CN117327821A - Kit for quantitatively detecting ultra-low-proportion rifampicin drug-resistant mutation and detection method - Google Patents
Kit for quantitatively detecting ultra-low-proportion rifampicin drug-resistant mutation and detection method Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 60
- 229940079593 drug Drugs 0.000 title claims abstract description 44
- 239000003814 drug Substances 0.000 title claims abstract description 44
- 230000035772 mutation Effects 0.000 title claims abstract description 43
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 title claims abstract description 28
- 229960001225 rifampicin Drugs 0.000 title claims abstract description 27
- 239000000523 sample Substances 0.000 claims abstract description 39
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 28
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 13
- 108700021469 Mycobacterium tuberculosis rpoB Proteins 0.000 claims abstract description 10
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 7
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 7
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 7
- 230000008685 targeting Effects 0.000 claims abstract description 4
- 241000187479 Mycobacterium tuberculosis Species 0.000 claims description 12
- 238000007847 digital PCR Methods 0.000 claims description 12
- 241000894006 Bacteria Species 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 9
- 101150090202 rpoB gene Proteins 0.000 claims description 8
- 101100038261 Methanococcus vannielii (strain ATCC 35089 / DSM 1224 / JCM 13029 / OCM 148 / SB) rpo2C gene Proteins 0.000 claims description 7
- 101150085857 rpo2 gene Proteins 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 206010064571 Gene mutation Diseases 0.000 claims description 5
- 108010006785 Taq Polymerase Proteins 0.000 claims description 3
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- 206010059866 Drug resistance Diseases 0.000 abstract description 20
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Abstract
The invention relates to a kit for quantitatively detecting ultra-low-proportion rifampicin drug-resistant mutation and a detection method. The kit comprises a primer and a probe for targeting a drug-resistant mutation site of mycobacterium tuberculosis rpoB and an internal reference gene, wherein a conserved region of the KatG gene is selected in a channel of the internal reference gene, and an upstream primer, a downstream primer and a probe sequence are designed; the gene region of the targeted mycobacterium tuberculosis rpoB is designed with an upstream primer sequence of a specific mutation site, a general downstream primer sequence and a probe sequence; specific mutation sites include rpoBL430P, rpoBH445Y, rpoBH445R, rpoBD435Y, rpoBH445D, rpoBH445L, rpoBS450L and rpoBS450W. The invention also discloses a method for detecting the nucleic acid sample by adopting the kit. The invention solves the clinical problems of difficult detection of low-proportion mutation and difficult drug resistance early warning in the early drug resistance stage at present.
Description
Technical Field
The invention belongs to the field of detection of drug-resistant gene mutation of mycobacterium tuberculosis, and particularly relates to a kit and a detection method for quantitatively detecting drug-resistant mutation of rifampicin with ultralow proportion, which are used for realizing high-risk drug-resistant patient monitoring and drug-resistant tuberculosis early warning by detecting ultralow frequency mutation in early drug-resistant stage.
Background
The drug-resistant tuberculosis has the characteristics of low cure rate and high death rate. The heterogeneous drug resistance refers to a state that drug-resistant bacteria and sensitive bacteria coexist in a sample, and the proportion of the drug-resistant bacteria is less than or equal to 1 percent. In clinical practice, once heterogeneous drug resistance occurs in flora in a patient with drug-sensitive tuberculosis, the proportion of drug-resistant bacteria tends to gradually rise and finally becomes dominant bacteria, and the patient changes from drug-sensitive tuberculosis to drug-resistant tuberculosis. Thus heterogeneous drug resistance is an early stage of drug-resistant tuberculosis occurrence and is also a critical window period for prevention and control of intervention. The method is used for dynamically monitoring and early warning the heterogeneous drug resistance level of high-risk cases of drug resistance tuberculosis, and is an important means for realizing early intervention and controlling drug resistance tuberculosis transmission. In the heterogeneous drug-resistant stage, the drug-resistant bacteria occupy low proportion, so that the detection difficulty is high, the quantitative detection is difficult to realize and the like.
The traditional phenotype drug resistance detection is a gold standard for judging tuberculosis drug resistance, heterogeneous drug resistance flora with the drug resistance ratio more than or equal to 1% can be detected theoretically, but in practice, more than 5% is generally needed for stable detection, and the defects of long time consumption, strict biosafety requirements on detection sites and the like exist, so that the rapid detection requirements are difficult to meet, and early drug resistance discovery and propagation prevention and control are not facilitated. With the rapid development of molecular detection technology, genotypic sensitization has begun to be applied in scientific research and clinic, which can be used to predict drug resistance by detecting specific regions of the mycobacterium tuberculosis genome. However, the sensitivity of the existing genotyping techniques to detect drug-resistant subpopulations is far lower than phenotypic drug-sensitivity.
For example, WHO recommended LiPA nucleic acid hybridization probe technology can detect a drug resistance ratio of 5% -50%, xpert-MTB/RIF can detect a drug resistance ratio of about 60%, and a melting curve method can detect a drug resistance ratio of 20% -30%. In recent years, the emerging nucleic acid mass spectrometry technology can detect drug-resistant proportion higher than 20%, but when detecting heterogeneous drug-resistant flora with a proportion of about 1% by high-throughput deep sequencing, 400 times of fragment coverage rate is needed, so that detection duration and cost are greatly increased. In summary, the prior art is difficult to detect heterogeneous drug-resistant flora with the drug-resistant proportion lower than 1%, and the requirements of drug-resistant early warning and dynamic monitoring of high-risk drug-resistant patients cannot be met. There is a need in the clinic for a stable, efficient and accurate technique to achieve stable detection of heterogeneous drug-resistant flora with drug-resistant subpopulations at a ratio of 0.5-1% and early intervention.
Disclosure of Invention
Aiming at the problems, the invention provides the kit and the detection method for quantitatively detecting the drug-resistant mutation of the rifampicin with ultralow proportion, which can exert the advantages of digital PCR sensitivity and quantification, realize the stable detection of the total copy number of 10-1000/microliter with the drug-resistant mutation proportion as low as 0.5%, and meet the detection requirement of early warning of clinical drug-resistant tuberculosis.
Therefore, the invention adopts the following technical scheme: the kit for quantitatively detecting the ultra-low proportion rifampicin drug-resistant mutation is characterized by comprising a primer and a probe for targeting a mycobacterium tuberculosis rpoB drug-resistant mutation site and an internal reference gene, wherein a conserved region of a KatG gene is selected in a channel of the internal reference gene, and an upstream primer, a downstream primer and a probe sequence are designed; the gene region of the targeted mycobacterium tuberculosis rpoB is designed with an upstream primer sequence of a specific mutation site, a general downstream primer sequence and a probe sequence; the specific mutation sites comprise rpoBL430P, rpoBH445Y, rpoBH445R, rpoBD 445Y, rpoBH445D, rpoBH445L, rpoBS450L and rpoBS450W.
Preferably, the primer sequence upstream of the specific mutation site introduces a mismatched base at the 2 nd or 3 rd base of the 3' end. In the invention, the mismatched base and the mismatched base at the 3' end work together, so that the amplification product rate of the primer in the template which is not complementary with the 3' end of the primer is obviously reduced, and the primer is normally amplified in the template which is complementary with the 3' end of the primer.
Preferably, the kit further comprises a digital PCR reaction reagent and a primer probe pair, wherein the digital PCR reaction reagent comprises a PCR buffer solution, taq DNA polymerase, HEX dye and nuclease-free water.
Preferably, the specific mutation site of the upstream primer sequence is designed in the gene region of the targeted mycobacterium tuberculosis rpoB, and specific information of the general downstream primer and probe sequence is as follows:
the invention also discloses a quantitative detection method for the ultra-low-proportion rifampicin drug-resistant mutation, and the kit is used for detecting the nucleic acid sample.
The KatG gene is used as an internal control site, 8 hot spot mutant types of the rifampicin resistant gene rpoB of the mycobacterium tuberculosis are detected, and whether the sample contains mycobacterium tuberculosis DNA and the ratio of the rifampicin resistant gene mutation is judged by calculating the ratio of each detected value to the internal reference detected value of the KatG gene.
Preferably, the present invention also includes a multiplex reaction system to extract genomic DNA from a sample by pretreatment.
Preferably, the reaction procedure of the detection system comprises 5min at 98 ℃; then 15s at 98 ℃, 1min at 60 ℃ and 40 cycles; and at 25℃for 10min.
The beneficial effects of the invention are as follows:
(1) The invention relates to a technology suitable for early detection of drug-resistant tuberculosis and dynamic monitoring of high-risk drug-resistant patients. Solves the clinical problems of difficult detection of low-proportion mutation and difficult drug resistance early warning in the early drug resistance stage at present.
(2) The invention not only realizes early detection of drug resistance, but also has higher detection rate for pathogen detection due to the high sensitivity of the kit designed based on the digital PCR platform.
(3) The invention has the advantages of quantification, sensitivity, rapidness and high flux, has low detection cost, is suitable for dynamic monitoring of high-risk drug-resistant patients, has strong operability, and is easy to popularize and apply.
Drawings
FIG. 1 is a linear correlation diagram of different mutation frequencies and detection values.
FIG. 2 is a graph showing the linear relationship between the amount of sample and the detection value (high concentration range).
FIG. 3 is a graph of the load versus the detection value (low concentration range).
Detailed Description
The invention will be further illustrated with reference to specific examples.
The invention provides a digital PCR kit for detecting main mutation sites of a rifampicin resistant gene rpoB of mycobacterium tuberculosis. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The kit comprises a primer and a probe for targeting a mycobacterium tuberculosis rpoB drug-resistant mutation site and an internal reference gene, wherein a conserved region of the KatG gene is selected in a channel of the internal reference gene, and an upstream primer, a downstream primer and a probe sequence are designed; the gene region of the targeted mycobacterium tuberculosis rpoB is designed with an upstream primer sequence of a specific mutation site, a general downstream primer sequence and a probe sequence; the specific mutation sites comprise rpoBL430P, rpoBH445Y, rpoBH445R, rpoBD 445Y, rpoBH445D, rpoBH445L, rpoBS450L and rpoBS450W.
Wherein the sequences of ID1-ID9 in the nucleotide sequence listing correspond to the rpoB wild type sequence, rpoB450L, rpoB450W, rpoB P, rpoB435Y, rpoB445Y, rpoB445R, rpoB445D and rpoB445L, respectively.
In one embodiment, the primer sequence upstream of the region-specific mutation site of the rpoB gene of the target gene introduces a mismatched base at the 2 nd or 3 rd base of the 3' end. The mismatched base and the mismatched base at the 3' end work together to obviously reduce the amplification product rate of the primer in the template which is not complementary with the 3' end, and the primer is amplified normally in the template which is complementary with the 3' end.
In one embodiment, the kit further comprises a digital PCR reaction reagent, a primer pair, wherein the digital PCR reaction reagent comprises a PCR buffer, taq DNA polymerase, HEX dye, and nuclease-free water.
The invention also provides a method for detecting the main gene locus of rifampicin resistance of mycobacterium tuberculosis for non-diagnostic purposes, which comprises the step of detecting a nucleic acid sample by using the digital PCR detection kit.
In the detection method of the invention, the sample is pretreated and extracted to obtain genomic DNA.
The invention adopts KatG gene as an internal control site, and detects 8 hot spot mutants of the rifampicin resistance gene rpoB of mycobacterium tuberculosis. And (3) judging whether the sample contains the mycobacterium tuberculosis DNA and the drug-resistant bacteria duty ratio carrying the rifampicin drug-resistant gene mutation by calculating the ratio of each position detection value to the KatG gene internal reference detection value.
1. Primer probe design
According to the main mutant information of the rifampicin resistant gene rpoB of the mycobacterium tuberculosis, designing a specific primer sequence of a corresponding site and a corresponding downstream primer and fluorescent marked probe sequence. The specific information is as follows:
2. digital PCR amplification system
3. Amplification procedure
98℃5min；【98℃15s，60℃1min】*40；25℃10min
4. Experimental results display
(1) And (5) evaluating the detection stability of the multiple systems.
Table 1 multiple system concentration gradient experimental data table for each target
Experimental results: in three independent multiple systems, the plasmid templates of all target sites are subjected to gradient dilution and then are detected, and all the target sites show good linear relations, so that the performance of each target in the multiple detection system is stable, and the specificity result of each target in the multiple system is good.
(2) rpoBS450L accounts for 85-90% of the rifampicin drug-resistant tuberculosis gene mutant type in China, and by taking the detection of rpoBS450L as an example, the detection performance of the kit comprises the detection limit value of the drug-resistant bacteria mutation ratio and the sample detection concentration range, and experimental verification is carried out. The wild type template and the mutant plasmid template are proportioned according to the following experimental requirements.
TABLE 2 detection Performance data sheet at different mutation ratios
| Mutation ratio | Copy/ul |
| 10.0％ | 494.22 |
| 5.0％ | 255.92 |
| 1.0％ | 41.19 |
| 0.5％ | 24.61 |
Experimental results: table 2 and FIG. 1 show that the detection value can be stably detected when the ratio of the target point is as low as 0.5% in the multiplex system, and that the detection points of each mutation ratio show high linear correlation, R 2 Greater than 0.999.
Table 3 precision test data table
Experimental results: the three concentration templates of high, medium and low are selected for precision experimental tests, and experimental data show that the detection CV values of targets of the multiple detection system disclosed by the invention are all lower than 10%, which indicates that the precision performance of the multiple system is good.
TABLE 4 multiplex System detection Limit test data (high concentration Range)
Experimental results: as shown in Table 4 and FIG. 2, the sample loading amount of the high-concentration template reaches 6 microliters, the detection value is 1382.628 copies/microliter, the linear range of the digital PCR platform is up to more than 10 ten thousand copies, and in the linear range, each concentration value and the sample loading amount show a linear relation, R 2 The value is greater than 0.99.
TABLE 5 multiplex System detection Limit test data (Low concentration Range)
Experimental results: as shown in Table 5 and FIG. 3, the invention can still stably detect the target gene value under the condition of ultralow template concentration, and still presents high linear correlation within 10 copies. Remarks: the gradient of S1 to S7, the template addition represents the dilution factor of each experimental group, and is not added as actual template volume.
(3) Clinical sample validation
The method comprises the steps of selecting 3 clinical isolates obtained by separating respiratory tract specimens of patients with drug-resistant tuberculosis, operating in a biological safety level 3 laboratory, wherein the 3 samples are phenotypic drug-sensitive drugs for indicating rifampicin resistance, but neither primary sequencing nor secondary sequencing indicates rifampicin resistance gene mutation, and detecting by the method, and finding that 2 samples have rifampicin resistance mutation rpoBS450L.
Table 6 comparison of different platform properties
| Sample numbering | The method of the invention | First generation sequencing platform | NGS platform |
| Sample 11 | Positive and negative | - | Negative of |
| Sample 14 | Negative of | - | Negative of |
| Sample 25 | Positive and negative | Negative of | - |
| Blank control | Negative of | Negative of | Negative of |
The results illustrate: in the template detection of low mutation frequency, the mutation frequency of 10% is theoretically the lower detection limit of the first generation sequencing platform, and compared with the first generation sequencing platform, the method has the detection performance of lower mutation frequency, and can be as low as 0.5%; the invention discloses a second generation sequencing platform, wherein the lower limit of mutation frequency and sequencing depth have a certain relation, and the problems of amplification deviation and the like exist, namely the stability of the detection result of the second generation sequencing platform is challenged to a certain degree. As shown in Table 6, according to the clinical diagnosis result as the gold standard, the detection result of sample 11/14/25 is superior to the first generation sequencing platform and the NGS platform.
The invention is based on the design concept of the base complementary pairing principle of the specific primer, can detect whether the sample contains the mycobacterium tuberculosis nucleic acid and whether the sample contains the rifampicin specific drug-resistant mutant, calculates the drug-resistant bacteria ratio carrying the rifampicin drug-resistant mutation, and realizes the quantitative detection of the heterogeneous drug-resistant ratio.
The invention greatly improves the problem of insufficient sensitivity of the existing early detection technology of drug-resistant tuberculosis, reduces the mutation proportion detected stably to 0.5%, has good stability when the concentration range of nucleic acid is 10-1000 copies/microliter, realizes quantitative detection of the mutation proportion, and has the advantages of short detection time, high flux, low cost and the like.
It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. Further, it is understood that various changes and modifications may be made by those skilled in the art after reading the teachings of the present invention, and such equivalents are intended to fall within the scope of the claims appended hereto.
Claims (8)
1. The kit for quantitatively detecting the ultra-low proportion rifampicin drug-resistant mutation is characterized by comprising a primer and a probe for targeting a mycobacterium tuberculosis rpoB drug-resistant mutation site and an internal reference gene, wherein a conserved region of a KatG gene is selected in a channel of the internal reference gene, and an upstream primer, a downstream primer and a probe sequence are designed; the gene region of the targeted mycobacterium tuberculosis rpoB is designed with an upstream primer sequence of a specific mutation site, a general downstream primer sequence and a probe sequence; the specific mutation sites comprise rpoBL430P, rpoBH445Y, rpoBH445R, rpoBD 445Y, rpoBH445D, rpoBH445L, rpoBS450L and rpoBS450W.
2. The kit for quantitatively detecting the ultra-low-proportion rifampicin resistant mutation according to claim 1, wherein the upstream primer sequence of the specific mutation site introduces a mismatched base at the 2 nd or 3 rd base of the 3' end.
3. The kit for quantitatively detecting the ultra-low-proportion rifampicin resistant mutation according to claim 2, further comprising a digital PCR reaction reagent and a primer pair, wherein the digital PCR reaction reagent comprises a PCR buffer, taq DNA polymerase, HEX dye and nuclease-free water.
4. A kit for quantitatively detecting a rifampicin resistant mutation in an ultralow proportion according to any one of claims 1 to 3, characterized in that the gene region of the targeted mycobacterium tuberculosis rpoB is designed with an upstream primer sequence of a specific mutation site, and specific information of a general downstream primer and a probe sequence is as follows:
5. a quantitative detection method for ultra-low proportion rifampicin drug-resistant mutation, which is characterized in that the kit of any one of claims 1-4 is adopted to detect nucleic acid samples.
6. The method according to claim 5, wherein the detection of 8 hot spot mutations of the rifampicin resistant gene rpoB of Mycobacterium tuberculosis is performed by using the KatG gene as an internal control site, and determining whether the sample contains Mycobacterium tuberculosis DNA and the ratio of the resistant bacteria carrying the rifampicin resistant gene mutation by calculating the ratio of each of the detected values to the internal reference detection value of the KatG gene.
7. The method according to claim 5 or 6, comprising a multiplex assay system, wherein the assay system comprises:
8. the method according to claim 7, wherein the reaction procedure of the detection system comprises 98℃for 5min; then 15s at 98 ℃, 1min at 60 ℃ and 40 cycles; and at 25℃for 10min.
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