CN107557435B - Preparation method and application of MC-ARMS-mMB triple technology in drug-resistant tubercle bacillus diagnostic kit - Google Patents
Preparation method and application of MC-ARMS-mMB triple technology in drug-resistant tubercle bacillus diagnostic kit Download PDFInfo
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Abstract
The invention discloses a preparation method and application of MC-ARMS-mMB triple technology in a drug-resistant tubercle bacillus diagnostic kit, and describes MC-ARMS-mMB (microcolony-amplification inhibition mutation system-multiple molecular beacon) triple technology, which is a diagnostic kit for rapidly culturing tubercle bacillus and drug-resistant bacteria thereof. Simultaneously, the preparation method of the diagnostic kit is elucidated; and scientifically expresses the clinical application of the Chinese medicinal preparation. Particularly, the clinical application value and the better development prospect of the diagnostic kit are pointed out.
Description
One, the technical field
The invention belongs to the field of the following contents:
(one) molecular biology category.
The field of bacterial biological characteristic research.
The field of infectious disease diagnostic medicine research.
Second, background Art
Molecular biology: the subject of the present application is the molecular beacon probe of mycobacterium tuberculosis. Specific sequences rpoB, katG315 and IS6110 of the tubercle bacillus are selected as target sequences to design primers and probes for detecting the tubercle bacillus.
(II) bacterial biological characteristic research: after the micro-colony culture of the tubercle bacillus, the inoculated bacteria are proved to be live bacteria or drug-resistant bacteria.
(III) infectious disease diagnostic medical research.
The treatment of tuberculosis has become a therapeutic challenge not only due to the innate high levels of resistance to antibiotics of tubercle bacillus, but also due to acquired mutations that have developed in recent years, making resistance more severe.
Because the diagnosis of the drug-resistant tuberculosis is complex, the treatment is difficult, the course of disease is long, and the medical cost is about 100 times of that of the common tuberculosis, the drug-resistant tuberculosis causes great harm to individuals, families and society. The World Health Organization (WHO) 2014 worldwide tuberculosis control report states that: worldwide, 3.5% of new cases of tuberculosis and 20.5% of cases that received antitubercular treatment suffered from multidrug resistant tuberculosis in 2013. That is, it is estimated that 48 million people have been stained for multidrug-resistant tuberculosis in 2013. On average, 9.0% of patients with multi-drug resistant tuberculosis suffer from wide drug resistant tuberculosis, China is one of three high-incidence areas of multi-drug resistant tuberculosis, about 10 thousands of patients with multi-drug resistant tuberculosis are newly developed every year, and the total number of the patients accounts for 1/4 of the whole world.
At present, few researchers for detecting drug-resistant mycobacterium tuberculosis at home and abroad combine phenotypic bacteria culture and genotype PCR. The MC-ARMS-mMB method established herein combines three technologies of micro colony culture (MC), amplification retardation mutation system primer (ARMS) and multi-molecular beacon probe (mMB) to rapidly detect the live tuberculosis bacteria and acquire the drug resistance of the live tuberculosis bacteria, and lays theoretical and technical foundation for developing a kit for rapidly detecting the tubercle bacillus and the drug resistance of the tubercle bacillus.
Third, the invention
The invention describes MC-ARMS-mMB (microcolony-amplification inhibition mutation system-multiple molecular beacon) triple technology, and relates to a diagnostic kit for rapidly culturing tubercle bacillus and drug-resistant bacteria thereof. Simultaneously, the preparation method of the diagnostic kit is elucidated; and scientifically expresses the clinical application of the Chinese medicinal preparation. Particularly, the clinical application value and the huge development prospect of the diagnostic kit are pointed out.
The characteristics of the invention
1. The growth of the tubercle bacillus is very slow, and the tubercle bacillus can grow visible colonies after 4-8 weeks, which is not beneficial to the diagnosis of tuberculosis and epidemiological investigation. The present invention provides a method for rapidly detecting the presence of tubercle bacillus in a biological sample and identifying the viability and death of tubercle bacillus. The method is characterized in that: the inoculated bacteria are live bacteria as evidenced by the formation of microcolonies after the tubercle bacillus is incubated for 12-72 hours.
2. Whether the bacteria in the sample are dead or alive, it cannot be judged whether the bacteria are mycobacterium tuberculosis or drug-resistant mycobacterium tuberculosis. Therefore, the invention provides a primer and a molecular beacon probe which have high specificity and high sensitivity and are used for detecting mycobacterium tuberculosis and drug resistance thereof. The method is characterized in that: the specific sequences IS6110, rpoB and katG of the tubercle bacillus are selected as target sequences to design a primer (polynucleotide sequence with SEQ ID N0.4, SEQ ID N0.5, SEQ ID N0.7, SEQ ID N0.8, SEQ ID N0.10 and SEQ ID N0.11) and a multiplex probe (polynucleotide sequence with SEQ ID N0.6, SEQ ID N0.9 and SEQ ID N0.12) to detect the tubercle bacillus and the drug-resistant strains thereof.
3. The polynucleotide sequence 2 IS PCR amplified to obtain the fragments of three tubercle bacillus of 92bp (IS6110), 191bp (rpoB) and 149bp (katG), which are respectively the polynucleotide sequences of SEQ ID N0.1, SEQ ID N0.2 and SEQ ID N0.3 of the sequence table. The method is characterized in that: completely identical to the original gene sequence of tubercle bacillus.
Application characteristics of (two) MC-ARMS-mMB triple technology
1. High specificity: the specific detection of rpoB, mutated Rifampicin of katG locus, isoniazid drug-resistant tubercle bacillus is 100%.
2. High sensitivity: the sensitivity of the MC-ARMS-mMB triple technology before micro colony culture is 3copies/mL, and the linear detection range is as follows: bacterial liquid concentration of 100～106cfu/mL; after micro-colony culture, the number of detectable copies is equal to 109copies/mL, the copy number is improved by 36 times compared with the copy number before culture; in terms of sensitivity, the sensitivity of the MC-ARMS-mMB triple technology after micro-colony culture reaches 109-1copies/mL, i.e., 1 copies/mL; as a result, the sensitivity of the MC-ARMS-mMB triple technology is 1copies/mL, and the clinical detection range is 100～106cfu/mL。
3. Accurate and quick: after specimen treatment, results were reported only within 12-18 hours.
4. Not only live bacteria of the mycobacterium tuberculosis are detected, but also the drug resistance condition of the mycobacterium tuberculosis to rifampicin is detected by adopting a plurality of different molecular beacon probes in the same PCR reaction, each probe is marked by different fluorescein, and the whole nuclear area of the MTB RNA polymerase rpoB or katG gene is covered after different probes are completely combined with a target sequence, so that drug-resistant strains of the mycobacterium tuberculosis can be synchronously detected.
5. Simple, conventional operation and easy mastering.
6. The method is convenient and has wide application range: 1) can detect various specimens such as sputum, pleural effusion, ascites, cerebrospinal fluid, urine, bone marrow and the like. 2) Used for the experimental diagnosis of tuberculosis. 3) Evaluation of the efficacy of antituberculosis treatments. 4) Especially the differential diagnosis of the initial patient. 5) The kit is used for quickly detecting the drug resistance of the tubercle bacillus. 6) Can be used for differential diagnosis of HIV high risk group. 7) Can detect a large number of smear missed patients and can be used for differential diagnosis of HIV high risk group.
7. The cost is relatively low, and the environment is not polluted.
Objects of the invention
Aiming at the defects of the existing technology for detecting mycobacterium tuberculosis, the invention aims to establish a microcolony rapid culture mycobacterium tuberculosis and provide a special primer and a molecular beacon for detecting mycobacterium tuberculosis and drug-resistant strains thereof and a preparation method of a kit thereof, so as to be used for clinical detection and scientific research.
The invention relates to a microcolony culture method, a target sequence RT-PCR primer, an ARMS primer, a DNA molecular beacon probe, a kit, a detection method and application thereof, which are designed for rapidly culturing and detecting tubercle bacillus. 1. The invention also provides a method for rapidly detecting whether the tubercle bacillus exists in the biological sample and identifying the death and the activity of the tubercle bacillus. The method comprises the following steps of,
(1) the biological specimen is inoculated on a cellulose acetate membrane attached to a 7H10 culture medium, and a micro-colony experiment is carried out before and after the culture to see whether micro-colonies are formed or not so as to determine the death and the activity of bacteria.
(2) Collecting cellulose acetate membrane inoculated with Bacillus tuberculosis and cultured for 12-72 hr, cutting with sterile scissors, placing into EP tube, adding 200ul DNA extractive solution
(3) Then placing the DNA template in boiling water for 10 minutes to prepare a DNA template,
(4) subjecting the DNA template obtained in step b) to real time PCR amplification treatment
(5) After the reaction is finished, data analysis is carried out by using software
Since the growth of tubercle bacillus is very slow, it takes about 12 hours to divide one generation, and it takes 4-8 weeks to grow visible colonies, which is not good for diagnosis and epidemiological investigation of tuberculosis. For the purpose of rapid examination, it is a feasible method to observe the growth of tubercle bacillus as early as possible by using a microscope.
After the formation of microcolonies is observed by the microcolony culture method, it is confirmed that the inoculated bacteria are viable bacteria, but it is not possible to judge whether they are Mycobacterium tuberculosis. Therefore, a specific sequence rpoB of the tubercle bacillus IS selected, and IS6110 IS used as a target sequence to design a primer and a probe for detecting the tubercle bacillus.
2. In order to achieve the aim of aiming at the characteristics of the tubercle bacillus, firstly, a corresponding target sequence is selected, then, a specific primer is designed aiming at the corresponding target sequence, and a specific target DNA fragment is amplified. Therefore, the primer design of the DNA sequences at both ends of the gene to be tested is also the gist of the present invention. The invention provides three pairs of primer sequences for detecting tubercle bacillus, which comprise the following steps:
TbIS6110 F:ACGCCTACGT CGCAGGATC
TbIS6110 R:GGGTCCAGAT GGCTTGCTC
Tb rpoB F:CGCTGTCGGG GTTGACACA
Tb rpoB R:GAGCCGATCA GACCGATGTTG
Tb katG F:GGTAAGGACGCGATCACCGG
Tb katG R:CCGTCCTTGG CGGTGTATT
3. the invention also provides three probes designed for detecting the three target sequences, wherein the sequences are
Tb IS6110 probe:FAM-CGCTCCGCTT CCACGATGGC CACCGAGCG-MGB-3'
Tb rpoB probe:VIC-CGACAGGTTG ACCCACAAGC GCCGACTGTCG-MGB-3'
Tb katG probe:NED-CTACGAGATCACCAGCGGCATCGAGGTCGTAG-MGB-3'
Wherein the loop portion is a sequence that is fully complementary to the target sequence.
It will be clear to one of ordinary skill in the art how to select the appropriate vector/transcriptional regulatory element (e.g., promoter, enhancer, etc.) and selectable marker gene.
4. Provides a host cell genetically engineered with polynucleotides containing mycobacterium tuberculosis IS6110, rpoB and katG, respectively. In the present invention, the tubercle bacillus IS6110, rpoB and katG polynucleotides or the recombinant vector containing the polynucleotides can be transformed or transduced into host cells to constitute genetically engineered host cells containing the polynucleotides or the recombinant vector. The term "host cell" refers to prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells such as salmonella typhimurium; fungal cells such as yeast; a plant cell; insect cells such as Drosophila S2 or Sf 9; animal cells such as CHO, COS or Bowes melanoma cells.
5. Methods for producing microcolony-molecular beacon detection kits are provided. Transformation of a host cell with a DNA sequence according to the invention or a recombinant vector containing said DNA sequence may be carried out by conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl2Methods, the steps used are well known in the art. Alternatively, MgCl is used2. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, or conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.
6. The invention also relates to the application of the detection kit in diagnosis, prevention and treatment of tuberculosis, and can also be used for epidemiological investigation.
Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure of the technology herein.
Description of the drawings
The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.
FIG. 1 shows the microcolony morphology of Mycobacterium tuberculosis of the present invention.
FIG. 2 IS a gel electrophoresis image of the amplified IS6110 and rpoB fragments of the Mycobacterium tuberculosis.
And annotating: m: marker (700bp, 600bp, 500bp, 400bp, 300bp, 200bp, 100bp), IS6110 for 1-3 tracks, rpoB for 4-6 tracks, 1 and 4 tracks: ddH2O; lanes 2 and 5: wild strain H37 Ra; lanes 3 and 6: mutant strain 1163.
FIG. 3 IS a gel electrophoresis image of the amplified Mycobacterium tuberculosis IS6110 and katG fragments of the present invention.
And annotating: the 4 tubercle bacillus IS6110 genes have obvious amplification bands at the positions of less than 100bp, the sizes of the bands are 92bp, and 13 non-tubercle bacillus have no amplification bands; in the katG gene electrophoresis, only the tubercle bacillus wild strains H37Ra and 1085 have obvious amplification bands of 149bp between 100 and 200bp, and the other strains have no amplification bands. The specificity of the two pairs of primers is good, and the katG-ARMS-P1 primer can detect whether the katG315 site of the tubercle bacillus is mutated.
FIG. 4A IS6110 gene specificity test results (IS6110 gene amplification curve).
And annotating: 17 strains only have 4 tubercle bacillus IS6110 genes with strong amplification signals, CT value IS less than 30, and 13 non-tubercle bacillus strains only have weak non-specific amplification, CT value IS more than 30.
FIG. 4B IS6110 gene specificity experimental results (statistical results for tubercle bacillus group and non-tubercle bacillus group).
FIG. 5A rpoB gene specificity test results (rpoB gene amplification curves).
And annotating: wild strain H37Ra was used as Wt group, 13 non-tubercle bacillus and rpoB drug-resistant gene mutant 1163 were used as NTM/Mut group, and only wild strain H37Ra had a significant amplification curve of rpoB gene.
FIG. 5B shows experimental results of rpoB gene specificity (statistical results of a tubercle bacillus wild group, tubercle bacillus resistant mutant bacteria and non-tubercle bacillus group).
FIG. 6A katG gene specificity test results (katG gene amplification curve).
And annotating: 17 strains only have 2 strains of tubercle bacillus wild strains, the katG gene has strong amplification signals, and the CT value is less than 35; and CT values of 2 mycobacterium tuberculosis drug-resistant strains and 13 nontuberculous bacillus strains are more than 35, and the strains are judged to be non-specific amplification.
FIG. 6B katG gene specificity experimental results (statistics of tubercle bacillus wild group and tubercle bacillus resistant mutant bacteria and nontubercle bacillus group).
Fifth, detailed description of the invention
Implementation case
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as sanlbrok et al: the conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.
Example 1 Microcolony culture method
The Mycobacterium tuberculosis standard strain H37Ra was used as a study object, and the standard turbidimetry of Mycoplasma is used as a reference to prepare 0.5McF (approximate bacterial concentration of 1.5X 10)8cfu/mL), 1.0McF (approximate bacterial concentration 3X 10)8cfu/mL), 2.0McF (approximate bacterial concentration 6X 10)8cfu/mL), observing the growth state of microcolonies after bacteria liquid with different concentrations are cultured on a filter membrane in a Middle brook7H10 solid culture medium containing penicillin for 14H and 24H, and searching the optimal conditions for microcolonies culture, wherein the results are shown in a figure 1.
Example 2: preparation of standards
1. By utilizing the genetic engineering technology, the DNA fragments with the sizes of IS6110(92bp) and rpoB (163bp) katG (149bp) are cloned on a PMD-19T vector, and the construction of a standard recombinant plasmid IS confirmed through PCR and sequencing identification.
2. Through gradient dilution of plasmid concentration, PCR reaction and 3% agarose gel electrophoresis as sensitivity test, the lower limit of the detection of tubercle bacillus by common PCR is confirmed to be 100copies/ml。
3. The diluted recombinant plasmids with different concentrations are used for real-time PCR reaction, a positive standard substance is constructed, a standard curve is formulated, and a foundation is laid for detecting the mycobacterium tuberculosis by a molecular beacon fluorescence quantitative PCR technology. Example 3: performing nucleic acid amplification by fluorescent quantitative PCR (molecular beacon) and detecting DNA of a sample by hybridization:
the method for detecting the presence or absence of mycobacterium tuberculosis in a biological sample according to the present invention comprises the steps of:
a) collecting a biological sample, carrying out microcolonic test b) preparing a DNA template c) carrying out fluorescent quantitative PCR amplification treatment on the DNA template obtained in the step b, wherein the primers and the probes are respectively as follows:
TbIS6110 F:ACGCCTACGT CGCAGGATC
TbIS6110 R:GGGTCCAGAT GGCTTGCTC
Tb rpoB F:CGCTGTCGGG GTTGACACA
Tb rpoB R:GAGCCGATCA GACCGATGTTG
Tb katG F:GGTAAGGACGCGATCACCGG
Tb katG R:CCGTCCTTGG CGGTGTATT
Tb IS6110 probe:FAM-CGCTCCGCTT CCACGATGGC CACCGAGCG-MGB-3'
Tb rpoB probe:VIC-CGACAGGTTG ACCCACAAGC GCCGACTGTCG-MGB-3'
Tb katG probe:NED-CTACGAGATCACCAGCGGCATCGAGGTCGTAG-MGB-3'
example 4 ARMS primer specificity verification
The wild strain H37Ra, the drug-resistant gene mutant strain and IS6110, rpoB and katG genes of the reference strain are simultaneously amplified by PCR, and the amplification products are subjected to 2% agarose gel electrophoresis at 140V for 30 min. IS6110 IS used as a specific insertion sequence of tubercle bacillus, the wild strain H37Ra and the drug-resistant gene mutant strain both have amplification products, rpoB and katG genes only have specific amplification products of the wild strain H37Ra, and the mutant strain has no amplification products, so that ARMS primer specificity IS judged according to agarose gel electrophoresis bands. According to the electrophoresis results (FIG. 2, FIG. 3), the ARMS primer specificity IS judged, the IS6110 gene product size IS 92bp, the rpoB gene product size IS 163bp, and the katG gene product size IS 149 bp.
Example 5 application study of MC-ARMS-mMB triple technology drug-resistant tubercle bacillus diagnostic kit
1. Verification of sensitivity of MC-ARMS-mMB triple technology application
(1) An IS6110 standard curve IS prepared. An IS6110 gene plasmid standard product IS constructed, the plasmid vector IS recombinant escherichia coli PUC-57, and the plasmid standard product IS constructed by Shanghai invitrogen company. IS6110 plasmid standard was diluted 10-fold with TE buffer to give final concentrations of 109-100And (3) performing fluorescence quantitative PCR reaction to draw a standard curve, wherein the loading amount of the plasmid standard substance is 1 mu L.
(2) The tubercle bacillus standard strain H37Ra and the multi-drug resistant strain are taken as two groups of target strains, and the rest 13 non-tubercle bacillus strains are taken as interfering strains. With the interfering strain 1.0McF suspension as background, the target strains were added to the suspension respectively so that the concentrations of the two groups of target strains were 1 × 10 respectively6cfu/mL，1×105cfu/mL，1×104cfu/mL，1×103cfu/mL，1×102cfu/mL，1×101cfu/mL，1×100Seven concentration gradients cfu/mL, extracting DNA from seven concentration bacterial suspensions of the two groups of bacterial strains by a boiling method before and after the microcolony culture respectively to perform fluorescent quantitative PCR, and analyzing an amplification curve and a CT value. The repeated times of the experiment are n-5, and the test result is analyzed by single-factor variance (when p is<0.05 considered that the difference was significant), the sensitivity of the system before and after the culture was compared.
2. Verification of specificity of MC-ARMS-mMB triple technology application:
(1) the following ATCC standard strains were purchased: the mycobacteria include Mycobacterium tuberculosis (H37Ra), Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium vaccae, Mycobacterium subspini and Mycobacterium avium; the nonmycobacteria include Streptococcus pneumoniae, Streptococcus pyogenes, Staphylococcus aureus, Pseudomonas diphtheria, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus epidermidis, Escherichia coli, and the like. The microcolony-molecular beacon method is adopted for detection. The bacteria are taken to prepare a bacterium liquid with certain turbidity, and then the bacterium liquid is diluted by different times.
(2) Taking a sterile glass tube, adding 2mL ddH into each glass tube2O, respectively picking 1-2 bacterial colonies by using sterile inoculating loops and dissolving in ddH2In O, after screwing the glass plug, shaking the glass plug on a vortex oscillator uniformly to prepare the glass plugmu.L of each suspension of 1.0McF was inoculated onto the surface of a filter membrane laid on a medium of Middle brook7H10 containing penicillin, and the membrane was spread evenly, and 2 bacterial culture tubes, i.e., 4 filter membranes, were inoculated to each strain and cultured in an incubator at 37 ℃ for 14 hours. And after the microcolonies are cultured for 14 hours, taking out the culture tubes from the thermostat, and performing transparent treatment on two filter membranes in one culture tube of each strain for observing the microcolonies. Two filters in another culture tube of each strain were taken out with sterile forceps, placed in a conical shape in a 1.5mL Eppendorf tube, and 600. mu.L of ddH was added thereto2And O, extracting DNA on the filter membrane by a boiling method to perform fluorescent quantitative PCR reaction. The optimized experimental conditions of each molecular beacon comprise that the working concentration of magnesium ions is 2.5mM-3.5mM, the annealing temperature is 52-60 ℃, and the concentrations of upstream and downstream primers and probes are selected to be 0.2-0.4 mu M. As can be seen from FIGS. 4A, 4B, 5A, 5B, 6A and 6B, the MC-ARMS-mMB triple technology can correctly detect the Mycobacterium tuberculosis wild strain H37Ra, non-Mycobacterium tuberculosis and drug-resistant Mycobacterium tuberculosis mutant (rifampicin and isoniazid) with a specificity of 100%.
Attached: DESCRIPTION OF THE SEQUENCES
1, information of SEQ ID NO: 1:
(1) sequence name of human Mycobacterium tuberculosis IS6110 gene sequence
(2) Sequence characteristics:
(a) length of 92bp
(b) Type nucleic acid
(c) Catenation and amphipathy
(d) Topological structure-linearity
(3) Molecular type cDNA
(4) Description of the sequence SEQ ID NO 1
1 ACGCCTACGT CGCAGGATCC TGGGCTGGCG GGTCGCTTCC ACGATGGCCA CCTCCATGGT
61 CCTCGACGCG ATCGAGCAAG CCATCTGGAC CC
Information of SEQ ID NO: 2:
(1) sequence name human Mycobacterium tuberculosis rpoB Gene sequence
(2) Sequence characteristics:
(a) length of 163bp
(b) Type nucleic acid
(c) Catenation and amphipathy
(d) Topological structure-linearity
(3) Molecular type cDNA
(4) Sequence description SEQ ID NO 2
1 CGCTGTCGGG GTTGACCCAC AAGCGCCGAC TGTCGGCGCT GGGGCCCGGC GGTCTGTCAC
61 GTGAGCGTGC CGGGCTGGAG GTCCGCGACG TGCACCCGTC GCACTACGGC CGGATGTGCC
121 CGATCGAAAC CCCTGAGGGG CCCAACATCG GTCTGATCGG CTC
3, information of SEQ ID NO: 3:
(1) sequence name human Mycobacterium tuberculosis katG Gene sequence
(2) Sequence characteristics:
(a) length of 149bp
(b) Type nucleic acid
(c) Catenation and amphipathy
(d) Topological structure-linearity
(3) Molecular type cDNA
(4) Sequence description SEQ ID NO 3
1 GGTAAGGACG CGATCACCAG CGGCATCGAG GTCGTATGGA CGAACACCCC GACGAAATGG
61 GACAACAGTT TCCTCGAGAT CCTGTACGGC TACGAGTGGG AGCTGACGAA GAGCCCTGCT
121 GGCGCTTGGC AATACACCGC CAAGGACGG
Information of SEQ ID NO: 4:
(1) sequence characteristics:
(a) length of 19bp
(b) Type nucleic acid
(c) Single chain of
(d) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Sequence description SEQ ID NO 4
TbIS6110 F:ACGCCTACGT CGCAGGATC
5, information of SEQ ID NO:
(1) sequence characteristics:
(a) length of 19bp
(b) Type nucleic acid
(c) Single chain of
(d) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Sequence description SEQ ID NO 5
TbIS6110 R:GGGTCCAGAT GGCTTGCTC
Information of SEQ ID NO. 6:
(1) sequence characteristics:
(a) the length is 29bp
(b) Type nucleic acid
(c) Single chain of
(d) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Sequence description SEQ ID NO 6
Tb IS6110 probe:FAM-CGCTCCGCTT CCACGATGGC CACCGAGCG-MGB-3'
Information of SEQ ID NO. 7:
(1) sequence characteristics:
(e) length of 19bp
(f) Type nucleic acid
(g) Single chain of
(h) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Sequence description SEQ ID NO 7
Tb rpoB F:CGCTGTCGGG GTTGACACA
Information of SEQ ID NO. 8:
(1) sequence characteristics:
(e) length of 19bp
(f) Type nucleic acid
(g) Single chain of
(h) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Sequence description SEQ ID NO 8
Tb rpoB R:GAGCCGATCA GACCGATGTTG
Information of SEQ ID NO. 9:
(1) sequence characteristics:
(e) length of 31bp
(f) Type nucleic acid
(g) Single chain of
(h) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Sequence description SEQ ID NO 9
Tb rpoB probe:VIC-CGACAGGTTG ACCCACAAGC GCCGACTGTCG-MGB-3'
10, information of SEQ ID NO: 10:
(1) sequence characteristics:
(i) length of 20bp
(j) Type nucleic acid
(k) Single chain of
(l) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Sequence description SEQ ID NO 10
Tb katG F:GGTAAGGACGCGATCACCGG
11, information of SEQ ID NO:
(1) sequence characteristics:
(i) length of 19bp
(j) Type nucleic acid
(k) Single chain of
(l) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Description of the sequence SEQ ID NO 11
Tb katG R:CCGTCCTTGG CGGTGTATT
12, information of SEQ ID NO:
(1) sequence characteristics:
(i) length of 32bp
(j) Type nucleic acid
(k) Single chain of
(l) Topological structure-linearity
(2) Molecular type oligonucleotide
(3) Description of the sequence SEQ ID NO 12
Tb katG probe:
NED-CTACGAGATCACCAGCGGCATCGAGGTCGTAG-MGB-3'。
Sequence listing
<110> Fujian medical university
Preparation method and application of MC-ARMS-mMB triple technology in drug-resistant tubercle bacillus diagnostic kit
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 92
<212> DNA
<213> SEQ ID NO:1
<400> 1
acgcctacgt cgcaggatcc tgggctggcg ggtcgcttcc acgatggcca cctccatggt 60
cctcgacgcg atcgagcaag ccatctggac cc 92
<210> 2
<211> 163
<212> DNA
<213> SEQ ID NO:2
<400> 2
cgctgtcggg gttgacccac aagcgccgac tgtcggcgct ggggcccggc ggtctgtcac 60
gtgagcgtgc cgggctggag gtccgcgacg tgcacccgtc gcactacggc cggatgtgcc 120
cgatcgaaac ccctgagggg cccaacatcg gtctgatcgg ctc 163
<210> 3
<211> 149
<212> DNA
<213> SEQ ID NO:3
<400> 3
ggtaaggacg cgatcaccag cggcatcgag gtcgtatgga cgaacacccc gacgaaatgg 60
gacaacagtt tcctcgagat cctgtacggc tacgagtggg agctgacgaa gagccctgct 120
ggcgcttggc aatacaccgc caaggacgg 149
<210> 4
<211> 19
<212> DNA
<213> SEQ ID NO:4
<400> 4
TbIS6110 F:acgcctacgt cgcaggatc 19
<210> 5
<211> 19
<212> DNA
<213> SEQ ID NO:5
<400> 5
TbIS6110 R:gggtccagat ggcttgctc 19
<210> 6
<211> 29
<212> DNA
<213> SEQ ID NO:6
<400> 6
Tb IS6110 probe:cgctccgctt ccacgatggc caccgagcg-MGB-3' 29
<210> 7
<211> 19
<212> DNA
<213> SEQ ID NO:7
<400> 7
Tb rpoB F:cgctgtcggg gttgacaca 19
<210> 8
<211> 21
<212> DNA
<213> SEQ ID NO:8
<400> 8
Tb rpoB R:gagccgatca gaccgatgtt g 21
<210> 9
<211> 31
<212> DNA
<213> SEQ ID NO:9
<400> 9
Tb rpoB probe: VIC-cgacaggttg acccacaagc gccgactgtc g-MGB-3' 31
<210> 10
<211> 20
<212> DNA
<213> SEQ ID NO:10
<400> 10
Tb katG F:ggtaaggacg cgatcaccgg 20
<210> 11
<211> 19
<212> DNA
<213> SEQ ID NO:11
<400> 11
Tb katG R:ccgtccttgg cggtgtatt 19
<210> 12
<211> 32
<212> DNA
<213> SEQ ID NO:12
<400> 12
Tb katG probe: NED-ctacgagatc accagcggca tcgaggtcgt ag-MGB-3' 32
Claims (3)
1. A composition of three pairs of primers and three molecular beacons designed for preparing MC-ARMS-mMB triple technology-cultured drug-resistant tubercle bacillus diagnostic kit and PCR amplification of target sequences selected by tubercle bacillus is characterized in that: the three pairs of primers are as follows:
TbIS6110 F: ACGCCTACGT CGCAGGATC TbIS6110 R: GGGTCCAGAT GGCTTGCTC，
Tb rpoB F: CGCTGTCGGG GTTGACACA Tb rpoB R: GAGCCGATCA GACCGATGTTG，
Tb katG F: GGTAAGGACGCGATCACCGG TbkatG R: CCGTCCTTGG CGGTGTATT；
the three molecular beacons are:
Tb IS6110 probe: FAM-CGCTCCGCTT CCACGATGGC CACCGAGCG-MGB-3'
Tb rpoB probe: VIC-CGACAGGTTG ACCCACAAGC GCCGACTGTCG-MGB-3'
Tb katG probe: NED-CTACGAGATCACCAGCGGCATCGAGGTCGTAG-MGB-3'
the sequence is that the loop part and the stem 3' end sequence are complementary with the target sequence in the co-stem structure, and the rest stem is complementary oligonucleotide unrelated to the target sequence.
2. The composition of claim 1, wherein: when the kit is prepared, the experimental optimization conditions of each molecular beacon comprise that the working concentration of magnesium ions is 2.5-3.5 mM, the annealing temperature is 52-60 ℃, and the concentrations of upstream and downstream primers and probes are selected to be 0.2-0.4 mu M.
3. A kit for detecting mycobacterium tuberculosis, comprising: the three pairs of primers and three molecular beacon probes as defined in claim 1, penicillin-containing Middle brook7H10 solid medium, rojen's medium, selective medium, cellulose acetate membrane, positive and negative control solutions, DNA extract, slide glass, 95% ethanol, absolute ethanol: phenol = 1: 10, adding 0.02-0.04ml of nucleic acid amplification mixed solution into a nucleic acid amplification and hybridization reaction tube,
the three pairs of primers and the three molecular beacon probe sequences are respectively as follows:
TbIS6110 F: ACGCCTACGT CGCAGGATC TbIS6110 R: GGGTCCAGAT GGCTTGCTC
Tb IS6110 probe: FAM-CGCTCCGCTT CCACGATGGC CACCGAGCG-MGB-3'
Tb rpoB F: CGCTGTCGGG GTTGACACA Tb rpoB R: GAGCCGATCA GACCGATGTTG
Tb rpoB probe: VIC-CGACAGGTTG ACCCACAAGC GCCGACTGTCG-MGB-3'
Tb katG F: GGTAAGGACGCGATCACCGG TbkatG R: CCGTCCTTGG CGGTGTATT
Tb katG probe: NED-CTACGAGATCACCAGCGGCATCGAGGTCGTAG-MGB-3'
the DNA extract solution is 50mmol/L NaOH, 10mmol/L Tris.HCl with pH8.0, 1% Triton X-100, 1% NP-40, 0.5mmol/L EDTA with pH 8.0.
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