), wheatgerm (wheatgerm), intestinal bacteria (E.coli) of frequent use.Utilize external Cell free expression system to give expression to pyrazinamidase, pyrazinamidase can change into pyrazine acid by catalysis pyrazinoic acid amide, and the latter and ferrous ammonium sulphate react and generate red-brown material.Judged the active height of pyrazinamidase by the depth of reaction solution color, detect mycobacterium tuberculosis according to this and whether resistance mutation occurs.External Cell free expression system is introduced the Drug Resistance Detection of pyrazinoic acid amide, not only there is gene test short advantage consuming time, have the advantage that Phenotypic examination can detect multidigit point medicament-resistant mutation and potential resistance site mutation simultaneously concurrently, avoid the generation of false negative result, there is better specificity and susceptibility.But due to amplification efficiency problem, the method can't well detect for the detection of pyrazinamide resistance of mycobacterium tuberculosis in clinical sample, can only be used for clinical separation strain.But from clinical sample, be separated mycobacterium tuberculosis, need the time reaching one month.
Summary of the invention
Technical problem to be solved by this invention is: provide a kind of primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof, utilizes this primer sets to detect the resistance to pyrazinoic acid amide property of medicine of mycobacterium tuberculosis, improves the efficiency of diagnosis of tuberculosis and treatment.
The invention discloses a kind of primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof, it is made up of following primer:
Quantitative PCR forward primer 1:5'CGGACGGATTTGTCGCTCAC3',
Quantitative PCR reverse primer 1:5'GCCCGATGAAGGTGTCGTAGAAG3',
Quantitative PCR forward primer 2:5'GGCGTCATGGACCCTATATCTGTGG3',
Quantitative PCR reverse primer 2:5'CGGTGAACAACCCGACCCAGCC3';
Vivoexpression forward primer 1:5'TAATACGACTCACTATAGGATACTCCCCCACAACAGCTTACAATACTCCCCC ACACAGCTTACAAATACTCCCCCAGTCGCCCGAACGTAAGGAGGACGT3',
Vivoexpression reverse primer 1:5'ACCGCCGCCAACAGTTCATCCCGGT3',
Vivoexpression forward primer 2:5'TAATACGACTCACTATAGGAGTCGCCCGAACGTAAGGAGGACGT3';
Vivoexpression reverse primer 2:5'ACCGCCGCCAACAGTTCATCCCGGT3';
Vivoexpression forward primer 3:5'TAATACGACTCACTATAGGAAGGAGATAATGCATCATCATCATCATCACAGT CGCCCGAACGTAAGGAGGACGT3';
Vivoexpression reverse primer 3:5'ACCGCCGCCAACAGTTCATCCCGGT3';
The method utilizing above-mentioned primer sets to detect the resistance to pyrazinoic acid amide property of medicine of mycobacterium tuberculosis is: whether the M. tuberculosis genes pack section containing total length pncA gene by fluorescent quantitative PCR detects in sample containing mycobacterium tuberculosis, described pncA gene is mycobacterium tuberculosis pyrazinamidase gene, just prove to contain mycobacterium tuberculosis in sample if there is positive amplification, then its detection of pyrazinamide resistance is detected; During detection, with the product of quantitative fluorescent PCR for template, adopt the primer containing vivoexpression element, again carry out pcr amplification total length pncA gene, add the element required for expressing; Then join in vitro expression systems by the PCR primer of amplification, the pyrazinoic acid amide enzymatic conversion pyrazinoic acid amide expressed by mensuration is the enzymic activity of pyrazinoic acid amide acid; Measured enzymic activity, whether total length pncA gene is contained on the one hand as aided verification quantitative fluorescent PCR product, compare with the mycobacterium tuberculosis type strain pyrazinoic acid amide enzymic activity adopting same method to express and to measure on the other hand, learn that in sample, whether mycobacterium tuberculosis there occurs medicament-resistant mutation, realizes the detection to its detection of pyrazinamide resistance accordingly.
Described detection method comprises the following steps:
(1) to extract and the DNA of mycobacterium tuberculosis in lysed sample, as the template that following PCR detects;
(2) according to pncA, GenBank sequence number is the feature of pyrazinamidase gene and upstream and downstream sequence thereof in the M. tuberculosis genes group described in U59967, design special primer, the primer adopted has and not to mate completely with any sequence in pncA gene reading frame and gained PCR primer contains the feature of total length pncA gene, and whether the M. tuberculosis genes pack section containing total length pncA gene by fluorescent quantitative PCR detects in sample containing mycobacterium tuberculosis;
(3) for sample mycobacterium tuberculosis being detected in step (2), adopt the primer containing vivoexpression element of design, the primer adopted has and not to mate completely with any sequence in pncA gene reading frame except initiator codon ATG and gained PCR primer contains the feature of total length pncA gene, with the quantitative fluorescent PCR product obtained in step (2) for template, again carry out the fragment that pcr amplification contains total length pncA gene, for pncA gene adds Expression element, make cell free in vitro expression system can carry out the expression of pyrazinamidase to the PCR primer added,
(4) PCR primer obtained in enrichment step (3) is also quantitative;
(5) add the PCR primer obtained in the step (4) of 1 ~ 20 microgram, adopt cell free in vitro expression system to express pyrazinamidase, select 1 to 24 hours expression times as required;
(6) get the vitro expression systems liquid after 1 ~ 50 microlitre expression pyrazinamidase, measure the activity of wherein contained pyrazinamidase; Enzyme on the one hand by recording with blank vivoexpression liquid under similarity condition is lived results contrast, judge that whether fluorescent quantitative PCR product is containing mycobacterium tuberculosis total length pncA fragment, pyrazinoic acid amide enzymic activity on the other hand by expressing standard pyrazinoic acid amide sensitive strain and Resistant strain pncA gene with same method compares, in judgement sample, whether the pncA gene of mycobacterium tuberculosis there occurs medicament-resistant mutation, realizes the detection to its detection of pyrazinamide resistance.
The vivoexpression element added in above-mentioned steps (3) adopts promotor, or adopts promotor and express enhanser;
The primer sequence of described promotor is: 5'TAATACGACTCACTATAGG3',
The primer sequence of described expression enhanser is: 5'ATACTCCCCCACAACAGCTTACAATACTCCCCCACACAGCTTACAAATACTCCC CC3'.
Vitro expression systems described in above-mentioned steps (3)-step (6) is wheatgerm vitro expression systems.
Judge in above-mentioned steps (6) method of fluorescent quantitative PCR product whether containing mycobacterium tuberculosis total length pncA fragment be do not add under similarity condition and method any DNA express after blank vivoexpression liquid in measured pyrazinamidase enzyme live add three times same method and condition under repeatedly measure its enzyme slip-knot really standard deviation as threshold value, measured sample enzyme work is judged as fluorescent quantitative PCR product not containing mycobacterium tuberculosis total length pncA fragment less than or equal to this threshold value, and fluorescence quantitative PCR detection result is false positive; Measured sample enzyme work is that fluorescent quantitative PCR product contains mycobacterium tuberculosis total length pncA fragment higher than the judgement of this threshold value, and fluorescence quantitative PCR detection result verification is positive.
In the middle judgement sample of above-mentioned steps (6), whether the pncA gene of mycobacterium tuberculosis there occurs the method for medicament-resistant mutation use is in kind deduct its enzyme activity determination result standard deviation of repeatedly expression of three times as threshold value with the pyrazinamidase enzyme work of pncA genetic expression in mycobacterium tuberculosis standard pyrazinoic acid amide sensitive strain under condition, measured sample enzyme work is judged as pyrazinoic acid amide resistance lower than this threshold value, higher than or equal this threshold value for pyrazinoic acid amide responsive.
The present invention is with in detection of pyrazinamide resistance testing process, the pyrazinoic acid amide enzymic activity contained in vivoexpression liquid, as aided verification quantitative fluorescent PCR product whether containing total length pncA gene, get rid of the non-specific amplification of quantitative fluorescent PCR, improve the specificity that in clinical sample, mycobacterium tuberculosis PCR detects.
Detection method provided by the invention compared with prior art, has following major advantage and effect:
1. the specificity of mycobacterium tuberculosis detection is high
There is the method for multiple PCR-based at present, comprise fluorescence quantifying PCR method, detect the mycobacterium tuberculosis in clinical sample.This fluorescence quantifying PCR method and other significant difference detecting mycobacterium tuberculosis PCR method are that the product increased is the tuberculosis pack section of the mycobacterium tuberculosis pncA gene including total length, all long than other PCR method of fragment of amplification, simultaneously to positive amplification sample, adopt external Cell free expression system to express pyrazinamidase, whether the mensuration that its enzyme is lived can assistant identification quantitative fluorescent PCR product be the pncA gene containing total length further.An authentication step due to organic combination, can get rid of the non-specific amplification result of quantitative fluorescent PCR, improves the specificity that mycobacterium tuberculosis detects.
2. the mycobacterium tuberculosis pyrazinoic acid amide Drug Resistance Detection time is short.
Traditional detection method of mycobacterium tuberculosis pyrazinamide drug resistance just can carry out drug sensitive experiment after needing to be separated to bacterium from clinical sample, and sense cycle reaches a wheat harvesting period.Present method can realize the detection of pyrazinamide resistance directly detecting mycobacterium tuberculosis from clinical sample, can by tubercule bacillus detects and detection of pyrazinamide resistance detects time shorten within one day, highly shortened mycobacterium tuberculosis pyrazinamide drug resistance detect needed for time.
3. mycobacterium tuberculosis pyrazinoic acid amide Drug Resistance Detection is highly sensitive.
Compared with current PCR-based Molecular Detection mycobacterium tuberculosis pyrazinamide drug resistance method, present method is by quantitative fluorescent PCR and vivoexpression PCR twice amplification template, mycobacterium tuberculosis sample a small amount of in clinical sample just meets the template amount needed for vivoexpression, substantially increases the sensitivity of Drug Resistance Detection.
4. can detect the detection of pyrazinamide resistance because base mutation any in mycobacterium tuberculosis pncA gene reading frame causes.
Current pncA detection method of gene mutation (comprising DNA sequencer) all can only judge whether resistance according to the mutational site relevant to resistance reported, for newfound base mutation, the result of whether resistance can not be provided, often need traditional confirming based on the phenotypic resistance detection method of cultivating.Because not mating completely with any sequence except initiator codon ATG in pyrazinamidase gene (pncA) reading frame of having of design of primers of the present invention and gained PCR primer contain the feature of total length pncA gene, the pyrazinamidase enzymic activity of vivoexpression can react the enzyme activity change that in whole pncA gene reading frame, any base mutation causes, can detect multisite mutation or potential medicament-resistant mutation, suitability is wider simultaneously.
In a word, mycobacterium tuberculosis detection in clinical sample detects with detection of pyrazinamide resistance and has organically combined by the present invention first.First detect in clinical sample whether have mycobacterium tuberculosis by quantitative fluorescent PCR, and then carry out the Resistance detection of pyrazinoic acid amide, these two kinds of current diagnosis of tuberculosis and the information needed for treatment can be supplied by Quick, drastically increase detection efficiency.
Accompanying drawing explanation
Fig. 1 is mycobacterium tuberculosis fluorescence quantitative and detection of pyrazinamide resistance joint-detection schematic diagram thereof in clinical sample.
Fig. 2 for used fluorescence quantification PCR primer is to 1, vivoexpression PCR primer pair 1 and the pncA gene relative position schematic diagram in M. tuberculosis genes group.
Fig. 3 is the typical amplification curve of mycobacterium tuberculosis reference culture quantitative fluorescent PCR.
Fig. 4 is the schematic diagram of detected through gel electrophoresis quantitative fluorescent PCR product and vivoexpression PCR primer.
Fig. 5 is the typical amplification curve detecting mycobacterium tuberculosis fluorescence quantitative PCR in Sputum samples.
Fig. 6 is for detecting mycobacterium tuberculosis reference culture H37Ra quantitative fluorescent PCR typical curve.
Fig. 7 is that in mycobacterium tuberculosis reference culture and sputum sample, mycobacterium tuberculosis adopts the enzyme activity determination colour developing result schematic diagram after wheat embryo cell-free expression system vivoexpression.
Fig. 8 is for passing through measured pyrazinoic acid amide Enzyme assay clinical sample drug sensitive experiment result.
When Fig. 9 is vivoexpression PCR, primer adds (vivoexpression forward primer 1) and does not add expression enhanser (the vivoexpression forward primer 2) comparative result to vivoexpression enzyme activity determination.
Figure 10 for used fluorescence quantification PCR primer is to 1, vivoexpression PCR primer pair 3 and the pncA gene relative position schematic diagram in M. tuberculosis genes group.
Figure 11 is the enzyme activity determination result adopting E. coli cell free expression system vivoexpression mycobacterium tuberculosis reference culture pyrazinamidase.
Figure 12 for used fluorescence quantification PCR primer is to 2, vivoexpression PCR primer pair 1 and the pncA gene relative position schematic diagram in M. tuberculosis genes group.
Figure 13 is the typical amplification curve adopting primer pair 2 Fluorescence quantitative PCR detection mycobacterium tuberculosis reference culture.
Figure 14 is the experimental result of carrying out wheat embryo cell-free expression system vivoexpression pyrazinoic acid amide Enzyme assay after adopting primer pair 2 fluorescent quantitative PCR.
Figure 15 is the experimental result of the primer dimer not containing goal gene being carried out wheat embryo cell-free expression system vivoexpression pyrazinoic acid amide Enzyme assay.
Embodiment
Primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof provided by the invention, utilize the resistance to pyrazinoic acid amide property of medicine of this primer sets to mycobacterium tuberculosis to detect, its testing process as shown in Figure 1.First fluorescent quantitative PCR technique is utilized whether to detect in clinical sample containing mycobacterium tuberculosis.Just to prove in sample containing mycobacterium tuberculosis if there is positive amplification, then need to its then detection of pyrazinamide resistance detect.When carrying out detection of pyrazinamide resistance detection, the quantitative fluorescent PCR product of above step is template, adopts the primer containing vivoexpression element, again carries out pcr amplification, adds the element required for expressing, as T7 promotor etc.Reclaim after the product of amplification is concentrated also quantitatively, join in vitro expression systems, the pyrazinoic acid amide enzymatic conversion pyrazinoic acid amide expressed by mensuration is the enzymic activity of pyrazinoic acid amide acid.Measured enzymic activity, enzyme on the one hand by recording with blank vivoexpression liquid under similarity condition is lived results contrast, judge that whether fluorescent quantitative PCR product is containing mycobacterium tuberculosis total length pncA fragment, compare with the mycobacterium tuberculosis standard sensitive strain adopting same method to express and to measure and persister pyrazinamidase Activity Results on the other hand, judge whether to there occurs medicament-resistant mutation, realize the detection of the detection of pyrazinamide resistance to mycobacterium tuberculosis in clinical sample.Time shown in figure, as just the roughly signal of total time needed for present method, can carry out increasing and decreasing and adjusting according to practical situation and needs.
Human body is except infecting except tuberculosis at the position such as hair, nail, and other all sites all can infect, but the most common with pulmonary tuberculosis.Therefore, in Clinical Laboratory, often need whether carry out detection to confirm tuberculosis containing mycobacterium tuberculosis in various clinical sample, wherein detect sputum the most common.
Be detected as example with mycobacterium tuberculosis and detection of pyrazinamide resistance thereof in clinical sputum below, in conjunction with specific embodiments and accompanying drawing the invention will be further described.
Embodiment 1:
Using pyrazinoic acid amide sensitive strain mycobacterium tuberculosis reference culture M.tuberculosisH37Ra (U.S.'s Culture Collection numbering: ATCC25177) contrast as pyrazinoic acid amide medicaments insensitive, standard pyrazinoic acid amide persister bacille Calmette-Guerin vaccine BCG (U.S.'s Culture Collection numbering: ATCC19274) contrast as pyrazinoic acid amide medicine resistance, be used for the measurement result of mycobacterium tuberculosis strains that these two are cultivated to set up the quantitative fluorescent PCR typical curve that to judge in actual sample whether containing mycobacterium tuberculosis and test present method and whether can distinguish standard pyrazinoic acid amide persister and sensitive strain.
Concrete steps are as follows:
1. cracking mycobacterium tuberculosis and its DNA of extraction:
(1) have the L-J substratum of mycobacterium tuberculosis reference culture M.tuberculosisH37Ra and BCG from cultivation, use transfering loop scraping one ring bacterium respectively, be dissolved in 2mLNaOH solution, after mixing, hatch 5min for 37 DEG C.
(2) centrifugal 10min, goes supernatant to stay precipitation.PBS washs, and repeats to wash 2 times, goes supernatant to stay precipitation to be checked.
(3) DNA extraction: add DNA extraction liquid in the above-mentioned sample handled well.Boiling water bath 10min, then directly utilizes lysate as template.
2. according to pyrazinamidase gene (pncA) reading frame and upstream and downstream sequence thereof in M. tuberculosis genes group, design special primer, primer should have and not mate completely with any sequence in pyrazinamidase gene (pncA) reading frame and gained PCR primer contains the feature of total length pncA gene, is detected mycobacterium tuberculosis by quantitative fluorescent PCR.In the present embodiment, the primer sequence of design and use is as follows:
Quantitative PCR forward primer 1:5'CGGACGGATTTGTCGCTCAC3';
Quantitative PCR reverse primer 1:5'GCCCGATGAAGGTGTCGTAGAAG3'.
Hereinafter referred to as fluorescence quantification PCR primer to 1, its position in M. tuberculosis genes group as shown in Figure 2.Its complete complementary region visible is positioned at the upstream and downstream region of pncA gene, not with any sequences match in pncA reading frame.
The MightyAmp that TaKaRa company produces is adopted during detection
tMforRealTime (
plus) test kit.Amplification system 20 μ L, wherein MIX adds 10 μ L, and upstream and downstream primer respectively adds 0.5 μ L, and template adds 2 μ L, and moisturizing is to cumulative volume 20 μ L.According to the instrument specification sheets of ABI company, setting response procedures and melting point curve routine analyzer.Reaction conditions: 98 DEG C of denaturation 2min; 98 DEG C of sex change 10s, 68 DEG C of annealing 65s, 85 DEG C gather fluorescence, 35 circulations.Typical pcr amplification curve as shown in Figure 3.The Gel electrophoresis results of amplified production as shown in Figure 4.The molecular band of amplified production is 918bp, meets expected design, and gained PCR primer should contain total length pncA gene.
3. get H37Ra genome production standard product, carry out quantitative fluorescent PCR after gradient dilution, obtain typical curve, result as shown in Figure 6.As seen from the figure, there is linear relationship between the Ct value of fluorescence quantitative PCR detection and H37Ra genomic template number logarithm, detection sensitivity can reach 10copies/ μ about l.
4. with the quantitative fluorescent PCR product in step 2 for template, use with promotor, or simultaneously with the primer of promotor and enhanser, carrying out pcr amplification, is that the product of gained in step 2 adds Cell free expression system element.The expression system adopted in the present embodiment is wheat embryo cell-free expression system, and according to this system features, the primer sequence of design and employing is as follows:
Vivoexpression forward primer 1:
5'TAATACGACTCACTATAGGATACTCCCCCACAACAGCTTACAATACTCCCCCACACAGCTTACAAATACTCCCCCAGTCGCCCGAACGTAAGGAGGACGT3'
Vivoexpression reverse primer 1:5'ACCGCCGCCAACAGTTCATCCCGGT3'
As shown in Figure 2, this has primer (vivoexpression primer pair 1) and not to mate completely with any sequence except initiator codon ATG in pyrazinamidase gene (pncA) reading frame and gained PCR primer contains the feature of total length pncA gene, and wherein forward primer contains T7 promotor 5'TAATACGACTCACTATAGG3' and a section of 5 ' UTR enhancer sequence of applicable wheat embryo cell-free expression system:
5'ATACTCCCCCACAACAGCTTACAATACTCCCCCACACAGCTTACAAATACTCCCCC3'
PCR reaction conditions: 98 DEG C of denaturation 30s; 98 DEG C of sex change 10s, 66 DEG C of annealing 1min, 72 DEG C of amplification 30s, 35 circulations, 72 DEG C of insulation 10min.The Gel electrophoresis results of vivoexpression pcr amplification product as shown in Figure 4.The molecular band of amplified production is 623bp, meets expected design, and gained PCR primer contains total length pncA gene.
After pcr amplification, the product obtained is concentrated:
(1) in the PCR primer obtained, 3M sodium-acetate (pH5.2) solution of 1/10 volume is added, Homogeneous phase mixing.
(2) the DNAmate solution of 4 μ L is added, Homogeneous phase mixing.
(3) add-20 DEG C of precooling dehydrated alcohols of 2.5 times of volumes, fully mix.
(4) 12,000r/min4 DEG C centrifugal 15 minutes.
(5) abandon solution, stay white precipitate.
(6) 70% ethanol 1mL of-20 DEG C of precoolings is added, washing precipitation of turning upside down.Repeated washing twice.
(7) 12,000r/min4 DEG C carefully discard ethanol, vacuum-drying after centrifugal 5 minutes.Add the water dissolution DNA of appropriate nuclease free.
5. adopt Nanodrop micro-spectrophotometer to measure the concentration reclaiming product.General concentration should reach 800ng/ μ L-1000ng/ μ L, reclaims the extinction ratio A260/A280 of product at wavelength 260nm and 280nm place within the scope of 1.70-1.85.
6. adopt the external acellular expression test kit of RTS100WheatgermCECFKit wheatgerm, express pyrazinamidase.Reaction system is placed in constant temperature blending instrument, 24 DEG C, 9000r/min, reaction 10-20h; Reaction system is in table 1.
7., due to after pyrazinoic acid amide is degraded to pyrazine acid, can react with ferrous ion and generate red-brown product, thus the colour-change of reaction solution can as the index of Resistance detection.After vivoexpression reaction terminates, get 20 μ L reaction mixtures and join in 150 μ L10mg/mL pyrazinoic acid amide (PZA) solution, hatch 3h for 37 DEG C.Then 10 μ L10% (W/V) ferrous ammonium sulphates are added, colour developing.The shown reddish-brown depth directly can be distinguished by naked eyes, also can be distinguished by spectrophotometric determination 460nm place light absorption value.
From Fig. 7, color reddish-brown after the reaction of standard sensitive strain H37Ra can be found out, illustrate from the pyrazinoic acid amide enzymic activity of its pncA genetic expression high, pyrazinoic acid amide can be made to play curative effect.And color is colourless or very light reddish-brown after the reaction of standard persister BCG, illustrate that from the pyrazinamidase activity of its pncA genetic expression substantially or low pyrazinoic acid amide can not play curative effect, shows as resistance.Under similarity condition, the cell free in vitro expression blank solution not adding vivoexpression pcr amplification product does not demonstrate any pyrazinoic acid amide enzymic activity, and it is minimum at the light absorption value at 460nm place.Obviously, if not containing total length pncA gene in quantitative fluorescent PCR product, so also total length pncA gene can not be contained in vivoexpression pcr amplification product, thus any pyrazinoic acid amide enzymic activity can not be demonstrated in expression liquid after vivoexpression.Therefore, cell free in vitro expresses the light absorption value that blank solution shows at 460nm place, can as an auxiliary judgment standard, if in the expression liquid after vivoexpression pyrazinoic acid amide enzymic activity lower than or equal blank absorption value, so total length pncA gene should not be contained in quantitative fluorescent PCR product, thus the false positive that some non-specific amplifications cause can be got rid of, improve the specificity of Fluorescence quantitative PCR detection mycobacterium tuberculosis.
Embodiment 2:
The present embodiment is with the 3 portions of clinical sputums (218 being diagnosed as tuberculosis patient gathered from Tuberculosis Control Institute of Wuhan City, 271, No. 8333) as test sample, test present method and whether can be suitable for detecting the detection of mycobacterium tuberculosis in clinical sputum and detection of pyrazinamide resistance carrying out preliminary checking.Concrete steps are as follows:
1. cracking and the DNA extracting mycobacterium tuberculosis in sputum;
(1) add 2mLNaOH solution respectively in a certain amount of sputum (1-5mL), after mixing, hatch 20min. for 37 DEG C
(2) centrifugal 10min, goes supernatant to stay precipitation.PBS washs, and repeats to wash 2 times, goes supernatant to stay precipitation to be checked.
(3) DNA extraction: add DNA extraction liquid pure water in the above-mentioned sample handled well.Boiling water bath 10min, then directly utilizes lysate as template.
2. adopting the primer sequence same with quantitative fluorescent PCR in embodiment 1, to whether there being mycobacterium tuberculosis in sputum being detected by quantitative fluorescent PCR.Quantitative PCR forward primer 1:5'CGGACGGATTTGTCGCTCAC3'; Quantitative PCR reverse primer 1:5'GCCCGATGAAGGTGTCGTAGAAG3'.
The MightyAmp that TaKaRa company produces is adopted during detection
tMforRealTime (
plus) test kit, the mycobacterium tuberculosis reference culture DNA extracted in synchronous parallel amplification embodiment 1 in contrast.Amplification system 20 μ L, wherein MIX adds 10 μ L, and upstream and downstream primer respectively adds 0.5 μ L, and template adds 2 μ L, and moisturizing is to cumulative volume 20 μ L.According to the specification sheets of the instrument of ABI company, setting response procedures and melting point curve routine analyzer.Reaction conditions: 98 DEG C of denaturation 2min; 98 DEG C of sex change 10s, 68 DEG C of annealing 65s, 85 DEG C gather fluorescence, 35 circulations.Three clinical sample amplifications as shown in Figure 5.Can find out, these three clinical samples have positive amplification, and detected result is containing mycobacterium tuberculosis in sputum.
3. adopt the primer sequence same with vivoexpression PCR in embodiment 1, for the positive amplification product of three in the present embodiment step 2, again carry out PCR, add the vivoexpression element same with embodiment 1 and product is concentrated.Also same PCR carried out respectively to the quantitative fluorescent PCR product of reference culture simultaneously and concentrate, as the contrast of sample results.
PCR reaction conditions: 98 DEG C of denaturation 30s; 98 DEG C of sex change 10s, 66 DEG C of annealing 1min, 72 DEG C of amplification 30s, 35 circulations, 72 DEG C of insulation 10min.
After pcr amplification, the product obtained is concentrated:
(1) in the PCR primer obtained, 3M sodium-acetate (pH5.2) solution of 1/10 volume is added, Homogeneous phase mixing.
(2) the DNAmate solution of 4 μ L is added, Homogeneous phase mixing.
(3) add-20 DEG C of precooling dehydrated alcohols of 2.5 times of volumes, fully mix.
(4) 12,000r/min4 DEG C centrifugal 15 minutes.
(5) abandon solution, stay white precipitate.
(6) 70% ethanol 1mL of-20 DEG C of precoolings is added, washing precipitation of turning upside down.Repeated washing twice.
(7) 12,000r/min4 DEG C carefully discard ethanol, vacuum-drying after centrifugal 5 minutes.Add the water dissolution DNA of appropriate nuclease free.
4. adopt Nanodrop micro-spectrophotometer to measure the concentration reclaiming product.General concentration should reach 800ng/ μ L-1000ng/ μ L, reclaims the extinction ratio A260/A280 of product at wavelength 260nm and 280nm place within the scope of 1.70-1.85.
5. adopt the external acellular expression test kit of RTS100WheatgermCECFKit wheatgerm, the PCR primer upper step obtained joins in vivoexpression solution system respectively, expresses pyrazinamidase.Reaction system is placed in constant temperature blending instrument, 24 DEG C, 9000r/min, reaction 10-20h; Reaction system is in table 2.
6. adopt the method same with embodiment 1, by under pyrazinamidase effect, after pyrazinoic acid amide is degraded to pyrazine acid, can react with ferrous ion the pyrazinoic acid amide enzymic activity that the reaction generating red-brown product measures expression respectively.After vivoexpression reaction terminates, get 20 μ L reaction mixtures and add in the pyrazinoic acid amide (PZA) of 150 μ L10mg/mL, hatch 3h for 37 DEG C.Then 10 μ L10% (W/V) ferrous ammonium sulphates are added, colour developing.The depth of reddish-brown directly can be distinguished by naked eyes, also can be distinguished by spectrophotometric determination 460nm place light absorption value.The colour developing result of the pyrazinoic acid amide enzymic activity expressed by the gene increased from three clinical samples and corresponding 460nm light absorption value are as shown in Figure 7.
7., due in whole experimentation, difference is tested and is repeated to produce certain error.In order to whether the difference judging actual sample and reference culture enzymic activity test result is because error causes, or because transgenation causes, the enzyme slip-knot fruit of replication reference culture can be passed through repeatedly, determine threshold value (Cutoff) by standard deviation.In the present embodiment, we adopt three reproducible results of embodiment 1 Plays sensitive strain H37Ra to determine threshold value.The detailed process that Cutoff value is determined is: using the 460nm average absorbance value of H37Ra tri-times as 1, the light absorption value of H37Ra unitary determination is carried out standard deviation (SD) as a result divided by the ratio of this average absorbance value calculate, threshold value divides into (1 – 3SD), as shown in Fig. 8 solid line.Due to pncA transgenation cause the major cause of pyrazinoic acid amide resistance be sudden change after pyrazinamidase live reduce, therefore, what be less than this threshold value can think that the pyrazinoic acid amide enzymic activity of sample is lower than sensitive control, be judged as pyrazinoic acid amide resistance, suitable with the pyrazinoic acid amide enzymic activity of H37Ra higher than can thinking of this threshold value, be judged as that pyrazinoic acid amide is responsive.Three reproducible results that our employing simultaneously does not add the blank vivoexpression liquid of any DNA determine that one judges whether quantitative fluorescent PCR product contains the threshold value of total length pncA gene.This threshold value deterministic process is using the 460nm average absorbance value of H37Ra tri-times as 1, the light absorption value of blank unitary determination is carried out standard deviation (SD) as a result divided by the ratio of this average absorbance value calculate, calculate the mean value A0 of blank ratio simultaneously, threshold value divides into (A0+3SD), and this result is as shown in Fig. 8 dotted line.
As can be seen from Figure 8, the measurement result of standard persister BCG and clinical sample 218,8333, is less than threshold value, shows as pyrazinoic acid amide resistance.And the pyrazinamidase work of clinical sample 271 is higher than threshold value, show as pyrazinoic acid amide sensitivity.Further PCR primer is checked order, the pncA gene of result display clinical sample 218 and 8333 has causes the transgenation of pyrazinoic acid amide resistance (to be respectively 89,151), and the pncA gene of clinical sample 271 does not have sudden change, consistent with the pncA sequence of standard sensitive strain H37Ra.This result illustrates simultaneously, and present method detects detection of pyrazinamide resistance, does not need to know mutational site information in advance, only need according to expressed pyrazinoic acid amide enzymic activity relatively and type strain whether change, just can judged result.Not mate completely with any sequence except initiator codon ATG in pyrazinamidase gene (pncA) reading frame and gained PCR primer contains the feature of total length pncA gene because design of primers of the present invention has, therefore the DNA product of pcr amplification can comprise may suddenling change of any position in pncA gene reading frame, can react from the pyrazinamidase enzymic activity of this amplified production vivoexpression the enzyme activity change that in whole pncA gene reading frame, any base mutation causes, multisite mutation or potential medicament-resistant mutation can be detected simultaneously.
From the result Fig. 8, it can also be seen that the pyrazinoic acid amide enzymic activity expressed by gene amplified from three strain clinical samples is all higher than the threshold value (A0+3SD) of blank, the pncA gene that should include total length in quantitative fluorescent PCR product is described, again demonstrates the result of quantitative fluorescent PCR.
The present embodiment result shows the resistance that method provided by the invention can be successfully used to measure mycobacterium tuberculosis in clinical sputum and pyrazinoic acid amide thereof.
Embodiment 3:
Need in the step that detection of pyrazinamide resistance detects for quantitative fluorescent PCR product adds vivoexpression element, as T7 promotor, express enhanser etc.The present embodiment is using pyrazinoic acid amide sensitive strain mycobacterium tuberculosis reference culture M.tuberculosisH37Ra (U.S.'s Culture Collection numbering: ATCC25177) contrast as pyrazinoic acid amide medicaments insensitive, standard pyrazinoic acid amide persister bacille Calmette-Guerin vaccine BCG (U.S.'s Culture Collection numbering: ATCC19274) as the contrast of pyrazinoic acid amide medicine resistance, two kinds of bacterial strains adopt respectively and carry out vivoexpression PCR with the primer reaching enhanser 5 ' UTR without a segment table.Then join in wheat embryo cell-free expression system, whether the expression enhanser in research primer can measure detection of pyrazinamide resistance impact.
Concrete steps are as follows:
1. adopt method cracking mycobacterium tuberculosis reference culture M.tuberculosisH37Ra and BCG same with embodiment 1 and extract its DNA.
2. adopt the fluorescence quantification PCR primer sequence same with embodiment 1 and method to carry out fluorescent quantitative PCR to the DNA extracted.
3. for the amplified production in the present embodiment step 2, adopt following two pairs of primers respectively, again carry out PCR, add applicable wheatgerm and express the original paper of vitro expression systems and product is concentrated.
Vivoexpression forward primer 1:
5'TAATACGACTCACTATAGGATACTCCCCCACAACAGCTTACAATACTCCCCCACACAGCTTACAAATACTCCCCCAGTCGCCCGAACGTAAGGAGGACGT3'；
Vivoexpression reverse primer 1:
5'ACCGCCGCCAACAGTTCATCCCGGT3'；
Vivoexpression forward primer 2:
5'TAATACGACTCACTATAGGAGTCGCCCGAACGTAAGGAGGACGT3'；
Vivoexpression reverse primer 2:
5'ACCGCCGCCAACAGTTCATCCCGGT3'。
Wherein forward primer 1 is containing T7 promotor 5'TAATACGACTCACTATAGG3' and a section of 5 ' the UTR enhancer sequence being applicable to wheat embryo cell-free expression system: 5'ATACTCCCCCACAACAGCTTACAATACTCCCCCACACAGCTTACAAATACTCCC CC3'; And forward primer 2 is containing T7 promotor, not containing 5 ' UTR enhanser.Two pairs of primers are all the same with the sequence that tuberculosis group is mated.
PCR reaction conditions: 98 DEG C of denaturation 30s; 98 DEG C of sex change 10s, 66 DEG C of annealing 1min, 72 DEG C of amplification 30s, 35 circulations, 72 DEG C of insulation 10min.
The method same with embodiment 1 is adopted to concentrate the DNA fragmentation of pcr amplification respectively.
4. adopt Nanodrop micro-spectrophotometer to measure the concentration reclaiming product.General concentration should reach 800ng/ μ L-1000ng/ μ L, reclaims the extinction ratio A260/A280 of product at wavelength 260nm and 280nm place within the scope of 1.70-1.85.
5. adopt the external acellular expression test kit of RTS100WheatgermCECFKit, and in embodiment 1, identical expression condition and method express pyrazinamidase and the pyrazinamidase work expressed by the detection of same Enzyme activity assay method.Result as shown in Figure 9.As can be seen from Figure 9 with the height of absorption value OD460 value ratio not with 5 ' UTR enhanser at 460nm wavelength after the standard sensitive strain H37Ra pyrazinoic acid amide enzyme reaction expressed by 5 ' UTR enhanser, illustrate that band 5 ' UTR enhanser can increase the expression of pyrazinamidase within the same time.And no matter standard persister BCG adds or do not add and express enhanser 5 ' UTR, the active difference of the pyrazinamidase recorded is little, and after reaction, color is that colourless or very light reddish-brown OD460 value is lower.But ' UTR, the enzyme of standard sensitive strain and persister difference of living is obvious, illustrates that no matter adding or do not add expression enhanser 5 ' UTR does not affect the judgement of this detection method for resistance with 5 for the primer no matter used.But with 5, ' UTR enhanser in same time, can distinguish sensitive strain and persister more significantly.
The present embodiment illustrate express that enhanser 5 ' UTR has can the effect of Enhanced expressing carboxamide dihydrochloride enzyme, even if this detection method also can be good at distinguishing sensitive strain and persister when not adding and expressing enhanser 5 ' UTR, design of primers can carry out the selection of vivoexpression element as required.
Embodiment 4:
Except adopting the external cell-free protein expression system of wheatgerm in above-described embodiment, this detection method principle can also use other vitro expression systems, as the cell free in vitro protein expression system of the rabbit reticulated red blood cells (rabbitreticulocyte) that often uses and intestinal bacteria (E.coli), carry out the sample to the fluorescence quantitative PCR detection positive, carry out the expression of pyrazinamidase, thus realize detection of pyrazinamide resistance detection.The present embodiment is using pyrazinoic acid amide sensitive strain mycobacterium tuberculosis reference culture M.tuberculosisH37Ra (U.S.'s Culture Collection numbering: ATCC25177) contrast as pyrazinoic acid amide medicaments insensitive, standard pyrazinoic acid amide persister bacille Calmette-Guerin vaccine BCG (U.S.'s Culture Collection numbering: ATCC19274) contrast as pyrazinoic acid amide medicine resistance, adopt intestinal bacteria (E.coli) Cell free expression system to express pyrazinamidase, realize detecting the resistance of mycobacterium tuberculosis pyrazinoic acid amide.
Concrete steps are as follows:
1. adopt method cracking mycobacterium tuberculosis reference culture M.tuberculosisH37Ra and BCG same with embodiment 1 and extract its DNA.
2. adopt the fluorescence quantification PCR primer sequence same with embodiment 1 and method to carry out fluorescent quantitative PCR to the DNA extracted.
3. for the amplified production in the present embodiment step 2, adopt following primer, again carry out PCR, add the expression original paper of applicable E. coli cell free vitro expression systems and product is concentrated.
The primer sequence adopted in the present embodiment is as follows:
Vivoexpression forward primer 3:
5'TAATACGACTCACTATAGGAAGGAGATAATGCATCATCATCATCATCACAGTCGCCCGAACGTAAGGAGGACGT3'；
Vivoexpression reverse primer 3:
5'ACCGCCGCCAACAGTTCATCCCGGT3'。
As Figure 10, this has primer and not to mate completely with any sequence except initiator codon ATG in pyrazinamidase gene (pncA) reading frame and gained PCR primer contains the feature of total length pncA gene, and wherein forward primer contains T7 promotor 5'TAATACGACTCACTATAGG3' and section of SD sequence: 5'AAGGAGATAATGCATCATCATCATCATCAC3' of applicable E. coli cell free expression system
The method same with embodiment 1 is adopted to concentrate the DNA fragmentation of pcr amplification respectively.
4. adopt Nanodrop micro-spectrophotometer to measure the concentration reclaiming product.General concentration should reach 800ng/ μ L-1000ng/ μ L, reclaims the extinction ratio A260/A280 of product at wavelength 260nm and 280nm place within the scope of 1.70-1.85.
5. adopt the external acellular expression test kit of RTS100E.coliHYKit, express pyrazinamidase.Reaction system is placed in constant temperature blending instrument, 30 DEG C, reaction 4-6h; Reaction system is in table 3.
6. adopt the method same with embodiment 1, by under pyrazinamidase effect, after pyrazinoic acid amide is degraded to pyrazine acid, can react with ferrous ion the pyrazinoic acid amide enzymic activity that the reaction generating red-brown product measures expression respectively.After vivoexpression reaction terminates, get 20 μ L reaction mixtures and add in the pyrazinoic acid amide (PZA) of 150 μ L10mg/mL, hatch 3h for 37 DEG C.Then 10 μ L10% (W/V) ferrous ammonium sulphates are added, colour developing.The result of experiment is distinguished by spectrophotometric determination 460nm place light absorption value.From Figure 11, can find out that the OD460 value of standard sensitive strain H37Ra is higher, illustrate from the pyrazinoic acid amide enzymic activity of its pncA genetic expression high, can be that pyrazinoic acid amide plays curative effect.And the OD460 value of standard persister BCG is lower, after reaction, color is colourless or very light reddish-brown, illustrates that from the pyrazinamidase activity of its pncA genetic expression substantially or low pyrazinoic acid amide can not play curative effect, shows as resistance.The present embodiment illustrates that the inventive method utilizes intestinal bacteria (E.coli) Cell free expression system still can obtain good detected result.Therefore, as required, the inventive method can select different vitro expression systems, and key is to express in the design of primers of PCR in vitro, needs to select to add the Expression element being applicable to different vitro expression systems.
Embodiment 5:
Except the quantitative PCR forward primer 1 that uses in above-described embodiment 1-4 and quantitative PCR reverse primer 1 this to except primer, can also design and adopt other fluorescence quantification PCR primer sequence to detect the mycobacterium tuberculosis in sputum, as long as primer has and not to mate completely with any sequence in pyrazinamidase gene (pncA) reading frame and gained PCR primer contains the feature of total length pncA gene, would not affect to pass behind vitro expression systems expression pyrazinamidase and measure enzyme and live, positive sample be carried out to the detection of detection of pyrazinamide resistance.The present embodiment is using pyrazinoic acid amide sensitive strain mycobacterium tuberculosis reference culture M.tuberculosisH37Ra (U.S.'s Culture Collection numbering: ATCC25177) contrast as pyrazinoic acid amide medicaments insensitive, standard pyrazinoic acid amide persister bacille Calmette-Guerin vaccine BCG (U.S.'s Culture Collection numbering: ATCC19274) as the contrast of pyrazinoic acid amide medicine resistance, adopt another to carry out mycobacterium tuberculosis and pyrazinoic acid amide medicine Resistance detection thereof to fluorescence quantification PCR primer to these two kinds of bacterial strains.
Concrete steps are as follows:
1. adopt method cracking mycobacterium tuberculosis reference culture M.tuberculosisH37Ra and BCG same with embodiment 1 and extract its DNA.
2. according to pyrazinamidase gene (pncA) reading frame and upstream and downstream sequence thereof in M. tuberculosis genes group, design special primer, primer should have and not mate completely with any sequence in pyrazinamidase gene (pncA) reading frame and gained PCR primer contains the feature of total length pncA gene, is detected mycobacterium tuberculosis by quantitative fluorescent PCR.In the present embodiment, the primer sequence of design and use is as follows:
Quantitative PCR forward primer 2:5'GGCGTCATGGACCCTATATCTGTGG3';
Quantitative PCR reverse primer 2:5'CGGTGAACAACCCGACCCAGCC3'.
Hereinafter referred to as fluorescence quantification PCR primer to 2, its position in M. tuberculosis genes group as shown in figure 12.Its complete complementary region visible is positioned at the upstream and downstream region of pncA gene, not with any sequences match in pncA reading frame.
The MightyAmp that TaKaRa company produces is adopted during detection
tMforRealTime (
plus) test kit.Amplification system 20 μ L, wherein MIX adds 10 μ L, and upstream and downstream primer respectively adds 0.5 μ L, and template adds 2 μ L, and moisturizing is to cumulative volume 20 μ L.According to the instrument specification sheets of ABI company, setting response procedures and melting point curve routine analyzer.Reaction conditions: 98 DEG C of denaturation 2min; 98 DEG C of sex change 10s, 68 DEG C of annealing 60s, 85 DEG C gather fluorescence, 35 circulations.Typical pcr amplification curve as shown in figure 13.
3. adopt the vivoexpression primer pair 1 identical with step 4 in embodiment 1 and PCR reaction conditions, for the positive amplification product in the present embodiment step 2, again carry out PCR, add the vivoexpression original paper same with embodiment 1 and adopt the method identical with step 4 in embodiment 1 to concentrate product.
4. adopt Nanodrop micro-spectrophotometer to measure the concentration reclaiming product.General concentration should reach 800ng/ μ L-1000ng/ μ L, reclaims the extinction ratio A260/A280 of product at wavelength 260nm and 280nm place within the scope of 1.70-1.85.
5. adopt the external acellular expression test kit of RTS100WheatgermCECFKit wheatgerm, the PCR primer that upper step obtains is joined in vivoexpression solution and system, express pyrazinamidase.Reaction system is placed in constant temperature blending instrument, 24 DEG C, 9000r/min, reaction 10-20h; Reaction system is in table 1.
6. adopt the method same with embodiment 1, by under pyrazinamidase effect, after pyrazinoic acid amide is degraded to pyrazine acid, can react with ferrous ion the pyrazinoic acid amide enzymic activity that the reaction generating red-brown product measures expression respectively.After vivoexpression reaction terminates, get 20 μ L reaction mixtures and add in the pyrazinoic acid amide (PZA) of 150 μ L10mg/mL, hatch 3h for 37 DEG C.Then 10 μ L10% (W/V) ferrous ammonium sulphates are added, colour developing.The result of experiment is distinguished by spectrophotometric determination 460nm place light absorption value.From Figure 14, can find out that the OD460 value of standard sensitive strain H37Ra is higher, illustrate from the pyrazinoic acid amide enzymic activity of its pncA genetic expression high, can be that pyrazinoic acid amide plays curative effect.And the OD460 value of standard persister BCG is lower, after reaction, color is colourless or very light reddish-brown, illustrates that from the pyrazinamidase activity of its pncA genetic expression substantially or low pyrazinoic acid amide can not play curative effect, shows as resistance.The present embodiment illustrates that this detection method has larger compatibility for fluorescence quantification PCR primer, as long as primer has and not to mate completely with any sequence in pyrazinamidase gene (pncA) reading frame and gained PCR primer contains the feature of total length pncA gene, can experimentally need appropriate design primer, not affect pyrazinoic acid amide Drug Resistance Detection result.
Embodiment 6:
Fluorescence quantitative PCR detection and pyrazinamidase vivoexpression mensuration enzyme two portions alive are the present invention includes, pyrazinamidase vivoexpression measures enzyme work except being used in detecting for detection of pyrazinamide resistance as described in above-described embodiment 1-5, a subsidiary function can also being played, whether containing mycobacterium tuberculosis total length pncA gene for verifying quantitative fluorescent PCR positive amplification product.For non-specific quantitative fluorescent PCR positive amplification, due to the pncA gene of product not containing mycobacterium tuberculosis, pyrazinoic acid amide enzymic activity can not be measured, thus the possible non-specific positive amplification of a part can be got rid of, improve the specificity of mycobacterium tuberculosis in fluorescence quantitative PCR detection sputum.The present embodiment adopts the quantitative fluorescent PCR forward primer 2 and reverse primer 2 that use in embodiment 5, do not add Mycobacterium tuberculosis DNA template, carry out 45 circulations to produce primer two polymer, this primer dimer is used for be verified the work of pyrazinamidase vivoexpression mensuration enzyme and can identifies that this does not contain the quantitative fluorescent PCR product of pncA gene.
Concrete steps are as follows:
1. adopt the quantitative fluorescent PCR forward primer 2 same with embodiment 5 and reverse primer 2:
Quantitative PCR forward primer 2:5'GGCGTCATGGACCCTATATCTGTGG3';
Quantitative PCR reverse primer 2:5'CGGTGAACAACCCGACCCAGCC3'.
Do not add any template, carry out pcr amplification reaction, the MightyAmp adopting TaKaRa company to produce during PCR reaction
tMforRealTime (
plus) test kit.Amplification system 20 μ L, wherein MIX adds 10 μ L, and upstream and downstream primer respectively adds 0.5 μ L, and moisturizing is to cumulative volume 20 μ L.According to the instrument specification sheets of ABI company, setting response procedures and melting point curve routine analyzer.Reaction conditions: 98 DEG C of denaturation 2min; 98 DEG C of sex change 10s, 68 DEG C of annealing 60s, 85 DEG C gather fluorescence, 45 circulations.
3. adopt the vivoexpression primer identical with step 4 in embodiment 1 and PCR reaction conditions, for the amplified production in the present embodiment step 2, again carry out PCR, add the vivoexpression original paper same with embodiment 1 and adopt the method identical with step 4 in embodiment 1 to concentrate product.
4. adopt Nanodrop micro-spectrophotometer to measure the concentration reclaiming product.General concentration should reach 800ng/ μ L-1000ng/ μ L, reclaims the extinction ratio A260/A280 of product at wavelength 260nm and 280nm place within the scope of 1.70-1.85.
5. adopt the external acellular expression test kit of RTS100WheatgermCECFKit wheatgerm, the PCR primer that upper step obtains is joined in vivoexpression solution and system, express pyrazinamidase.Establish one not add any DNA wheat embryo cell-free expression system solution in contrast simultaneously.Reaction system is placed in constant temperature blending instrument, 24 DEG C, 9000r/min, reaction 10-20h; Reaction system is in table 1.
6. adopt the method same with embodiment 1, by under pyrazinamidase effect, after pyrazinoic acid amide is degraded to pyrazine acid, can react with ferrous ion the pyrazinoic acid amide enzymic activity that the reaction generating red-brown product measures expression respectively.After vivoexpression reaction terminates, get 20 μ L reaction mixtures and add in the pyrazinoic acid amide (PZA) of 150 μ L10mg/mL, hatch 3h for 37 DEG C.Then 10 μ L10% (W/V) ferrous ammonium sulphates are added, colour developing.The result of experiment is by spectrophotometric determination 460nm place light absorption value.As shown in figure 15, add and have compared with the reaction mixture of DNA contrasts with the blank reaction mixture not adding any DNA, light absorption value does not have difference.PncA gene not containing total length in quantitative fluorescent PCR product is described, is judged as non-specific amplification, is primer dimer due to what add in the present embodiment, proves that this result is correct obviously.
The present embodiment illustrates and adopts vivoexpression pyrazinamidase, and whether its enzyme mensuration of living can assistant identification quantitative fluorescent PCR product be the pncA gene containing total length further.An authentication step due to organic combination, can get rid of the non-specific amplification result of quantitative fluorescent PCR, improves the specificity that mycobacterium tuberculosis detects.
Subordinate list
Table 1
Table 2
Table 3
<110> Wuhan Virology Institute,Chinan academy of Sciences
Mycobacterium tuberculosis and detection of pyrazinamide resistance associated detecting method in <120> clinical sample
<140>
<141>
<160>1
<170>
<210>1
<211>20
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>1
CGGACGGATTTGTCGCTCAC
<210>2
<211>23
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>2
GCCCGATGAAGGTGTCGTAGAAG
<210>3
<211>100
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>3
TAATACGACTCACTATAGGATACTCCCCCACAACAGCTTACAATACTCCCCCACACAGCTTACAAATACTCCCCCAGTCGCCCGAACGTAAGGAGGACGT
<210>4
<211>25
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>4
ACCGCCGCCAACAGTTCATCCCGGT
<210>5
<211>25
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>5
GGCGTCATGGACCCTATATCTGTGG
<210>6
<211>22
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>6
CGGTGAACAACCCGACCCAGCC
<210>7
<211>44
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>7
TAATACGACTCACTATAGGAGTCGCCCGAACGTAAGGAGGACGT
<210>8
<211>25
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>8
ACCGCCGCCAACAGTTCATCCCGGT
<210>9
<211>74
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>9
TAATACGACTCACTATAGGAAGGAGATAATGCATCATCATCATCATCACAGTCGCCCGAACGTAAGGAGGACGT
<210>10
<211>25
<212>DNA
<213> artificial sequence
<220>
<221>
<222>
<223>
<400>10
ACCGCCGCCAACAGTTCATCCCGGT
Claims (7)
1., for detecting a primer sets for mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof, it is characterized in that being made up of following primer:
Quantitative PCR forward primer 1:5'CGGACGGATTTGTCGCTCAC3',
Quantitative PCR reverse primer 1:5'GCCCGATGAAGGTGTCGTAGAAG3',
Quantitative PCR forward primer 2:5'GGCGTCATGGACCCTATATCTGTGG3',
Quantitative PCR reverse primer 2:5'CGGTGAACAACCCGACCCAGCC3';
Vivoexpression forward primer 1:5'TAATACGACTCACTATAGGATACTCCCCCACAACAGCTTACAATACTCCCCC ACACAGCTTACAAATACTCCCCCAGTCGCCCGAACGTAAGGAGGACGT3',
Vivoexpression reverse primer 1:5'ACCGCCGCCAACAGTTCATCCCGGT3',
Vivoexpression forward primer 2:5'TAATACGACTCACTATAGGAGTCGCCCGAACGTAAGGAGGACGT3';
Vivoexpression reverse primer 2:5'ACCGCCGCCAACAGTTCATCCCGGT3';
Vivoexpression forward primer 3:5'TAATACGACTCACTATAGGAAGGAGATAATGCATCATCATCATCATCACAGT CGCCCGAACGTAAGGAGGACGT3';
Vivoexpression reverse primer 3:5'ACCGCCGCCAACAGTTCATCCCGGT3';
The method utilizing above-mentioned primer sets to detect the resistance to pyrazinoic acid amide property of medicine of mycobacterium tuberculosis is: whether the M. tuberculosis genes pack section containing total length pncA gene by fluorescent quantitative PCR detects in sample containing mycobacterium tuberculosis, described pncA gene is mycobacterium tuberculosis pyrazinamidase gene, just prove to contain mycobacterium tuberculosis in sample if there is positive amplification, then its detection of pyrazinamide resistance is detected; During detection, with the product of quantitative fluorescent PCR for template, adopt the primer containing vivoexpression element, again carry out pcr amplification total length pncA gene, add the element required for expressing; Then join in vitro expression systems by the PCR primer of amplification, the pyrazinoic acid amide enzymatic conversion pyrazinoic acid amide expressed by mensuration is the enzymic activity of pyrazinoic acid amide acid; Measured enzymic activity, whether total length pncA gene is contained on the one hand as aided verification quantitative fluorescent PCR product, compare with the mycobacterium tuberculosis type strain pyrazinoic acid amide enzymic activity adopting same method to express and to measure on the other hand, learn that in sample, whether mycobacterium tuberculosis there occurs medicament-resistant mutation, realizes the detection to its detection of pyrazinamide resistance accordingly.
2. the primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof according to claim 1, is characterized in that described detection method comprises the following steps:
(1) to extract and the DNA of mycobacterium tuberculosis in lysed sample, as the template that following PCR detects;
(2) according to pncA, GenBank sequence number is the feature of pyrazinamidase gene and upstream and downstream sequence thereof in the M. tuberculosis genes group described in U59967, design special primer, the primer adopted has and not to mate completely with any sequence in pncA gene reading frame and gained PCR primer contains the feature of total length pncA gene, and whether the M. tuberculosis genes pack section containing total length pncA gene by fluorescent quantitative PCR detects in sample containing mycobacterium tuberculosis;
(3) for sample mycobacterium tuberculosis being detected in step (2), adopt the primer containing vivoexpression element of design, the primer adopted has and not to mate completely with any sequence in pncA gene reading frame except initiator codon ATG and gained PCR primer contains the feature of total length pncA gene, with the quantitative fluorescent PCR product obtained in step (2) for template, again carry out the fragment that pcr amplification contains total length pncA gene, for pncA gene adds Expression element, make cell free in vitro expression system can carry out the expression of pyrazinamidase to the PCR primer added,
(4) PCR primer obtained in enrichment step (3) is also quantitative;
(5) add the PCR primer obtained in the step (4) of 1 ~ 20 microgram, adopt cell free in vitro expression system to express pyrazinamidase, select 1 to 24 hours expression times as required;
(6) get the vitro expression systems liquid after 1 ~ 50 microlitre expression pyrazinamidase, measure the activity of wherein contained pyrazinamidase; Enzyme on the one hand by recording with blank vivoexpression liquid under similarity condition is lived results contrast, judge that whether fluorescent quantitative PCR product is containing mycobacterium tuberculosis total length pncA fragment, pyrazinoic acid amide enzymic activity on the other hand by expressing standard pyrazinoic acid amide sensitive strain and Resistant strain pncA gene with same method compares, in judgement sample, whether the pncA gene of mycobacterium tuberculosis there occurs medicament-resistant mutation, realizes the detection to its detection of pyrazinamide resistance.
3. the primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof according to claim 2, is characterized in that the vivoexpression element added in step (3) adopts promotor, or adopts promotor and express enhanser;
The primer sequence of described promotor is: 5'TAATACGACTCACTATAGG3',
The primer sequence of described expression enhanser is: 5'ATACTCCCCCACAACAGCTTACAATACTCCCCCACACAGCTTACAAATACTCCC CC3'.
4. the primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof according to claim 2, is characterized in that the vitro expression systems described in step (3)-step (6) is wheatgerm vitro expression systems.
5. the primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof according to claim 2, it is characterized in that judging in step (6) method of fluorescent quantitative PCR product whether containing mycobacterium tuberculosis total length pncA fragment be do not add under similarity condition and method any DNA express after blank vivoexpression liquid in measured pyrazinamidase enzyme live add three times same method and condition under repeatedly measure its enzyme slip-knot really standard deviation as threshold value, measured sample enzyme work is judged as fluorescent quantitative PCR product not containing mycobacterium tuberculosis total length pncA fragment less than or equal to this threshold value, fluorescence quantitative PCR detection result is false positive, measured sample enzyme work is that fluorescent quantitative PCR product contains mycobacterium tuberculosis total length pncA fragment higher than the judgement of this threshold value, and fluorescence quantitative PCR detection result verification is positive.
6. the primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof according to claim 2, it is characterized in that the pncA gene of mycobacterium tuberculosis in judgement sample in step (6) whether there occurs method that medicament-resistant mutation uses be in kind live with the pyrazinamidase enzyme of pncA genetic expression in mycobacterium tuberculosis standard pyrazinoic acid amide sensitive strain under condition deduct three times its enzyme activity determination result standard deviation of repeatedly expression as threshold value, measured sample enzyme work is judged as pyrazinoic acid amide resistance lower than this threshold value, higher than or equal this threshold value for pyrazinoic acid amide responsive.
7. the primer sets for detecting mycobacterium tuberculosis and the resistance to pyrazinoic acid amide property of medicine thereof according to claim 2, it is characterized in that with in detection of pyrazinamide resistance testing process, the pyrazinoic acid amide enzymic activity contained in vivoexpression liquid, as aided verification quantitative fluorescent PCR product whether containing total length pncA gene, get rid of the non-specific amplification of quantitative fluorescent PCR, improve the specificity that in clinical sample, mycobacterium tuberculosis PCR detects.
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