CN104450877A - Method for detecting four tuberculosis rifampicin, isoniazide and fluoroquinolones-resistant genes - Google Patents
Method for detecting four tuberculosis rifampicin, isoniazide and fluoroquinolones-resistant genes Download PDFInfo
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Abstract
The invention relates to a method for detecting four tuberculosis rifampicin, isoniazide and fluoroquinolones-resistant genes. The method comprises the following steps: designing primers capable of covering full lengths of GyrA, lnhA, katG and rpoB genes; merging the four gene primers and forming a primer pool; amplifying amplicons capable of covering full lengths of drug-resistance-related genes; establishing complete an SV-MTB library; and detecting and acquiring drug-resistant sites. By the method, multiple mutation sites of a multiple-drug-resistant strain can be detected at one time and tuberculosis patients can be scientifically, safely and accurately guided to take the medicine.
Description
Technical field
The invention belongs to biological medicine detection field, more specifically, relate to a kind of Rimactazid, fluoroquinolones four Drug Resistance for Tuberculosis gene testers.
Background technology
At present, in diagnosis of tuberculosis medical treatment, tubercule bacillus drug-resistant test or an important technology, but also there are some problems in the detection method of traditional Drug Resistance for Tuberculosis genes involved:
1. the detection method of traditional Drug Resistance for Tuberculosis genes involved can only detect a small amount of specific site simultaneously, and cannot judge its emergent properties.
2. the Resistant strain in the clinical sample of reality is much resistance, and may there is the bacterial strain of several resistance to different pharmaceutical in strains.And, due to the distribution proportion heterogeneity of bacterial strain, traditional resistance loci detection method can detect the mutational site that jump signal is higher, namely the dominant group in bacterial strain, in this case, provide pharmacological agent to have significantly that sb.'s illness took a favorable turn early stage based on traditional technique in measuring, but along with the decline of dominant strain, inferior position strains is known from experience and is slowly converted into dominant strain, and the treatment later stage there will be significantly that sb.'s illness took a turn for the worse.Can not science, safety, instruct tuberculosis patient medication accurately.
Summary of the invention
For above-mentioned defect of the prior art, the invention provides a kind of for Rimactazid, fluoroquinolones four Drug Resistance for Tuberculosis gene testers, the method disposablely can detect multiple mutational sites of Drug-fast case bacterial strain, and can more science, safety, instruct tuberculosis patient medication accurately.
The invention provides a kind of Rimactazid, fluoroquinolones four Drug Resistance for Tuberculosis gene testers, it comprises the following steps:
(1) design covers the primer of gyrA, lnhA, katG, rpoB full length gene;
(2) 4 kinds of gene primers in step (1) are merged formation primer pool;
(3) primer pool in step (2) is utilized to amplify the amplicon that can cover drug resistance related gene total length;
(4) further end reparation carried out to the amplicon described in step (3), add joint, amplified library and quantitatively, set up complete S V-MTB library;
(5) method by once contacting makes SV-MTB library form water in oil structure, then checks order with ionPGM, detects resistance site.
Resistance site described in step (5) is the resistance site that SV-MTB detects different pharmaceutical simultaneously, and described resistance site comprises: the resistances such as GyrA-_94, GyrA-_88, GyrA-_89, GyrA-_90, GyrA-_91, rpoB_516, rpoB_511, rpoB_513, rpoB_522, rpoB_526, rpoB_531, rpoB_533, katG_315, inhA_-15, inhA_-8 are correlated with focus.
Preferably, carry out the detection of resistant strains site and comprise the following steps:
A: by the standard compliant sample DNA of Quality Control, obtains the Ampliseq PCR primer of four genes;
B: by described in the Ampliseq-PCR product described in steps A and step (3) amplicon carry out end reparation, add joint, amplified library and quantitatively, set up complete S V-MTB library;
E:(5) method by once contacting makes SV-MTB library form water in oil structure, then checks order with ion PGM, detects resistance site.
Preferably, the primer that PCR is used in step comprises:
The primer numbers of gyrA is respectively: gyrA_8.1.75513, gyrA_8.1.51886, gyrA_8.1.82296, gyrA_8.2.58628, gyrA_8.2.29245, gyrA_8.2.49325, gyrA_8.2.94533, gyrA_8.2.1239, gyrA_8.3.158404, gyrA_8.3.159760, gyrA_8.3.104094, gyrA_8.3.113730, gyrA_8.4.22908, gyrA_8.4.67530, gyrA_8.4.47561, gyrA_8.4.24855, gyrA_8.5.10674, gyrA_8.5.14302,
The primer numbers of InhA is respectively: InhA_5.1.57946, InhA_5.1.69702, InhA_5.1.55270, InhA_5.1.88153, InhA_5.2.101468, InhA_5.2.77605, InhA_5.2.4266;
The primer numbers of katG is respectively: katG_2.1.99813, katG_2.1.50357, katG_2.1.1237 .3.130345, katG_2.3.100427, katG_2.4.25702, katG_2.4.39974, katG_2.4.116783, katG_2.4.112440;
The primer numbers of rpoB is respectively: rpoB_1.1.113180, rpoB_1.1.148688, rpoB_1.1.81682, rpoB_1.2.113638, rpoB_1.2.37694, rpoB_1.2.19193, rpoB_1.2.142863, rpoB_1.2.128222, rpoB_1.3.65890, rpoB_1.3.48836, rpoB_1.3.50710, rpoB_1.4.72080, rpoB_1.4.86222, rpoB_1.4.9838, rpoB_1.4.125079, rpoB_1.4.112057, rpoB_1.5.99561, rpoB_1.5.102258, rpoB_1.5.104523, rpoB_1.5.30503, rpoB_1.6.138705, rpoB_1.6.142676, rpoB_1.6.123078, rpoB_1.6.43945, rpoB_1.6.91215.
Preferably, the step setting up complete S V-MTB library comprises the following steps:
A: increase in target area: primer is divided into two parts, and same sample divides two pipes to increase separately;
B: part removes primer: the PCR primer of primer pool different for same sample in step a is merged into a pipe, carries out removal primer;
C: add joint: the library of not increased in reaction system, then the library purifying that will do not increase;
D: amplification is carried out with quantitative to the library obtained in c.
In the present invention, the sequence of gyrA is: GyrA (7295 ... 9847)
atgacagacacgacgttgccgcctgacgactcgctcgaccggatcgaaccggttgacatcgagcaggagatgcagcgcagctacatcgactatgcgatgagcgtgatcgtcggccgcgcgctgccggaggtgcgcgacgggctcaagcccgtgcatcgccgggtgctctatgcaatgttcgattccggcttccgcccggaccgcagccacgccaagtcggcccggtcggttgccgagaccatgggcaactaccacccgcacggcgacgcgtcgatctacgacagcctggtgcgcatggcccagccctggtcgctgcgctacccgctggtggacggccagggcaacttcggctcgccaggcaatgacccaccggcggcgatgaggtacaccgaagcccggctgaccccgttggcgatggagatgctgagggaaatcgacgaggagacagtcgatttcatccctaactacgacggccgggtgcaagagccgacggtgctacccagccggttccccaacctgctggccaacgggtcaggcggcatcgcggtcggcatggcaaccaatatcccgccgcacaacctgcgtgagctggccgacgcggtgttctgggcgctggagaatcacgacgccgacgaagaggagaccctggccgcggtcatggggcgggttaaaggcccggacttcccgaccgccggactgatcgtcggatcccagggcaccgctgatgcctacaaaactggccgcggctccattcgaatgcgcggagttgttgaggtagaagaggattcccgcggtcgtacctcgctggtgatcaccgagttgccgtatcaggtcaaccacgacaacttcatcacttcgatcgccgaacaggtccgagacggcaagctggccggcatttccaacattgaggaccagtctagcgatcgggtcggtttacgcatcgtcatcgagatcaagcgcgatgcggtggccaaggtggtgatcaataacctttacaagcacacccagctgcagaccagctttggcgccaacatgctagcgatcgtcgacggggtgccgcgcacgctgcggctggaccagctgatccgctattacgttgaccaccaactcgacgtcattgtgcggcgcaccacctaccggctgcgcaaggcaaacgagcgagcccacattctgcgcggcctggttaaagcgctcgacgcgctggacgaggtcattgcactgatccgggcgtcggagaccgtcgatatcgcccgggccggactgatcgagctgctcgacatcgacgagatccaggcccaggcaatcctggacatgcagttgcggcgcctggccgcactggaacgccagcgcatcatcgacgacctggccaaaatcgaggccgagatcgccgatctggaagacatcctggcaaaacccgagcggcagcgtgggatcgtgcgcgacgaactcgccgaaatcgtggacaggcacggcgacgaccggcgtacccggatcatcgcggccgacggagacgtcagcgacgaggatttgatcgcccgcgaggacgtcgttgtcactatcaccgaaacgggatacgccaagcgcaccaagaccgatctgtatcgcagccagaaacgcggcggcaagggcgtgcagggtgcggggttgaagcaggacgacatcgtcgcgcacttcttcgtgtgctccacccacgatttgatcctgttcttcaccacccagggacgggtttatcgggccaaggcctacgacttgcccgaggcctcccggacggcgcgcgggcagcacgtggccaacctgttagccttccagcccgaggaacgcatcgcccaggtcatccagattcgcggctacaccgacgccccgtacctggtgctggccactcgcaacgggctggtgaaaaagtccaagctgaccgacttcgactccaatcgctcgggcggaatcgtggcggtcaacctgcgcgacaacgacgagctggtcggtgcggtgctgtgttcggccggcgacgacctgctgctggtctcggccaacgggcagtccatcaggttctcggcgaccgacgaggcgctgcggccaatgggtcgtgccacctcgggtgtgcagggcatgcggttcaatatcgacgaccggctgctgtcgctgaacgtcgtgcgtgaaggcacctatctgctggtggcgacgtcagggggctatgcgaaacgtaccgcgatcgaggaatacccggtacagggccgcggcggtaaaggtgtgctgacggtcatgtacgaccgccggcgcggcaggttggttggggcgttgattgtcgacgacgacagcgagctgtatgccgtcacttccggcggtggcgtgatccgcaccgcggcacgccaggttcgcaaggcgggacggcagaccaagggtgttcggttgatgaatctgggcgagggcgacacactgttggccatcgcgcgcaacgccgaagaaagtggcgacgataatgccgtggacgccaacggcgcagaccagacgggcaattaa
Shown in the primer information table specific as follows of gyrA:
The sequence of lnhA involved in the present invention is: InhA (1674134 ... 1675154)
atgacaggactgctggacggcaaacggattctggttagcggaatcatcaccgactcgtcgatcgcgtttcacatcgcacgggtagcccaggagcagggcgcccagctggtgctcaccgggttcgaccggctgcggctgattcagcgcatcaccgaccggctgccggcaaaggccccgctgctcgaactcgacgtgcaaaacgaggagcacctggccagcttggccggccgggtgaccgaggcgatcggggcgggcaacaagctcgacggggtggtgcattcgattgggttcatgccgcagaccgggatgggcatcaacccgttcttcgacgcgccctacgcggatgtgtccaagggcatccacatctcggcgtattcgtatgcttcgatggccaaggcgctgctgccgatcatgaaccccggaggttccatcgtcggcatggacttcgacccgagccgggcgatgccggcctacaactggatgacggtcgccaagagcgcgttggagtcggtcaacaggttcgtggcgcgcgaggccggcaagtacggtgtgcgttcgaatctcgttgccgcaggccctatccggacgctggcgatgagtgcgatcgtcggcggtgcgctcggcgaggaggccggcgcccagatccagctgctcgaggagggctgggatcagcgcgctccgatcggctggaacatgaaggatgcgacgccggtcgccaagacggtgtgcgcgctgctgtctgactggctgccggcgaccacgggtgacatcatctacgccgacggcggcgcgcacacccaattgctctag
The primer information of lnhA is as shown in following table:
The sequence of katG involved in the present invention is: katG (2153864 ... 2156128)
gtgcccgagcaacacccacccattacagaaaccaccaccggagccgctagcaacggctgtcccgtcgtgggtcatatgaaataccccgtcgagggcggcggaaaccaggactggtggcccaaccggctcaatctgaaggtactgcaccaaaacccggccgtcgctgacccgatgggtgcggcgttcgactatgccgcggaggtcgcgaccatcgacgttgacgccctgacgcgggacatcgaggaagtgatgaccacctcgcagccgtggtggcccgccgactacggccactacgggccgctgtttatccggatggcgtggcacgctgccggcacctaccgcatccacgacggccgcggcggcgccgggggcggcatgcagcggttcgcgccgcttaacagctggcccgacaacgccagcttggacaaggcgcgccggctgctgtggccggtcaagaagaagtacggcaagaagctctcatgggcggacctgattgttttcgccggcaactgcgcgctggaatcgatgggcttcaagacgttcgggttcggcttcggccgggtcgaccagtgggagcccgatgaggtctattggggcaaggaagccacctggctcggcgatgagcgttacagcggtaagcgggatctggagaacccgctggccgcggtgcagatggggctgatctacgtgaacccggaggggccgaacggcaacccggaccccatggccgcggcggtcgacattcgcgagacgtttcggcgcatggccatgaacgacgtcgaaacagcggcgctgatcgtcggcggtcacactttcggtaagacccatggcgccggcccggccgatctggtcggccccgaacccgaggctgctccgctggagcagatgggcttgggctggaagagctcgtatggcaccggaaccggtaaggacgcgatcaccagcggcatcgaggtcgtatggacgaacaccccgacgaaatgggacaacagtttcctcgagatcctgtacggctacgagtgggagctgacgaagagccctgctggcgcttggcaatacaccgccaaggacggcgccggtgccggcaccatcccggacccgttcggcgggccagggcgctccccgacgatgctggccactgacctctcgctgcgggtggatccgatctatgagcggatcacgcgtcgctggctggaacaccccgaggaattggccgacgagttcgccaaggcctggtacaagctgatccaccgagacatgggtcccgttgcgagataccttgggccgctggtccccaagcagaccctgctgtggcaggatccggtccctgcggtcagccacgacctcgtcggcgaagccgagattgccagccttaagagccagatccgggcatcgggattgactgtctcacagctagtttcgaccgcatgggcggcggcgtcgtcgttccgtggtagcgacaagcgcggcggcgccaacggtggtcgcatccgcctgcagccacaagtcgggtgggaggtcaacgaccccgacggggatctgcgcaaggtcattcgcaccctggaagagatccaggagtcattcaactccgcggcgccggggaacatcaaagtgtccttcgccgacctcgtcgtgctcggtggctgtgccgccatagagaaagcagcaaaggcggctggccacaacatcacggtgcccttcaccccgggccgcacggatgcgtcgcaggaacaaaccgacgtggaatcctttgccgtgctggagcccaaggcagatggcttccgaaactacctcggaaagggcaacccgttgccggccgagtacatgctgctcgacaaggcgaacctgcttacgctcagtgcccctgagatgacggtgctggtaggtggcctgcgcgtcctcggcgcaaactacaagcgcttaccgctgggcgtgttcaccgaggcctccgagtcactgaccaacgacttcttcgtgaacctgctcgacatgggtatcacctgggagccctcgccagcagatgacgggacctaccagggcaaggatggcagtggcaaggtgaagtggaccggcagccgcgtggacctggtcttcgggtccaactcggagttgcgggcgcttgtcgaggtctatggcgccgatgacgcgcagccgaagttcgtgcaggacttcgtcgctgcctgggacaaggtgatgaacctcgacaggttcgacgtgcgctga
The primer information of katG is as shown in following table:
RpoB sequence involved in the present invention is:
ttggcagattcccgccagagcaaaacagccgctagtcctagtccgagtcgcccgcaaagttcctcgaataactccgtacccggagcgccaaaccgggtctccttcgctaagctgcgcgaaccacttgaggttccgggactccttgacgtccagaccgattcgttcgagtggctgatcggttcgccgcgctggcgcgaatccgccgccgagcggggtgatgtcaacccagtgggtggcctggaagaggtgctctacgagctgtctccgatcgaggacttctccgggtcgatgtcgttgtcgttctctgaccctcgtttcgacgatgtcaaggcacccgtcgacgagtgcaaagacaaggacatgacgtacgcggctccactgttcgtcaccgccgagttcatcaacaacaacaccggtgagatcaagagtcagacggtgttcatgggtgacttcccgatgatgaccgagaagggcacgttcatcatcaacgggaccgagcgtgtggtggtcagccagctggtgcggtcgcccggggtgtacttcgacgagaccattgacaagtccaccgacaagacgctgcacagcgtcaaggtgatcccgagccgcggcgcgtggctcgagtttgacgtcgacaagcgcgacaccgtcggcgtgcgcatcgaccgcaaacgccggcaaccggtcaccgtgctgctcaaggcgctgggctggaccagcgagcagattgtcgagcggttcgggttctccgagatcatgcgatcgacgctggagaaggacaacaccgtcggcaccgacgaggcgctgttggacatctaccgcaagctgcgtccgggcgagcccccgaccaaagagtcagcgcagacgctgttggaaaacttgttcttcaaggagaagcgctacgacctggcccgcgtcggtcgctataaggtcaacaagaagctcgggctgcatgtcggcgagcccatcacgtcgtcgacgctgaccgaagaagacgtcgtggccaccatcgaatatctggtccgcttgcacgagggtcagaccacgatgaccgttccgggcggcgtcgaggtgccggtggaaaccgacgacatcgaccacttcggcaaccgccgcctgcgtacggtcggcgagctgatccaaaaccagatccgggtcggcatgtcgcggatggagcgggtggtccgggagcggatgaccacccaggacgtggaggcgatcacaccgcagacgttgatcaacatccggccggtggtcgccgcgatcaaggagttcttcggcaccagccagctgagccaattcatggaccagaacaacccgctgtcggggttgacccacaagcgccgactgtcggcgctggggcccggcggtctgtcacgtgagcgtgccgggctggaggtccgcgacgtgcacccgtcgcactacggccggatgtgcccgatcgaaacccctgaggggcccaacatcggtctgatcggctcgctgtcggtgtacgcgcgggtcaacccgttcgggttcatcgaaacgccgtaccgcaaggtggtcgacggcgtggttagcgacgagatcgtgtacctgaccgccgacgaggaggaccgccacgtggtggcacaggccaattcgccgatcgatgcggacggtcgcttcgtcgagccgcgcgtgctggtccgccgcaaggcgggcgaggtggagtacgtgccctcgtctgaggtggactacatggacgtctcgccccgccagatg
The primer information of rpoB is as shown in following table:
The invention solves deficiency of the prior art, the beneficial effect reached:
1. the multiple drug resistant gene of one-time detection.The present invention one-time detection can go out multiple site, comprises the resistance to snack made with traditional Chinese medicines of difference of same medicine and the resistance site of different pharmaceutical, and can judge its emergent properties, can detect multiple mutational sites of Drug-fast case bacterial strain.
2. detect complete drug resistant gene, brand-new resistant mutational site can be found, the present invention covers the total length of four genes such as GyrA, lnhA, katG, rpoB, the sudden change of all sites within the scope of this gene can be detected, the resistance site of open report and brand-new mutational site can be detected, be conducive to the discovery in brand-new resistance site.
3. the present invention can detect that the high frequency in bacterial strain suddenlys change and low frequency sudden change (being low to moderate one of percentage) simultaneously, and advantage Resistant strain mutational site and inferior position Resistant strain mutational site, can more scientific, safer, instruct in conjunction with patient's medication more accurately.
4. the present invention is optimized experimental technique, and to the MPure magnetic bead consumption in DNA purge process in experimentation of the present invention, in library construction process, the usage quantity of template and the amount etc. in higher level library are optimized, and the detected result made reaches optimum.
Accompanying drawing explanation
Below in conjunction with the drawings and specific embodiments, the present invention is further detailed explanation.
Fig. 1 multidigit point detects result schematic diagram.
The resistance site result schematic diagram that Fig. 2 is new.
The low frequency sudden change schematic diagram in the resistance site that Fig. 3 SV-MIB detects.
Detected result after optimizing in Fig. 4 DNA purge process.
Embodiment
In order to make those skilled in the art person understand the present invention program better, and enable above-mentioned purpose of the present invention, feature and advantage become apparent more, below in conjunction with embodiment and embodiment accompanying drawing, the present invention is further detailed explanation.
The following steps are comprised for for Rimactazid, fluoroquinolones four Drug Resistance for Tuberculosis gene testers in the embodiment of the present invention 1:
One, DNA quality inspection
1) with Qubit, DNA concentration is detected.
2) whether clip size of carrying out the DNA agilent that concentration is too low detects DNA and degrades.
3) measure 260/280 value of DNA, require to be greater than 1.6,260/280 value of DNA is in the present embodiment greater than 1.6; If when actual experiment, DNA not up to standard measures 260/280 value again after 0.5X Mpure magnetic beads for purifying.
Two, library is built
1) object region amplification
A. primer is divided into pool1, pool2, same sample divides two pipes to increase separately; Increase each group component (totally 10 μ l); Genome consumption is 0.1ng/pool.Reagent is as shown in table 1.
Table 1
| Reagent | Consumption (μ l) |
| 2 primer pool | 5 |
| HIFI | 2 |
| GDNA | x |
| Water | 3-x |
B.PCR condition is as shown in table 2:
Table 2
The PCR primer increased above directly can be carried out next step operation, also can be placed in-20 DEG C of preservations.
2) part removes primer
The PCR primer of the different primer pool of same sample in previous step is merged into a pipe, adds the fupa of 2 μ l, inhale with 10 μ l pipettors (range is adjusted to 10 μ l) and play mixing 10 times; Mixture is put into ABI96 hole PCR amplification instrument to react, reaction conditions is as following table 3 again.
Table 3
| Temperature | Time |
| 50℃ | 10m |
| 55℃ | 10m |
| 60℃ | 20m |
| 10℃ | 1h |
Above enzyme cut after product need to carry out next step operation at once.
3) Barcode joint is connected
A. reaction system is as table 4(totally 30 μ l):
Table 4
Reaction conditions: as table 5.
Table 5
| Temperature | Time |
| 22℃ | 30m |
| 72℃ | 10m |
| 10℃ | 1h |
B. the library (1.5 x Agencourt AMPure XP Reagent) that rear purifying does not increase has been reacted
4) amplified library is with quantitative
A：Q-PCR
Reaction system (total amount 20 μ l) is as shown in table 6.
Table 6
| Reagent | Consumption |
| 2 SYBR MIX | 10μl |
| PRIMER(ADAPTER | 1μl |
| TEMPLATE | 5μl |
| H2O | 4μl |
Reaction system in table 6 is added library standard substance Criterion curve, the concentration of library standard substance is: 30pmol, 3 pmol, 0.3 pmol, 0.03 pmol, 0.003 pmol.
Reaction conditions is as shown in table 7.
Table 7
After obtaining Ct value, Ct value and lgC(log concentration with standard substance) do typical curve, draw the concentration of various sample according to typical curve, be diluted to 16pmol.
B：Equalizer
A. by 2) in b in (reaction complete after the purifying library of not increasing) this step, after washing twice with 70% ethanol, beads is resuspended with 50 μ l accidental platinumPCR Super Mix High Fidelity, adds the Equalizer Primers of 2 μ l;
B. suction is played mixing and is placed on standing 2min on magnetic frame, and collect supernatant and increase in PCR pipe, amplification condition is as shown in table 8.
Table 8
| Stage | Temperature (DEG C) | Time |
| hold | 98 | 2m |
| 7cycle | 98 | 15s |
| 60 | 1m | |
| hold | 10 | 1h |
C. amplification after library add 25 μ l(0.5X) Agencourt AMPure XP Reagent, mix rear leave standstill 5 minutes.
D. get the Agencourt AMPure XP Reagent that supernatant liquor joins 60 μ l, mix latter standing 5 minutes.
E. leaving standstill is placed on magnetic frame, and after liquid clarification, removing supernatant, washes 2 times with 150 μ l70% ethanol.
F. leave standstill 2min after the ethanol volatilization completely of 70%, add the resuspended beads of LTE or ddH2O of 30 μ l, leave standstill 5 minutes.
G. be placed on magnetic frame by product, supernatant is the library after the amplification obtained.
H. get 2 μ l increase library dilute with 198 μ l after reagent(buffer:reagent=200:1) mix after, measure library concentration with Qubit.
Three. library emulsification PCR and enrichment
1, open Ion One Touch 2Instrument, click screen upper " Open Lid ".
2, the collection tube in test kit is put into corresponding centrifugal tube seat, by collection tube crane span structure in corresponding groove, close centrifugal lid.
3, the handle above Ion One Touch 2Instrument heating cover is pulled open, open heat lid.
4, amplification plate is installed and is placed into corresponding position, handle is withdrawn into original position, heat of closing lid.
5, be stuck in by the hose of ligation amplification plate in the respective grooves of Ion One Touch 2Instrument, slowly vertically inserted in centrifugal lid by the syringe needle of the other end, do not insert sideling, if can not add centrifugal lid, shutdown is started shooting again.
6, in Ion One Touch 2Instrument oil pipe, add the Ion PGM OT2 OIL provided in test kit, be approximately added to 1/2nd places.
7, in Ion One Touch 2Instrument Recovery pipe, the Ion PGM Recovery Solution provided in test kit is added.
8, the waste liquid in waste liquid tank is discharged.
9, according to configuration amplification liquid in table 9.
Table 9
| Component | Volume (μ l) |
| Ion PGM Reagent | 500 |
| Ion PGM ReagentB | 300 |
| Ion PGM Enzyme Mix | 50 |
| Ion PGM Ion Sphere Particles | 100 |
| The library of having diluted | 25 |
| H2O | 25 |
| Cumulative volume | 1000 |
10. will increase liquid vortex 5s, gently throw away core barrel, be thrown in centrifuge tube by tube wall solution.
1000 μ l amplification liquid are slowly injected in Ion PGM Plus Reaction Filter by 11., are more slowly injected in Ion PGM Plus Reaction Filter by 750 μ l reaction oils, do not produce bubble.
12. repeat previous step, are slowly injected into by 750 μ l reaction oils in Ion PGM Plus Reaction Filter, do not produce bubble.
Ion PGM Plus Reaction Filter turns by 13., and be inserted in the reactive tank of Ion One Touch 2Instrument, run Ion One Touch 2Instrument, select corresponding test kit, and step completes before guaranteeing, in oil pipe, reaction solution is sufficient.
14. reaction process about 5 hours 19 minutes, after having run, click the next of screen, and continue centrifugal 10 minutes.
15. centrifugal complete after, removed by collection tube, remove supernatant, often pipe remains 50 μ l, by remaining liquid rifle head piping and druming mixing, is moved on in new 1.5ml centrifuge tube by the liquid rotating in two pipes.
Solution needed for 16. configuration ES, solution component is as shown in table 10.
Table 10
| Component | Volume (μ l) |
| 1M NaOH | 40 |
| Tween20 | 280 |
| Cumulative volume | 320 |
17. configuration Myone Beads, after vibration 30s, draw in 50 μ l to 1.5ml centrifuge tubes, to be placed on magnetic frame after 2min, abandon supernatant, add 1ml Ion One Touch Wash Solution, vibration 5s, of short duration centrifugal, centrifuge tube is placed on 2min on magnetic frame, abandon supernatant, add 130 μ l Ion PGM Myone Beads Capture Solution, vortex mixes.
18. take out eight platoons, are placed on Ion One Touch 2 ES eight platoon groove, and are placed on the high order end of eight platoon grooves, according to adding different solution in table 11.
Table 11
| Well sequence number | Reagent to dispense in well |
| 1 | ISP sample (100μl) |
| 2 | 130μl of my one beads |
| 3 | 300μl of Ion One Touch Wash Solution |
| 4 | 300μl of Ion One Touch Wash Solution |
| 5 | 300μl of Ion One Touch Wash Solution |
| 6 | empty |
| 7 | 300μl of freshly-prepared Melt-Off Solution |
| 8 | empty |
19. rifle heads are positioned on the end of rifle head arm, and PCR is placed in the aperture on the left side, run about 35 minutes of Ion One Touch 2 ES, after operation, PCR tubule is taken out, adds 3 μ l control ISP, piping and druming mixing, centrifugal 5 minutes, remove supernatant, remain 3 μ l.
20. add 3 μ l Ion PGM Sequencing Primer, carry out PCR according to the reaction conditions in table 12.
Table 12
| Temperature (DEG C) | Time |
| 95 | 2m |
| 37 | 2m |
| 25 | Keep |
After 21.PCR terminates, add 11 μ l Ion PGM Sequencing 200V2 poiymerase, preserve under putting into 4 DEG C of conditions, prepare to carry out computer experiment.
Four, upper machine order-checking.
Through order-checking, in the method for the invention can the multiple drug resistant gene of one-time detection, its result is as shown in table 13.
Table 13 one-time detection multiple drug resistant gene result table
The method that the present invention relates to one-time detection can go out multiple site, comprises the resistance to snack made with traditional Chinese medicines of difference of unified medicine and the resistance site of different pharmaceutical, and can judge its emergent properties, can detect multiple mutational sites of Drug-fast case bacterial strain.Its result is as shown in table 14.
The resistance site detected result table (* wherein in form: be common resistance site, #: unknown mutation site, can find brand-new resistance site) that table 14 is new
In the present embodiment, concrete, detect 300 bacterial strain samples, the resistance site of wherein traditional method checking place is detected by method of the present invention 100%, in addition, also detects more resistance site (result as shown in Figure 1).
As shown in Figure 2, detect complete drug resistant gene, brand-new resistant mutational site can be found, present invention covers the total length of four genes such as GyrA, lnhA, katG, rpoB, the sudden change of all sites within the scope of this gene can be detected, the resistance site of open report and brand-new mutational site can be detected, be conducive to the discovery in brand-new resistance site.
As shown in Figure 3, the present invention can detect that the high frequency in bacterial strain suddenlys change and low frequency sudden change (being low to moderate one of percentage) simultaneously, and advantage Resistant strain mutational site and inferior position Resistant strain mutational site, can more scientific, safer, instruct in conjunction with patient's medication more accurately.Its result is as shown in table 15.
Table 15 detects the sudden change of bacterial strain medium-high frequency and low frequency sudden change result table simultaneously
As shown in Figure 4, the present invention is optimized experimental technique, to the MPure magnetic bead consumption in DNA purge process in experimentation of the present invention, in library construction process, the usage quantity of template and the amount etc. in higher level library are optimized, and the detected result made reaches optimum.
The above; be only the specific embodiment of the present invention, protection scope of the present invention is not limited thereto, and is anyly familiar with those skilled in the art in the technical scope that the present invention discloses; the change that can expect easily or replacement, all should be encompassed within protection scope of the present invention.Therefore, the protection domain that protection scope of the present invention should define with claim is as the criterion.
Claims (4)
1. Rimactazid, fluoroquinolones four Drug Resistance for Tuberculosis gene testers, it comprises the following steps:
(1) design covers the primer of gyrA, lnhA, katG, rpoB full length gene;
(2) 4 kinds of gene primers in step (1) are merged formation primer pool;
(3) primer pool in step (2) is utilized to amplify the amplicon that can cover drug resistance related gene total length;
(4) further end reparation carried out to the amplicon described in step (3), add joint, amplified library and quantitatively, set up complete S V-MTB library;
(5) method by once contacting makes SV-MTB library form water in oil structure, then checks order with ionPGM, detects resistance site;
Resistance site described in step (5) is the resistance site that SV-MTB detects different pharmaceutical simultaneously, and described resistance site comprises: the resistances such as GyrA-_94, GyrA-_88, GyrA-_89, GyrA-_90, GyrA-_91, rpoB_516, rpoB_511, rpoB_513, rpoB_522, rpoB_526, rpoB_531, rpoB_533, katG_315, inhA_-15, inhA_-8 are correlated with focus.
2. Rimactazid according to claim 1, fluoroquinolones four Drug Resistance for Tuberculosis gene testers, is characterized in that, carries out the detection of resistant strains site and comprise the following steps:
A: by the standard compliant sample DNA of Quality Control, obtains the Ampliseq PCR primer of four genes;
B: by described in the Ampliseq-PCR product described in steps A and step (3) amplicon carry out end reparation, add joint, amplified library and quantitatively, set up complete S V-MTB library;
E:(5) method by once contacting makes SV-MTB library form water in oil structure, then checks order with ion PGM, detects resistance site.
3. Rimactazid according to claim 2, fluoroquinolones four Drug Resistance for Tuberculosis gene testers, it is characterized in that, the primer that PCR is used in step comprises:
The primer numbers of gyrA is respectively: gyrA_8.1.75513, gyrA_8.1.51886, gyrA_8.1.82296, gyrA_8.2.58628, gyrA_8.2.29245, gyrA_8.2.49325, gyrA_8.2.94533, gyrA_8.2.1239, gyrA_8.3.158404, gyrA_8.3.159760, gyrA_8.3.104094, gyrA_8.3.113730, gyrA_8.4.22908, gyrA_8.4.67530, gyrA_8.4.47561, gyrA_8.4.24855, gyrA_8.5.10674, gyrA_8.5.14302,
The primer numbers of InhA is respectively: InhA_5.1.57946, InhA_5.1.69702, InhA_5.1.55270, InhA_5.1.88153, InhA_5.2.101468, InhA_5.2.77605, InhA_5.2.4266;
The primer numbers of katG is respectively: katG_2.1.99813, katG_2.1.50357, katG_2.1.12373, katG_2.1.119397, katG_2.2.102638, katG_2.2.46452, katG_2.2.110719, katG_2.3.111113, katG_2.3.4130, katG_2.3.6329, katG_2.3.134245, katG_2.3.130345, katG_2.3.100427, katG_2.4.25702, katG_2.4.39974, katG_2.4.116783, katG_2.4.112440,
The primer numbers of rpoB is respectively: rpoB_1.1.113180, rpoB_1.1.148688, rpoB_1.1.81682, rpoB_1.2.113638, rpoB_1.2.37694, rpoB_1.2.19193, rpoB_1.2.142863, rpoB_1.2.128222, rpoB_1.3.65890, rpoB_1.3.48836, rpoB_1.3.50710, rpoB_1.4.72080, rpoB_1.4.86222, rpoB_1.4.9838, rpoB_1.4.125079, rpoB_1.4.112057, rpoB_1.5.99561, rpoB_1.5.102258, rpoB_1.5.104523, rpoB_1.5.30503, rpoB_1.6.138705, rpoB_1.6.142676, rpoB_1.6.123078, rpoB_1.6.43945, rpoB_1.6.91215.
4. Rimactazid according to claim 1, fluoroquinolones four Drug Resistance for Tuberculosis gene testers, it is characterized in that, the step setting up complete S V-MTB library comprises the following steps:
A: increase in target area: primer is divided into two parts, and same sample divides two pipes to increase separately;
B: part removes primer: the PCR primer of primer pool different for same sample in step a is merged into a pipe, carries out removal primer;
C: add joint: the library of not increased in reaction system, then the library purifying that will do not increase;
D: amplification is carried out with quantitative to the library obtained in c.
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| CN110499377A (en) * | 2019-09-11 | 2019-11-26 | 深圳市芯思微生物科技有限公司 | A kind of primer and probe, kit and application detecting mycobacterium tuberculosis and its drug resistant gene |
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| CN117187425A (en) * | 2023-11-03 | 2023-12-08 | 迪飞医学科技(南京)有限公司 | AS-RAPID tuberculosis drug-resistant duplex detection primer probe group, kit and application |
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