CN111172303A - Mycobacterium tuberculosis drug resistance detection kit and mycobacterium tuberculosis drug resistance detection method - Google Patents
Mycobacterium tuberculosis drug resistance detection kit and mycobacterium tuberculosis drug resistance detection method Download PDFInfo
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Abstract
The invention provides a tubercle bacillus drug resistance detection kit and a method, wherein the kit comprises a tubercle bacillus drug resistance detection reagent, the tubercle bacillus drug resistance detection reagent comprises a sequencing primer aiming at a tubercle bacillus drug resistance gene, and the tubercle bacillus drug resistance gene comprises one or more of rpoB, katG, inhA-promoter, inhA-structural, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl genes. Further, the kit also contains a tubercle bacillus nucleic acid detection reagent: primer pair 1 for IS6110, primer pair 2 for IS6110 and probe primer for IS 6110. The kit can rapidly detect the tubercle bacillus nucleic acid in a sample, further detect the drug resistance of a positive sample, has good sensitivity, specificity and accuracy, can simultaneously carry out mutation detection on 48 sites of 17 drug resistance genes of common antituberculosis drugs and deletion detection of a gene spacer segment, and can more accurately and comprehensively guide the drug use of tuberculosis.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection, and particularly relates to a tubercle bacillus drug resistance detection kit and a tubercle bacillus drug resistance detection method.
Background
Mycobacterium tuberculosis, commonly known as Mycobacterium tuberculosis, is the causative agent of tuberculosis. Mycobacterium tuberculosis is a slender and slightly bent bacillus which can invade susceptible organisms through respiratory tract, digestive tract or skin injury to cause tuberculosis of various tissues and organs, wherein pulmonary tuberculosis is caused through the respiratory tract at most. If the patient is infected with mycobacterium tuberculosis and has drug resistance to one or more antituberculosis drugs, the drug-resistant tuberculosis is obtained. The WHO 2008 report shows that the total drug resistance rate of the global tuberculosis is 20.0 percent, the multi-drug resistance rate is 5.3 percent, and the estimated global multi-drug resistant tuberculosis is 50 ten thousand cases, wherein 27 drug-resistant high-burden countries which are identified by the WHO account for 85 percent of the total number of cases. The state is one of the high-burden countries of drug-resistant tuberculosis, according to the estimation of the world health organization, about 1/4-1/5 of patients with multi-drug-resistant tuberculosis occur in China, the prevalence of the drug-resistant tuberculosis is relatively serious, and the results of the national tuberculosis drug-resistant baseline investigation conducted in 2007-2008 show that the multi-drug resistance rate of the patients with pulmonary tuberculosis in China is 8.3%, so that the estimated result shows that 12 thousands of new patients with multi-drug resistance in China each year account for 24.0% of the total number of new patients in the world each year and rank the second place in the world.
Traditional diagnosis of tuberculosis relies fundamentally on Acid Fast Bacilli (AFB) smear microscopy and selection of mycobacterial cultures on solid media, but these methods all have limitations. First, AFB smears are less sensitive (30-40%) and their accuracy is often limited. Secondly, although culture-based methods have high accuracy and have long been considered as the gold standard for the diagnosis of tuberculosis in the laboratory, the long turnaround time (3-8 weeks) for mycobacterial cultures often results in delayed diagnosis. The reverse dot hybridization method for molecular detection has low sensitivity, complex operation and unreliable result. Commercially available real-time Polymerase Chain Reaction (PCR) detection methods, such as the Abbott real-time MTB assay and the GeneXpert MTB/RIF Ultra, can provide timely and accurate diagnosis of tuberculosis but are expensive. However, most of these methods are for detecting M.tuberculosis, GeneXpert MTB/RIF can detect drug-resistant tuberculosis, but only one drug-resistant gene of rifampicin, which results in patients who are resistant to other drugs or resistant to other drug-resistant genes of rifampicin drugs being undetectable. The molecular reverse dot hybridization method can also detect, but at present, only ten sites of 5 drug-resistant genes of four drugs (isoniazid, rifampicin, ethambutol and streptomycin) can be detected, and the drug-resistant genes except the four drugs can not be detected.
Disclosure of Invention
Based on the above, the invention provides a tubercle bacillus drug resistance detection kit and a tubercle bacillus drug resistance detection method, wherein the tubercle bacillus drug resistance detection kit can be used for carrying out drug resistance detection on a tubercle bacillus nucleic acid positive sample, and comprises mutation detection on 48 sites of 17 drug resistance genes of common first-line second-line antituberculosis drugs and deletion detection of a gene spacer region fragment. Furthermore, the kit can also rapidly detect the tubercle bacillus nucleic acid in the sample with good sensitivity, specificity and accuracy.
The specific technical scheme is as follows:
a tubercle bacillus drug resistance detection kit comprises a tubercle bacillus drug resistance detection reagent, wherein the tubercle bacillus drug resistance detection reagent comprises a sequencing primer aiming at a tubercle bacillus drug resistance gene, and the tubercle bacillus drug resistance gene comprises one or more of rpoB, katG, inhA-promoter, inhA-structral, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl genes; the sequencing primer sequence aiming at the rpoB gene is shown as SEQ ID NO.8 and SEQ ID NO. 9; the sequencing primer sequence aiming at the katG gene is shown as SEQ ID NO.10 and SEQ ID NO. 11; the sequencing primer sequence aiming at the inhA-promoter gene is shown as SEQ ID NO.12 and SEQ ID NO. 13; the sequencing primer sequence aiming at the inhA-structural gene is shown as SEQ ID NO.14 and SEQ ID NO. 15; the sequencing primer sequence aiming at the furA gene is shown as SEQ ID NO.16 and SEQ ID NO. 17; the sequencing primer sequence aiming at the embB gene is shown as SEQ ID NO.18 and SEQ ID NO. 19; the sequencing primer sequences aiming at the ubiA gene are shown as SEQ ID NO.20 and SEQ ID NO. 21; the sequencing primer sequence aiming at the pncA gene is shown as SEQ ID NO.22 and SEQ ID NO. 23; the sequencing primer sequences aiming at the rpsA gene are shown as SEQ ID NO.24 and SEQ ID NO. 25; the sequencing primer sequence aiming at the gyrA gene is shown as SEQ ID NO.26 and SEQ ID NO. 27; the sequencing primer sequence aiming at the gyrB gene is shown as SEQ ID NO.28 and SEQ ID NO. 29; the sequencing primer sequence aiming at the eis gene is shown as SEQ ID NO.30 and SEQ ID NO. 31; the sequencing primer sequences aiming at the rpsL gene are shown as SEQ ID NO.32 and SEQ ID NO. 33; the sequencing primer sequence aiming at the rrs gene is shown as SEQ ID NO.34 and SEQ ID NO. 35; the sequencing primer sequence aiming at the tlyA gene is shown as SEQ ID NO.36 and SEQ ID NO. 37; the sequencing primer sequence aiming at the rplC gene is shown as SEQ ID NO.38 and SEQ ID NO. 39; the sequencing primer sequence aiming at the rrl gene is shown as SEQ ID NO.40 and SEQ ID NO. 41.
In some embodiments, the mycobacterium tuberculosis drug resistance detection kit further contains a mycobacterium tuberculosis nucleic acid detection reagent, wherein the mycobacterium tuberculosis nucleic acid detection reagent comprises a primer pair 1 aiming at IS6110, a primer pair 2 aiming at IS6110 and a probe primer aiming at IS 6110; the primer pair 1 aiming at IS6110 comprises IS6110-FW1 with the sequence shown as SEQ ID NO.1 and IS6110-RV1 with the sequence shown as SEQ ID NO. 2; the primer pair 2 aiming at IS6110 comprises IS6110-FW2 with the sequence shown as SEQ ID NO.3 and IS6110-RV2 with the sequence shown as SEQ ID NO. 4; the probe primer sequence aiming at IS6110 IS shown as SEQ ID NO.5, the 5 'end of the probe primer IS modified with a fluorescent group, and the 3' end of the probe primer IS modified with a quenching group.
In some embodiments, the probe primer for IS6110 IS modified with a fluorophore FAM at the 5 'end and a quencher BHQ1 at the 3' end.
In some embodiments, the tubercle bacillus nucleic acid detection reagent further comprises an internal quality control template and a probe primer aiming at the internal quality control template; the internal quality control template contains a mouse RAB3A oncogene fragment and an IS6110 primer pair 2 binding region; the sequence of the internal quality control template is shown as SEQ ID NO. 6; the probe primer sequence for the internal quality control template IS shown as SEQ ID NO.7, the 5 'end of the probe primer IS modified with a fluorescent group, the 3' end of the probe primer IS modified with a quenching group, and the fluorescent group and the quenching group are different from the fluorescent group and the quenching group of the probe primer for IS 6110.
In some embodiments, the 5 'end of the probe primer for the internal quality control template is modified with a fluorescent group LC610, and the 3' end is modified with a quenching group BBQ.
In some embodiments, the mycobacterium tuberculosis drug resistance detection kit further comprises a DNA purification reagent, and/or a Probe PCR Master Mix; the DNA purification reagent is Ampure xp Beads.
The invention also provides a library construction method for detecting the drug resistance of the tubercle bacillus.
The specific technical scheme is as follows:
a method for constructing a library for detecting drug resistance of tubercle bacillus comprises the following steps:
(1) collecting a sputum sample, extracting DNA, and performing PCR amplification detection on nucleic acid of the mycobacterium tuberculosis by using the mycobacterium tuberculosis drug resistance detection kit;
(2) and (2) constructing a sequencing library of the DNA of the nucleic acid positive specimen of the mycobacterium tuberculosis in the step (1) by using the mycobacterium tuberculosis drug resistance detection kit:
A. purifying DNA of the nucleic acid positive sputum specimen of the mycobacterium tuberculosis in the step (1);
B. using the DNA purified in the step A as a template, and carrying out PCR amplification by using sequencing primer pairs of sequencing sites in rpoB, katG, inhA-promoter, inhA-structure, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl genes;
C. purifying the amplification product of step B;
D. labeling the purified product in the step C with genome DNA;
E. and amplifying and purifying the library to obtain a sequencing library.
In some of these embodiments, the step (1) detection system comprises the following components: IS6110-FW 10.8-1 μ M; IS6110-RV 10.8-1 mu M; IS6110-FW 240-41 μ M; IS6110-RV 240-41 mu M; IS6110 probe primer 3.5-4 μ M; internal quality control template 1.8-2.2X 10-10ng/mul; the internal quality control template probe primer is 3.5-4 mu M.
In some of these embodiments, the step (1) detection system comprises the following components: IS6110-FW 10.9 μ M; IS6110-RV 10.9 mu M(ii) a IS6110-FW 240.5 μ M; IS6110-RV 240.5 mu M; IS6110 probe primer 4. mu.M; internal quality control template 2 x 10-10ng/mul; the quality control template probe primer is 4 mu M.
In some embodiments, the step (1) detection procedure is as follows: 2min at 95 ℃; 15 cycles: 5s at 95 ℃ and 30s at 72 ℃; 42 cycles of: collecting fluorescence at 95 ℃ for 5s and 6 ℃ for 30s and at 6 ℃ for 30 s; 30s at 40 ℃.
In some embodiments, the PCR amplification procedure in step B is as follows: 10s at 98 ℃; 10 cycles, annealing temperature drop 0.5 ℃ after each cycle: 10s at 98 ℃, 30s at 65.5 ℃ and 45s at 72 ℃; 30 cycles: 10s at 98 ℃, 30s at 60.5 ℃ and 45s at 72 ℃; 7min at 72 ℃.
In some embodiments, the genomic DNA labeling procedure in step D is as follows: 5min at 55 ℃ and keeping at 10 ℃.
In some of these embodiments, the procedure for amplifying the library in step E is as follows: 3min at 72 ℃; 30s at 98 ℃; 13 cycles: 10s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃; 5min at 72 ℃.
The invention also provides a method for detecting the drug resistance of the mycobacterium tuberculosis.
The specific technical scheme is as follows:
a method for detecting drug resistance of tubercle bacillus comprises the following steps: the library is constructed by the method, and the constructed library is subjected to concentration determination and then is sequenced by a sequencer.
Compared with the prior art, the invention has the following beneficial effects:
the mycobacterium tuberculosis drug resistance detection kit comprises sequencing primers of 48 sites of 17 drug resistance genes of common first-line second-line antituberculosis drugs, wherein the sequencing primers are obtained by the inventor after a large amount of research and optimization, PCR amplification can be carried out under the same condition, the construction and sequencing of a sequencing library are facilitated, the operation steps are reduced, and the lowest detection lower limit is low. The kit has wide drug resistance detection range, covers the prior commonly used antituberculosis drugs, and can more accurately and comprehensively guide the drug administration for tuberculosis.
The kit for detecting the drug resistance of the tubercle bacillus can also detect the tubercle bacillus nucleic acid at the same time, quickly detect the tubercle bacillus nucleic acid firstly, and then detect the drug resistance of a specimen with positive tubercle bacillus nucleic acid. The nucleic acid detection reagent for the bacillus in the kit can simultaneously detect the external primer with higher melting temperature and the internal primer with lower melting temperature in a single tube, thereby ensuring high sensitivity and specificity of detection.
Furthermore, the tubercle bacillus nucleic acid detection reagent also contains a homologous internal quality control template, the internal quality control template contains a mouse RAB3A oncogene fragment and an IS6110 primer pair 2 combination region, the condition of a PCR link sample can be monitored more effectively, the PCR link sample IS easily distinguished from a true negative result, the accuracy of a detection result IS further improved, the tubercle bacillus nucleic acid in the sample can be detected quickly and accurately, the subsequent drug resistance detection of a tubercle bacillus positive sample IS ensured, and the tubercle bacillus nucleic acid detection reagent IS low in price and easy to popularize and use.
Detailed Description
In order that the invention may be more readily understood, reference will now be made to the following more particular description of the invention, examples of which are set forth below. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. These embodiments are provided so that this disclosure will be thorough and complete. It is to be understood that the experimental procedures in the following examples, where specific conditions are not noted, are generally in accordance with conventional conditions, or with conditions recommended by the manufacturer. The various reagents used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The instrument comprises the following steps: nucleic acid quantifier Qubit 3.0 (Qubit); PCR instrument (ABI Veriti); fragment detector agent 2100 (agilent); NovaSeq6000(Illumina) gene sequencer.
Reagent: the library reagents Nextera XT DNA Sample Preparation Kit (Illumina) and Nextera XT Index Kit v2(96indexs) (Illumina); purification reagent
XP Reagent (Beckman); NovaSeq 5000/6000S2 Reagent Kit (Illumina); quality control of concentration
dsDNA HS Assay Kit (Qubit); fragment quality control DNA 1000kit (Agilent).
Primer: synthesized by Shanghai Czeri bioengineering, Inc.
Example 1 Mycobacterium tuberculosis drug resistance detection kit
The kit for detecting the drug resistance of the mycobacterium tuberculosis comprises a mycobacterium tuberculosis nucleic acid detection reagent and a mycobacterium tuberculosis drug resistance detection reagent:
(1) the tubercle bacillus nucleic acid detection reagent comprises a primer pair 1 aiming at IS6110, a primer pair 2 aiming at IS6110, a probe primer aiming at IS6110, an internal quality control template, a probe primer aiming at the internal quality control template, QuanntiNova ProbePCR Master Mix and DEPC water.
The primer pair 1 aiming at IS6110 comprises IS6110-FW1 with the sequence shown as SEQ ID NO.1 and IS6110-RV1 with the sequence shown as SEQ ID NO. 2; the primer pair 2 aiming at IS6110 comprises IS6110-FW2 with the sequence shown as SEQ ID NO.3 and IS6110-RV2 with the sequence shown as SEQ ID NO. 4; the probe primer sequence aiming at IS6110 IS shown as SEQ ID NO.5, the 5 'end of the probe primer IS modified with a fluorescent group FAM, and the 3' end of the probe primer IS modified with a quenching group BHQ 1; the internal quality control template contains a mouse RAB3A oncogene fragment and an IS6110 primer pair 2 binding region; the sequence of the internal quality control template is shown as SEQ ID NO. 6; the probe primer sequence aiming at the internal quality control template is shown as SEQ ID NO.7, the 5 'end of the probe primer is modified with a fluorescent group LC610, and the 3' end of the probe primer is modified with a quenching group BBQ.
The above sequence information is shown in table 1:
TABLE 1 sequence information
(2) The tubercle bacillus drug resistance detection reagent comprises a sequencing primer aiming at a tubercle bacillus drug resistance gene and Ampurexp Beads.
The drug resistance gene of the tubercle bacillus comprises one or more genes of rpoB, katG, inhA-promoter, inhA-structural, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl; the sequencing primer sequence aiming at the rpoB gene is shown as SEQ ID NO.8 and SEQ ID NO. 9; the sequencing primer sequence aiming at the katG gene is shown as SEQ ID NO.10 and SEQ ID NO. 11; the sequencing primer sequence aiming at the inhA-promoter gene is shown as SEQ ID NO.12 and SEQ ID NO. 13; the sequencing primer sequence aiming at the inhA-structural gene is shown as SEQ ID NO.14 and SEQ ID NO. 15; the sequencing primer sequence aiming at the furA gene is shown as SEQ ID NO.16 and SEQ ID NO. 17; the sequencing primer sequence aiming at the embB gene is shown as SEQ ID NO.18 and SEQ ID NO. 19; the sequencing primer sequences aiming at the ubiA gene are shown as SEQ ID NO.20 and SEQ ID NO. 21; the sequencing primer sequence aiming at the pncA gene is shown as SEQ ID NO.22 and SEQ ID NO. 23; the sequencing primer sequences aiming at the rpsA gene are shown as SEQ ID NO.24 and SEQ ID NO. 25; the sequencing primer sequence aiming at the gyrA gene is shown as SEQ ID NO.26 and SEQ ID NO. 27; the sequencing primer sequence aiming at the gyrB gene is shown as SEQ ID NO.28 and SEQ ID NO. 29; the sequencing primer sequence aiming at the eis gene is shown as SEQ ID NO.30 and SEQ ID NO. 31; the sequencing primer sequences aiming at the rpsL gene are shown as SEQ ID NO.32 and SEQ ID NO. 33; the sequencing primer sequence aiming at the rrs gene is shown as SEQ ID NO.34 and SEQ ID NO. 35; the sequencing primer sequence aiming at the tlyA gene is shown as SEQ ID NO.36 and SEQ ID NO. 37; the sequencing primer sequence aiming at the rplC gene is shown as SEQ ID NO.38 and SEQ ID NO. 39; the sequencing primer sequence aiming at the rrl gene is shown as SEQ ID NO.40 and SEQ ID NO. 41.
The sequence information of the detection sites and sequencing primers of the drug-resistant gene of the mycobacterium tuberculosis is shown in table 2:
TABLE 2 detection sites and sequencing primer sequence information of drug-resistant genes of Mycobacterium tuberculosis
the internal quality control template (IC template) IS pUC57 plasmid containing mouse Rab3a oncogene and Mycobacterium tuberculosis IS6110 sequence, and can be expressed in DH5 α Escherichia coli strain, and its preparation method comprises the steps of extracting plasmid DNA of frozen stock of DH5 α IS6110 IC with QIAprep Spin Miniprep kit (Qiagen Pte Ltd.; catalog No. 27104), performing PCR amplification on the plasmid DNA to obtain 346bp internal quality control template, purifying with QIAquick PCR purification kit (Qiagen Pte Ltd.; catalog No. 28106), and diluting to 5 × 10 EB buffer solution-9ng/. mu.l to prepare the working concentration of IC template.
The result judgment standard of the kit when used for detecting the mycobacterium tuberculosis nucleic acid is shown in the following table 3:
TABLE 3 judgment standards for the results of the kit for detecting Mycobacterium tuberculosis nucleic acids
Note: IS6110 positive: the sample has a ct value and an S-shaped curve; IS6110 negative: the samples had no ct values or no sigmoid curves. Positive internal quality control template: the sample has an S-shaped curve of the ct value and the IC; negative internal quality control template: sample IS6110 has no ct value or sigmoid curve.
Example 2 library construction method for drug resistance detection of tubercle bacillus
The embodiment of the invention relates to a method for constructing a library for detecting drug resistance of tubercle bacillus, which utilizes the kit of embodiment 1 to construct the library, and comprises the following steps:
(1) collecting a sputum specimen, extracting DNA, and then carrying out PCR amplification detection on nucleic acid of the mycobacterium tuberculosis:
A. adding 100 mu L of digestive juice into 1mL of sputum sample, vortexing, shaking, mixing uniformly, and then incubating for 10 minutes at 65 ℃; B. centrifuging the mixed solution obtained in the step A at 13200g for 10 minutes, and removing the supernatant; C. adding 100 mu of LTris-HCl solution into the precipitate obtained in the step B, uniformly mixing by vortex oscillation, centrifuging for 10 minutes at 13200g, and removing the supernatant; D. adding 100 μ L of lysate to the precipitate obtained in step C, followed by incubation at 65 ℃ for 45 minutes; E. adding 100 mu L of Tris-HCl solution into the mixed solution obtained in the step D, and uniformly mixing to obtain a DNA solution; F. and (3) configuring a PCR amplification system according to the table 4, adding 5 mu L of the DNA solution obtained in the step E into the system, shaking and mixing uniformly, and then carrying out PCR amplification detection according to the amplification program in the table 5.
TABLE 4 PCR amplification System
| Components | Final concentration | Volume of |
| 2×QuantiNova | 1× | 15μl |
| IS6110-FW1 | 0.9mM | 1μl |
| IS6110-RV1 | 0.9μM | 1μl |
| IS6110-FW2 | 40.5μM | 1μl |
| IS6110-RV2 | 40.5μM | 1μl |
| Quality control template | 2×10-10ng/μl | 1μl |
| IS6110 probe primer | 4μM | 1.5μl |
| Quality control template probe primer | 4μM | 1.5μl |
| DEPC water | -- | 2μl |
| Total volume | -- | 25μl |
TABLE 5 PCR amplification procedure
And (3) after the detection is finished, judging the result according to the judgment standard of the embodiment 3, and selecting a positive sample of the tubercle bacillus nucleic acid.
(2) Constructing a sequencing library of the DNA of the nucleic acid positive specimen of the bacillus tuberculosis in the step 1:
A. purifying DNA of the nucleic acid positive sputum specimen of the mycobacterium tuberculosis in the step 1: 1) use of
3.0, measuring the concentration by a Fluorometer, and recording concentration data; 2) taking a proper amount of sample, and using ddH2Supplementing O to 100ul, adding 180ul (2 x) Ampure xp Beads into the sample, mixing well, centrifuging, standing at room temperature for 5min, standing on a magnetic frame until the sample is clear, and removing the supernatant; 3) adding 200 μ L of fresh 80% ethanol, incubating for 30s, clarifying, and removing supernatant; 4) repeating the previous step once, standing at room temperature and drying in the air; 5) add 20. mu.L of ddH2O eluting the sample; 6) use of
3.0Fluorometer assay concentration; recording the purified concentration data;
B. and B, performing PCR amplification by using the DNA obtained by purification in the step A as a template and sequencing primer pairs of sequencing sites in rpoB, katG, inhA-promoter, inhA-structure, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl genes:
1) the following 5-tube amplification primers were configured for each sample:
TABLE 6 amplification primer 1
TABLE 7 amplification primers 2
TABLE 8 amplification primers 3
TABLE 9 amplification primers 4
TABLE 10 amplification primers 5
2) The following 5-tube PCR amplification system was provided for each sample
TABLE 11 PCR amplification System
| Reagent | Volume per unit (μ L) |
| DNA | 2 |
| Q5 High-Fidelity 2X Master Mix | 12.5 |
| Primer(10uM) | 2.5 |
| H2O | 8 |
| Total | 25 |
3) Carrying out PCR amplification on the system in the step 2) in the following procedure
TABLE 12 PCR amplification System
C. Purifying the amplification product of step B: 1) adding 45ul Ampure xp Beads into the sample, mixing thoroughly, microcentrifuging, standing at room temperature for 5min, standing on a magnetic rack until the sample is clear, and removing the supernatant; 2) adding 200 μ L of fresh 80% ethanol, incubating for 30s, clarifying, and removing supernatant; 3) repeating the previous step once, standing at room temperature and drying in the air; 4) add 20. mu.L of ddH2Eluting with oxygen; 5) use of
3.0Fluorometer to determine the concentration and record the concentration data of each PCR product; 6) 5 tubes of PCR products are put into the same EP tube according to the same mass pooling, and the subsequent steps are carried out;
D. and C, performing genomic DNA labeling on the purified product in the step C:
1) concentration determination and dilution: use of
3.0Fluorometer to measure the concentration and record the concentration data of the pooling sample; diluting the sample to 1ng/ul with nuclease-free water, measuring the concentration again, and recording the concentration data; using the diluted sample to perform the subsequent steps;
2) the following reagents were added to the reaction system in order:
TABLE 13 reaction reagents
| Reagent | Volume per unit (μ L) |
| Labeled DNA buffer | 10 |
| DNA(1ng) | x |
| ddH2O | 5-x |
| Amplicon labeling mixture | 5 |
| Total | 20 |
3) Place on a pre-programmed amplification apparatus and run the following labeling program:
TABLE 14 Mark program
4) Adding 5ul of NT into each hole, blowing and beating the mixed solution up and down, and performing microcentrifugation;
5) incubating at room temperature for 5 min;
E. amplifying and purifying the library to obtain a sequencing library:
1) adding the following reagents into a reaction system, blowing, uniformly mixing and microcentrifuging:
TABLE 15 reaction reagents
| Reagent | Volume per unit (μ L) |
| DNA | 25 |
| Label 1(i7) | 5 |
| Label 2(i5) | 5 |
| Nextera PCR premix | 15 |
| Total | 50 |
2) Place on a pre-programmed amplificator and run the PCR program:
TABLE 16 PCR procedure
3) And (3) purification: adding 30ul (0.6X) Ampure xp Beads into the sample, mixing well, microcentrifugation, standing at room temperature for 5min, standing on a magnetic frame until clarification, and removing the supernatant; adding 200 μ L of fresh 80% ethanol, incubating for 30s, clarifying, and removing supernatant; repeating the previous step once, standing at room temperature and drying in the air; the sequencing library was obtained by adding 20. mu.L of RSB elution library.
Example 3 drug resistance detection method for tubercle bacillus
The method for detecting drug resistance of mycobacterium tuberculosis in the embodiment comprises the following steps of constructing a library by using the method in the embodiment 2, measuring the concentration of the constructed library, and sequencing by using a sequencer, wherein the method comprises the following specific steps:
1. library concentration determination and 2100 determination
(1) Use of
3.0Fluorometer library concentration determination, Length determination Using an agilent 2100bioAnalyzer；
(2) Recording the concentration data in a detection sample information recording table;
(3) the library was stored at-20 ℃.
2. Detection on machine
(1) Computer reagent information recording and preparing
recording the information of the reagent used in the computer in a 'molecular pathology reagent use record table';
taking out SBS Reagent cards and Cluster Reagent cards stored at-20 deg.c, and thawing in water.
③ taking out the Flow Cell preserved at 4 ℃, and balancing for 30min at room temperature.
(2) Instrument cleaning
preparing 1000.0mL of cleaning solution containing 0.05% of Tween-20, and pouring 400mL of cleaning solution into SBS WashCartridge;
preparing 20.0mL of 0.25% sodium hypochlorite solution, and adding 5.0mL of the sodium hypochlorite solution into a No.17 hole of the Cluster Wash Cartidge;
and thirdly, replacing the Wash Flow Cell to clean the instrument.
(3) Preparing computer library
the library was diluted to 1.8nM with PH 8.5Tris-HCl as follows;
② 0.2N NaOH (10 μ L of 2NNaOH stock solution +90 μ LH2O) is prepared;
③ evenly mixing the 1.8nM mixed library with 0.2N NaOH, and carrying out room temperature denaturation for 8 min;
adding pH 8Tris-HCl for dilution;
fifthly, transferring all the solution to a Library Tube, and performing machine sequencing.
The method of example 2 was used to detect tubercle bacillus nucleic acid in 65 sputum samples, wherein the 65 sputum samples were known to be infected with tubercle bacillus (53 samples were positive for tubercle bacillus, 12 samples were negative for tubercle bacillus), and the types of drug resistance gene mutation in the tubercle bacillus positive samples were known.
Further using the method described in example 2 to construct sequencing library for the sample positive for the nucleic acid of the mycobacterium tuberculosis, and using the method described in example 3 to perform sequencing, the sequencing results are shown in the following table:
TABLE 17 sequencing results of nucleic acid positive specimens of Mycobacterium tuberculosis
According to the detection of the kit, 53 samples in 65 samples are positive, 12 samples are negative, and the detection result is completely consistent with the known result (the specific data is omitted), so that the nucleic acid detection reagent for the mycobacterium tuberculosis in the kit has good sensitivity and specificity, and the detection accuracy is up to 100%.
As can be seen from the results in Table 1, the kit of the invention can successfully detect the mutation conditions of the 17 drug-resistant genes of the tubercle bacillus in the sample, and the detection result is completely consistent with the known result, so that the kit has good specificity and accuracy, and can more accurately and more comprehensively guide the administration of the tubercle bacillus.
The technical features of the above embodiments can be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the above embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the scope of the present description should be considered as being described in the present specification.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> Guangzhou gold region medical examination group GmbH, Guangzhou gold region medical examination center GmbH
<120> tubercle bacillus drug resistance detection kit and tubercle bacillus drug resistance detection method
<130>2019-12-28
<160>41
<170>PatentIn version 3.3
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Claims (10)
1. The tubercle bacillus drug resistance detection kit is characterized by comprising a tubercle bacillus drug resistance detection reagent, wherein the tubercle bacillus drug resistance detection reagent comprises a sequencing primer aiming at a tubercle bacillus drug resistance gene, and the tubercle bacillus drug resistance gene comprises one or more of rpoB, katG, inhA-promoter, inhA-structural, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl genes; the sequencing primer sequence aiming at the rpoB gene is shown as SEQ ID NO.8 and SEQ ID NO. 9; the sequencing primer sequence aiming at the katG gene is shown as SEQ ID NO.10 and SEQ ID NO. 11; the sequencing primer sequence aiming at the inhA-promoter gene is shown as SEQ ID NO.12 and SEQ ID NO. 13; the sequencing primer sequence aiming at the inhA-structural gene is shown as SEQ ID NO.14 and SEQ ID NO. 15; the sequencing primer sequence aiming at the furA gene is shown as SEQ ID NO.16 and SEQ ID NO. 17; the sequencing primer sequence aiming at the embB gene is shown as SEQ ID NO.18 and SEQ ID NO. 19; the sequencing primer sequences aiming at the ubiA gene are shown as SEQ ID NO.20 and SEQ ID NO. 21; the sequencing primer sequence aiming at the pncA gene is shown as SEQ ID NO.22 and SEQ ID NO. 23; the sequencing primer sequences aiming at the rpsA gene are shown as SEQ ID NO.24 and SEQ ID NO. 25; the sequencing primer sequence aiming at the gyrA gene is shown as SEQ ID NO.26 and SEQ ID NO. 27; the sequencing primer sequence aiming at the gyrB gene is shown as SEQ ID NO.28 and SEQ ID NO. 29; the sequencing primer sequence aiming at the eis gene is shown as SEQ ID NO.30 and SEQ ID NO. 31; the sequencing primer sequences aiming at the rpsL gene are shown as SEQ ID NO.32 and SEQ ID NO. 33; the sequencing primer sequence aiming at the rrs gene is shown as SEQ ID NO.34 and SEQ ID NO. 35; the sequencing primer sequence aiming at the tlyA gene is shown as SEQ ID NO.36 and SEQ ID NO. 37; the sequencing primer sequence aiming at the rplC gene is shown as SEQ ID NO.38 and SEQ ID NO. 39; the sequencing primer sequence aiming at the rrl gene is shown as SEQ ID NO.40 and SEQ ID NO. 41.
2. The tubercle bacillus drug resistance detection kit according to claim 1, characterized by further comprising tubercle bacillus nucleic acid detection reagents, wherein the tubercle bacillus nucleic acid detection reagents comprise a primer pair 1 aiming at IS6110, a primer pair 2 aiming at IS6110 and a probe primer aiming at IS 6110;
the primer pair 1 aiming at IS6110 comprises IS6110-FW1 with the sequence shown as SEQ ID NO.1 and IS6110-RV1 with the sequence shown as SEQ ID NO. 2; the primer pair 2 aiming at IS6110 comprises IS6110-FW2 with the sequence shown as SEQ ID NO.3 and IS6110-RV2 with the sequence shown as SEQ ID NO. 4; the probe primer sequence aiming at IS6110 IS shown as SEQ ID NO.5, the 5 'end of the probe primer IS modified with a fluorescent group, and the 3' end of the probe primer IS modified with a quenching group.
3. The tubercle bacillus drug resistance detection kit according to claim 2, wherein the tubercle bacillus nucleic acid detection reagent further comprises an internal quality control template and a probe primer aiming at the internal quality control template; the internal quality control template contains a mouse RAB3A oncogene fragment and an IS6110 primer pair 2 binding region; the sequence of the internal quality control template is shown as SEQ ID NO. 6; the probe primer sequence for the internal quality control template IS shown as SEQ ID NO.7, the 5 'end of the probe primer IS modified with a fluorescent group, the 3' end of the probe primer IS modified with a quenching group, and the fluorescent group and the quenching group are different from the fluorescent group and the quenching group of the probe primer for IS 6110.
4. The tubercle bacillus drug resistance detection kit according to any one of claims 1 to 3, wherein the tubercle bacillus drug resistance detection kit further comprises a DNA purification reagent, and/or a Probe PCR Master Mix; the DNA purification reagent is Ampure xp Beads.
5. A library construction method for tubercle bacillus drug resistance detection is characterized by comprising the following steps:
(1) collecting a sputum sample, extracting DNA, and then carrying out PCR amplification detection on nucleic acid of mycobacterium tuberculosis by using the mycobacterium tuberculosis drug-resistance detection kit of any one of claims 2-4;
(2) constructing a sequencing library of the DNA of the nucleic acid positive specimen of the Bacillus tuberculosis in the step (1) by using the kit for detecting drug resistance of the Bacillus tuberculosis as claimed in claim 1 or 4:
A. purifying DNA of the nucleic acid positive sputum specimen of the mycobacterium tuberculosis in the step (1);
B. using the DNA purified in the step A as a template, and carrying out PCR amplification by using sequencing primer pairs of sequencing sites in rpoB, katG, inhA-promoter, inhA-structure, furA, embB, ubiA, pncA, rpsA, gyrA, gyrB, eis, rpsL, rrs, tlyA, rplC and rrl genes;
C. purifying the amplification product of step B;
D. labeling the purified product in the step C with genome DNA;
E. and amplifying and purifying the library to obtain a sequencing library.
6. The method for constructing a library for detecting tubercle bacillus drug resistance according to claim 5, wherein the detection system of step (1) comprises the following components: IS6110-FW 10.8-1 μ M; IS6110-RV 10.8-1 mu M; IS6110-FW 240-41 μ M; IS6110-RV 240-41 mu M; IS6110 probe primer 3.5-4 μ M; internal quality control template 1.8-2.2X 10-10ng/mul; the internal quality control template probe primer is 3.5-4 mu M.
7. The method for constructing a library for detecting tubercle bacillus drug resistance according to claim 5, wherein the detection procedure of step (1) is as follows: 2min at 95 ℃; 15 cycles: 5s at 95 ℃ and 30s at 72 ℃; 42 cycles of: collecting fluorescence at 95 ℃ for 5s and 6 ℃ for 30s and at 6 ℃ for 30 s; 30s at 40 ℃.
8. The method for constructing a library for detecting tubercle bacillus drug resistance according to any of claims 5-7, wherein the PCR amplification procedure in step B is as follows: 10s at 98 ℃; 10 cycles, annealing temperature drop 0.5 ℃ after each cycle: 10s at 98 ℃, 30s at 65.5 ℃ and 45s at 72 ℃; 30 cycles: 10s at 98 ℃, 30s at 60.5 ℃ and 45s at 72 ℃; 7min at 72 ℃.
9. The method for constructing a library for detecting tubercle bacillus drug resistance according to any of claims 5 to 7, wherein the genomic DNA labeling in step D is performed by the following procedure: 5min at 55 ℃ and keeping at 10 ℃.
10. The method for constructing a library for detecting tubercle bacillus drug resistance according to any of claims 5 to 7, wherein the procedure for amplifying the library in step E is as follows: 3min at 72 ℃; 30s at 98 ℃; 13 cycles: 10s at 95 ℃, 30s at 55 ℃ and 30s at 72 ℃; 5min at 72 ℃.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342307A (en) * | 2020-11-09 | 2021-02-09 | 盛世中方(北京)生物科技有限公司 | Mycobacterium tuberculosis drug resistance detection kit and method based on NGS technology |
| CN113025752A (en) * | 2021-03-24 | 2021-06-25 | 广州金域医学检验中心有限公司 | Internal reference gene, kit and detection method for PCR detection of 2019-nCoV and SARS virus |
| CN113388690A (en) * | 2021-07-02 | 2021-09-14 | 海南医学院 | Primer, probe and kit for detecting mycobacterium tuberculosis and gene mutation sites related to drug resistance of therapeutic drugs |
| CN113493848A (en) * | 2021-07-23 | 2021-10-12 | 杭州圣庭医疗科技有限公司 | Mycobacterium tuberculosis drug-resistant type identification method based on nanopore sequencer |
| CN115992272A (en) * | 2022-10-17 | 2023-04-21 | 杭州遂真生物技术有限公司 | Composition for detecting mycobacterium tuberculosis and drug-resistant gene thereof and integrated kit |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561350A (en) * | 2015-01-28 | 2015-04-29 | 深圳华大基因研究院 | Kit and application thereof |
| CN105950726A (en) * | 2016-05-16 | 2016-09-21 | 杭州市疾病预防控制中心 | Mycobacterium tuberculosis pyrazinamide molecule drug sensitivity detection method |
| CN107385049A (en) * | 2017-08-04 | 2017-11-24 | 首都医科大学附属北京胸科医院 | For expanding the primer pair combination product of drug resistance of Mycobacterium tuberculosis related gene |
-
2019
- 2019-12-31 CN CN201911415507.2A patent/CN111172303A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561350A (en) * | 2015-01-28 | 2015-04-29 | 深圳华大基因研究院 | Kit and application thereof |
| CN105950726A (en) * | 2016-05-16 | 2016-09-21 | 杭州市疾病预防控制中心 | Mycobacterium tuberculosis pyrazinamide molecule drug sensitivity detection method |
| CN107385049A (en) * | 2017-08-04 | 2017-11-24 | 首都医科大学附属北京胸科医院 | For expanding the primer pair combination product of drug resistance of Mycobacterium tuberculosis related gene |
Non-Patent Citations (2)
| Title |
|---|
| MD MAHMUDUL ISLAM;H.M.ADNAN HAMEED;JULIUS MUGWERU;CHIRANJIBI CHHOTARAY;CHANGWEI WANG;YAOJU TAN;JIANXIONG LIU;XINJIE LI;SHOUYONG TA: "Drug resistance mechanisms and novel drug targets for tuberculosis therapy" * |
| 郑晓静;杜博平;贾红彦;侯继增;杨超;高汉青;杜凤娇;邢爱英;李自慧;曹廷明;张宗德;李琦;: "结核分枝杆菌对氨基水杨酸耐药相关基因的筛选及鉴定" * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112342307A (en) * | 2020-11-09 | 2021-02-09 | 盛世中方(北京)生物科技有限公司 | Mycobacterium tuberculosis drug resistance detection kit and method based on NGS technology |
| CN113025752A (en) * | 2021-03-24 | 2021-06-25 | 广州金域医学检验中心有限公司 | Internal reference gene, kit and detection method for PCR detection of 2019-nCoV and SARS virus |
| CN113388690A (en) * | 2021-07-02 | 2021-09-14 | 海南医学院 | Primer, probe and kit for detecting mycobacterium tuberculosis and gene mutation sites related to drug resistance of therapeutic drugs |
| CN113493848A (en) * | 2021-07-23 | 2021-10-12 | 杭州圣庭医疗科技有限公司 | Mycobacterium tuberculosis drug-resistant type identification method based on nanopore sequencer |
| CN115992272A (en) * | 2022-10-17 | 2023-04-21 | 杭州遂真生物技术有限公司 | Composition for detecting mycobacterium tuberculosis and drug-resistant gene thereof and integrated kit |
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