WO2020052356A1 - 一种同时检测结核分枝杆菌利福平与异烟肼耐药基因点突变的方法及其试剂盒 - Google Patents

一种同时检测结核分枝杆菌利福平与异烟肼耐药基因点突变的方法及其试剂盒 Download PDF

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WO2020052356A1
WO2020052356A1 PCT/CN2019/098074 CN2019098074W WO2020052356A1 WO 2020052356 A1 WO2020052356 A1 WO 2020052356A1 CN 2019098074 W CN2019098074 W CN 2019098074W WO 2020052356 A1 WO2020052356 A1 WO 2020052356A1
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reaction tube
mutant
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刘利成
王华贵
邹弈君
陈璐
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江苏宏微特斯医药科技有限公司
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

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  • the invention relates to the technical field of molecular biology, in particular to a method and a kit for simultaneously detecting point mutations of Mycobacterium tuberculosis rifampicin and isoniazid resistance genes.
  • tuberculosis commonly known as tuberculosis
  • tuberculosis is a pathogen that causes tuberculosis, which can invade various organs in the body, but tuberculosis is the most common clinically.
  • Tuberculosis has become a very important infectious disease globally.
  • One third of the world's population is infected with Mycobacterium tuberculosis.
  • China is one of the 22 countries with severe TB epidemics in the world, with the number of cases ranking second in the world, and 46% of drug-resistant tuberculosis patients account for 46% of tuberculosis patients in China.
  • the development of drug resistance has greatly increased the difficulty of treating tuberculosis. This makes it very necessary to test patients for TB resistance mutation sites before TB treatment, so as to carry out reasonable medication.
  • the resistance types of first-line drugs in domestic drug-resistant tuberculosis patients are as follows: the two drugs with the highest rate of single drug resistance and any drug resistance among newly treated patients are isotonic smoke Hydrazine (28.2%) and streptomycin (20.7%), and the combinations with the highest multidrug resistance and multidrug resistance are: isoniazid + streptomycin (13.7%), isoniazid + rifampicin (5.4 %), Rifampicin + streptomycin (3.7%), isoniazid + rifampicin + streptomycin (2.9%), isoniazid + ethambutol (5.8%); drug resistance in retreated patients One drug whose rate has increased significantly is rifampicin.
  • the main gene related to rifampicin resistance is rpoB. 80% of rifampicin resistant strains are caused by mutations in the rpoB gene, and more than 95% of rpoB mutations occur in a highly conserved 81bp core region. Among them, mutations usually occur at codons 531, 526, and 516, and the main mutation forms are S531L, H526Y, and D516V.
  • the main genes related to isoniazid resistance are katG and inhA.
  • the mutation frequency of the 315 locus of the katG gene is the highest, and 30% -60% of isoniazid resistant strains will have a mutation at codon 315.
  • the S315T Mutation causes the enzyme to lose its ability to activate isoniazid; mutation of the C base at position -15 of the promoter region of the inhA gene to a T base is also an important point mutation in resistance to isoniazid resistant strains.
  • the existing methods for detecting resistance mutation sites of Mycobacterium tuberculosis include: chip method, MGB probe method, and fluorescence quantitative PCR melting curve method.
  • chip method although the detection rate is high, the cost is also high; MGB detection Needle method relies on only one probe to distinguish, and sometimes results are not accurate; the interpretation of the results of the fluorescence quantitative PCR melting curve method needs to refer to the peak shape and number of melting peaks and the Tm value, which increases the requirements for basic laboratory personnel. .
  • the ARMS system is a commonly used analysis technique for gene mutation. Its genotyping test usually includes two complementary PCR reactions, using the same DNA template and the same common primer and reaction conditions. The only difference is that the ARMS primers paired with the common primer are different. This allows the two reactions to selectively amplify specific DNA templates. It can perform genotyping quickly and economically, and has high accuracy and cost performance. Various improvements to the ARMS system have also spawned a variety of highly accurate methods for detecting point mutations.
  • the invention provides a method for simultaneously detecting a point mutation of a Mycobacterium tuberculosis rifampicin and an isoniazid resistance gene.
  • the method is based on an ARMS fluorescent quantitative PCR method.
  • the resistance to the drug resistance is separately used.
  • the ⁇ Ct value is the Ct value of the mutant reaction tube at the mutation site and the wild-type reaction tube Ct The difference in values.
  • the method uses a Taqman probe, which is designed to detect the amplification efficiency of the ARMS primer on the template within the amplified fragment of the ARMS primer.
  • the point mutation of the drug resistance gene is a rifampicin resistance point mutation rpoB 516A> T, rpoB 526C> T ⁇ G, rpoB 531C> G ⁇ T, isoniazid resistance point Mutations katG 315 G> C ⁇ A and InhA-15 C> T.
  • one multiple reaction combination is rpoB 526 C> T ⁇ G and katG 315 G> C ⁇ A two mutation sites and a reaction solution of the internal reference gene; the other multiple reaction combination is rpoB 516 A> T RpoB, 531, C> G ⁇ T and InhA-15, C> T three reaction sites, rpoB, 516, A> T and 531, C> G ⁇ T share a downstream primer.
  • the internal reference gene is gene IS6110.
  • the primer sequences for detecting the point mutation and the reference gene are as follows:
  • the probe sequence for detecting the point mutation and the reference gene is:
  • rpoB 526 C> T ⁇ G uses FAM detection channel
  • katG 315 G> C ⁇ A uses Cy5 detection channel
  • internal reference gene uses ROX channel
  • rpoB 516 A> T uses FAM detection channel
  • rpoB 531C > G ⁇ T uses VIC detection channel
  • InhA-15 C> T uses ROX channel.
  • the sample needs to be re-extracted for detection; if the internal reference gene has an amplification signal, 1) targeting 516A> T and 531C> G ⁇ T of the rpoB gene Point, only whether there are mutations in the wild type and these two sites, but not at which sites are the mutations:
  • mutant-type reaction tube has an amplification signal and the wild-type reaction tube has no amplification signal, it is determined to be a mutation homozygous type; if both the mutant-type reaction tube and the wild-type reaction tube have amplification signals, and the ⁇ Ct value is ⁇ Ct ⁇ -6, also determined as mutation homozygous;
  • both the mutant reaction tube and the wild type reaction tube have amplification signals: for rpoB 526 C> T ⁇ G, if the ⁇ Ct value of the two reaction tubes is 0 ⁇ Ct ⁇ 12, it is determined as rpoB 526 C> T ⁇ G Heterozygous mutation; for katG 315 G> C ⁇ A, if the ⁇ Ct value of the two reaction tubes is 0 ⁇ Ct ⁇ 18, it is determined to be katG 315 G> C ⁇ A hybrid mutation; for inhA-15 C> T, if If the ⁇ Ct value of the two reaction tubes is 0 ⁇ Ct ⁇ 15, it is determined as inhA-15C> T heterozygous mutant.
  • the present invention provides a kit for detecting point mutations in Mycobacterium tuberculosis rifampicin and isoniazid resistance genes based on ARMS fluorescence quantitative PCR, said kit according to any one of the methods described above. Use.
  • the primers of the ARMS method of the invention have strong specificity, high sensitivity, low reagent cost, and low interference between each primer in a multiplex PCR reaction, which greatly improves the sensitivity of the method of the invention; the classic detection gene of tuberculosis introduced in the invention As an internal reference group, IS6110 can guarantee highly reliable detection results.
  • the operation is simple, and the main resistance sites of first-line drugs for tuberculosis can be detected through multiple tests in a short time, which greatly reduces the testing costs and costs, and the time required for testing.
  • the method of the present invention is straightforward to judge, and the requirements for technicians using the method are greatly reduced, which is convenient for the promotion and application of the method of the present invention.
  • Figure 1 shows the amplification curves of rpoB, 516, A> T, rpoB, 531, C> G, T, InhA-15, and C> T in 1 # clinical samples.
  • A1 rpoB 516 and A> T mutant type.
  • Figure 2 shows rpoB, 526, C> T ⁇ G and katG, 315, G> C, and A> C ⁇ A amplification curves of 1 # clinical samples, and A1: rpoB 526 and C> T ⁇ G mutations.
  • Curve of A type primer amplification A2: rpoB 526 C> T ⁇ G wild type primer amplification curve; B1: katG 315 G> C ⁇ A mutant primer amplification curve, B2: katG 315 G> C ⁇ A Curve of wild type primer amplification; C1: amplification curve of internal reference gene.
  • Figure 3 shows the amplification curve of rpoB, 516, A, T, rpoB, C, G, T, InhA-15, C, and T mutations in clinical sample # 2, where A1: rpoB, 516, and A> T mutant primers.
  • Figure 4 shows rpoB, 526, C> T ⁇ G and katG, 315, G> C, and A> C ⁇ A amplification curves of 2 # clinical samples, where A1: rpoB, 526, and C> T ⁇ G mutations.
  • Curve of A type primer amplification A2: rpoB 526 C> T ⁇ G wild type primer amplification curve; B1: katG 315 G> C ⁇ A mutant primer amplification curve, B2: katG 315 G> C ⁇ A Curve of wild type primer amplification; C1: amplification curve of internal reference gene.
  • PCR buffer, dNTP, dUTP, UDG, and DNA polymerase were purchased from Shenzhen Feipeng Biological Co., Ltd.
  • the primers and probes used were synthesized by Shanghai Biological Engineering Technology Service Co., Ltd., and a standard plasmid extraction kit Purchased from Beijing Tiangen Biotechnology Co., Ltd.
  • Clinical samples are DNA extracted from sputum provided by the hospital.
  • Table 1 Primer sequence information for detecting five point mutations and reference genes
  • Table 2 Probe sequence information for detecting five point mutations and reference genes
  • Samples are two mutant standard plasmids of katG, 315, G> C ⁇ A and wild type standard plasmids, and mixed plasmids in which mutant standard plasmids and wild type standard plasmids are mixed at a certain ratio: 50% mutations, 10% mutations , 1% mutation, 0.1% mutation, the specific mixing method is shown in the following table:
  • the fluorescence PCR amplification system is:
  • the ARMS primers were evaluated in terms of the difference in amplification efficiency between the primer pair for the mutant template and the wild type template and the sensitivity when the primer amplified the corresponding type of template: mutant ARMS primers K315-F1 ⁇ K315-F2 ⁇ K315- When F3 ⁇ K315-F4 was used to amplify the mutant template and the wild type template, the Ct difference between the two types of templates was ⁇ Ct ⁇ 8, the difference in amplification efficiency was small, which was not conducive to the type distinction, and did not meet the type distinction. Requirements.
  • K315-F5 ⁇ K315-F6 ⁇ K315-F8 ⁇ K315-F9 ⁇ K315-F10 ⁇ K315-F11 is used to amplify a low-concentration template (10copies / reaction and 10 2 copies / reaction)
  • the mutant template is amplified, but Ct value is high, Ct> 33 (10copies / reaction), no amplification of wild type template; when amplifying high concentration template ( ⁇ 10 3 copies / reaction), the Ct difference between the two types of template is 8 ⁇ Ct ⁇ 12, the difference in amplification efficiency is obvious; when the mixed template is amplified, the 0.1% mutant template has no amplification signal and cannot distinguish between the 0.1% mutation and the wild type, so these mutant ARMS primers do not meet the requirements.
  • K315-F7 (which is K315-F in the preferred primer)
  • the template concentration is 10 copies / reaction, 10 2 copies / reaction and 10 3 copies / reaction
  • the amplified mutant template has amplification and Ct ⁇ 32
  • amplified No wild-type template was amplified; at template concentrations ⁇ 10 4 copies / reaction, the Ct difference between the mutant template and the wild-type template was ⁇ Ct> 12, and the amplification efficiency was significantly different.
  • the mutant template When the mixed template was amplified, 0.1%
  • the mutant template also has an amplification signal; when triple-amplified with the detection of the 526 locus of the rpoB gene and the internal reference gene IS6110, this primer can still distinguish between mutant, wild-type, and 0.1% mutant types; that is, K315 -F
  • This mutant ARMS primer has both good discrimination effect and high sensitivity for amplifying mutant templates, and is not disturbed in multiple reactions, so it is preferably katG 315 G in the present invention.
  • the difference in Ct between the two types of templates is ⁇ Ct>17; but when compared with rpoB
  • the primer could not distinguish between the wild type and the 0.1% mutant type, which did not meet the requirements.
  • W-K315-F7 was amplifying 10copies / reaction template, the Ct difference between mutant template and wild-type template was ⁇ Ct ⁇ 6, the difference in amplification efficiency was small, and it was impossible to effectively distinguish the types.
  • W-K315-F5 and W-K315-F6 were used to amplify the mutant template and the wild type template, the mutant template did not have an amplification signal, the wild type template had an amplification signal and Ct ⁇ 31, and the amplification efficiency was significantly different; but When performing triple amplification with the detection of the 526 locus of the rpoB gene and the internal reference gene IS6110, the binding efficiency of the primers is affected. When amplifying a low-concentration template, the amplification efficiency of the mutant template and the wild-type template are not significantly different. , Cannot be distinguished. W-K315-F4 (which is W-K315-F in the preferred primer).
  • the mutant template When a mutant template and a wild type template are amplified, the mutant template has no amplification signal, and the wild type template has an amplification signal and Ct ⁇ 31. (10copies / reaction), the amplification efficiency is significantly different; when triple amplification is performed with the detection of the 526 locus of the rpoB gene and the internal reference gene IS6110, the primer binding efficiency is not affected.
  • the mutant template has no amplification
  • the wild type template has amplification and Ct ⁇ 32
  • at high concentration template ⁇ 10 4 copies / reaction
  • the Ct difference of the type template is ⁇ Ct> 16, and the type can still be effectively distinguished, so it is preferably the wild-type ARMS primer with katG 315 G> C ⁇ A in the present invention.
  • the ARMS primers for the other four loci were screened using the above-mentioned katG 315 and G> C ⁇ A primer selection principles and methods.
  • the preferred mutant ARMS primer for rpoB 516 A> T is 516-AF13, the preferred wild type ARMS primer is W-516-AF8; the preferred mutant ARMS primer for rpoB 531 C> G ⁇ T is 531-F5, and wild type ARMS is preferred
  • the primer is W-531-F1; rpoB 526 C> T ⁇ G is the preferred mutant ARMS primer 526-F1, and the wild type ARMS primer is W-526-F2;
  • InhA-15 is the preferred mutant ARMS primer for C> T It is Ih-R16, and the wild type ARMS primer is preferably W-Ih-R5.
  • the selected primers are shown in the following table:
  • Primer probes are as described in the preferred primer probes in Tables 1 and 2;
  • the samples are rpoB, 526, C> T ⁇ G, katG, 315, G> C ⁇ A, and the mutant standard plasmid and the wild-type standard plasmid at the three positions of the internal reference gene IS6110 are mixed in a corresponding manner. Plasmids, of which there are two types of mutant plasmids at 526 sites: 526-T1 (C> T) and 526-T2 (C> G).
  • the wild-type plasmid of 526 is rpoB-W; katG 315 and G> C ⁇ A.
  • the mutant plasmids are K315-T1 (G> C) and K315-T2 (G> A), the wild-type plasmid is K315-W; the standard plasmid of the internal reference gene IS6110 is IS; and the mutant plasmid and wild-type plasmid are used according to certain Proportional mixed plasmid: 50% mutation, 10% mutation, 1% mutation, 0.1% mutation.
  • the specific mixing mode is shown in the following table:
  • the fluorescence PCR amplification system is:
  • the detection result is consistent with the actual genotype of the template; when the homozygous mutant plasmid of three sites is mixed with templates -2 and -4, when the template is 10copies / reaction, 10 2 copies / reaction, 10 Mutation reaction wells were amplified at 3 copies / reaction, but not wild-type reaction wells.
  • the template was 10 4 copies / reaction, 10 5 copies / reaction, 10 6 copies / reaction, the mutation reaction well was different from wild type.
  • the ⁇ Ct value of the reaction well is ⁇ Ct ⁇ -6. It can be judged that rpoB 526 C> G is a homozygous mutation.
  • the test result and the actual genotype of the template Character When amplifying the three-site wild-type plasmid mixed template, when the template is 10copies / reaction, 10 2 copies / reaction, 10 3 copies / reaction, 10 4 copies / reaction, the wild-type reaction wells are amplified, and the mutant type There is no amplification in the reaction wells.
  • the ⁇ Ct value of the mutant reaction well and the wild type reaction well is ⁇ Ct> 12, and it can be determined that rpoB 526 C> T ⁇ G is wild.
  • the detection results are consistent with the actual genotype of the template.
  • the template -1, -2, -3, and -4 of the three types of wild-type and mutant-type plasmids are amplified, the ⁇ Ct value of the mutant-type reaction well and the wild-type reaction well is 0 ⁇ Ct ⁇ 12.
  • rpoB 526 C> T ⁇ G is a heterozygous mutation, and the detection result is consistent with the actual genotype of the template.
  • Cy5 channel (katG 315 G> C ⁇ A site detection channel): when homozygous mutant plasmid mixed template-1 and mutant plasmid mixed template-2 are amplified at three sites, when the template is 10copies / Reaction, 10 2 copies / reaction, 10 3 copies / reaction, amplification of mutant reaction wells, no amplification of wild-type reaction wells, when the template is 10 4 copies / reaction, 10 5 copies / reaction, 10 6 copies / reaction At this time, the ⁇ Ct value of the mutant response well and the wild-type response well is ⁇ Ct ⁇ -6. It can be determined that katG 315 G> C is a homozygous mutation, and the detection result is consistent with the actual genotype of the template.
  • wild-type reaction wells when amplifying three-site wild-type plasmid mixed templates, wild-type reaction wells when the template is 10copies / reaction, 10 2 copies / reaction, 10 3 copies / reaction, 10 4 copies / reaction, 10 5 copies / reaction There is amplification, but there is no amplification in the mutant reaction well.
  • the template is 10 6 copies / reaction
  • the ⁇ Ct value of the mutant reaction well and the wild type reaction well is ⁇ Ct> 19, and it can be determined that katG 315 G> C ⁇ A is wild. The detection results are consistent with the actual genotype of the template.
  • the ⁇ Ct value of the mutant-type reaction well and the wild-type reaction well is 0 ⁇ Ct ⁇ 18, which can be judged katG 315 G> C ⁇ A is a heterozygous mutation, and the detection result is consistent with the actual genotype of the template.
  • ROX channel detection channel of the internal reference gene IS6110: In the mixed template -1, -2, -3, and -4 of the mutant plasmid amplified at three sites, in the ROX channel of the mutant reaction well: when the template When there are 10copies / reaction, 10 2 copies / reaction, 10 3 copies / reverse, 10 4 copies / reaction, 10 5 copies / reaction, 10 6 copies / reaction, there are amplification signals, and the sensitivity can reach 10 copies / reaction.
  • Primer probes are as described in the preferred primer probes in Tables 1 and 2;
  • the samples are rpoB, 516, A> T, rpoB, 531, C> G ⁇ T, and InhA-15, and C> T three mutant standard plasmids and wild type standard plasmids are mixed in a corresponding manner.
  • the 516 and 531 sites are constructed on one plasmid, so the wild-type plasmids of both are rpoB-W, and the double mutant plasmids of both are rpoB-T1 (516A> T, 531C> G) and rpoB.
  • 516A> T, 531C> T 516 mutant type and 531 wild type plasmid is 516-T
  • 516 wild type and 531 mutant type plasmid are 531-T1 (531C> G) and 531-T2 ( 531 (C> T)
  • InhA-15 The mutant plasmid of C> T is InhA-T, and the wild-type plasmid is InhA-W; and the plasmid mixed with the wild-type plasmid at a certain ratio: 50% mutation, 10% mutation, 1% mutation, 0.1% mutation, the specific mixing mode is shown in the following table:
  • the fluorescence PCR amplification system is:
  • the ⁇ Ct value in the FAM channel in the mutant reaction well and the wild type reaction well is ⁇ Ct ⁇ 10, and the mutant reaction well reacts with the wild type
  • the ⁇ Ct value in the VIC channel in the well is ⁇ Ct ⁇ 8; from this, it can be determined that there are mutations in the two positions of 516 and 531, and the detection result is consistent with the actual genotype of the template.
  • wild-type and mutant plasmid mixed templates -2 and -3 were amplified at three sites, when the template was 0.1% mutation, 1% mutation, and 10% mutation, there was no amplification in the FAM channel of the mutant reaction well.
  • the VIC of the mutant reaction well and the wild type reaction well There are amplifications in the channels, and the ⁇ CT value in the VIC channels of the two reaction wells is ⁇ CT ⁇ 5; it can be determined that there are mutations in the two positions of 516 and 531, and the detection results are consistent with the actual genotype of the template.
  • wild-type and mutant plasmid mixed template-4 was amplified at three sites, both the mutant reaction well and the wild-type reaction well were amplified in the FAM channel, and the ⁇ Ct value in the FAM channel of the two reaction wells was ⁇ Ct.
  • the VIC channels of the mutant reaction well and the wild type reaction well are amplified in the VIC channel, and the ⁇ Ct value in the VIC channel of the two reaction wells is ⁇ Ct ⁇ 8; thus it can be judged that there are mutations in the two positions of 516 and 531
  • the test results are consistent with the actual genotype of the template.
  • FAM and VIC channels in mutant reaction wells when amplifying three-site wild-type plasmid mixed templates when the template is 10copies / reaction, 10 2 copies / reaction, 10 3 copies / reaction, 10 4 copies / reaction There is no amplification in the FAM and VIC channels in the wild-type reaction wells; when the template is 10 5 copies / reaction and 10 6 copies / reaction, the FAM channel in the mutant reaction well and the wild-type reaction well Both were amplified, and the ⁇ Ct value of the two reaction wells was ⁇ Ct> 11, and the VIC channels of the mutant and wild type reaction wells were also amplified, and the ⁇ Ct value of the two reaction wells was ⁇ Ct>16; It can be determined that the two sites of rpoB 516 A> T and 531 C> G ⁇ T are wild-type, and the detection results are consistent with the actual genotype of the template.
  • InhA-15 C> T detection channel when homozygous mutant plasmids of three sites are mixed with templates -1, -2, and -3, when the template is 10 copies / reaction, 10 2 copies / Reaction, 10 3 copies / reaction, 10 4 copies / reaction, 10 5 copies / reaction when the mutant reaction wells are amplified, but wild-type reaction wells are not amplified; when the template is 10 6 copies / reaction, the mutant and The wild-type reaction wells were amplified, and the ⁇ Ct value was ⁇ Ct>15; from this, it can be judged that InhA-15 C> T is a homozygous mutation, and the detection result is consistent with the actual genotype of the template.
  • the template When amplifying the three-site wild-type plasmid mixed template, when the template is 10copies / reaction, 10 2 copies / reaction, 10 3 copies / reaction, 10 4 copies / reaction, the wild-type reaction wells are amplified, and the mutant type There is no amplification in the reaction wells.
  • the template is 10 5 copies / reaction and 10 6 copies / reaction, the ⁇ Ct value of the mutant reaction well and the wild type reaction well is ⁇ Ct> 15, and it can be determined that InhA-15 C> T is wild type. The test results are consistent with the actual genotype of the template.
  • Example 5 The detection system of five drug resistance mutation sites of rifampicin and isoniazid in the present invention is used to detect two clinical samples of known genotypes
  • the actual genotypes of clinical samples 1 # and 2 # are: 1 # is a rifampicin-resistant isoniazid katG 315 mutation, and 2 # is a rifampicin-resistant isoniazid mutation InhA;
  • the fluorescence PCR amplification system is:
  • rpoB 526 C> T ⁇ G and katG 315 G> C ⁇ A test results of the two resistance sites and the internal reference gene IS6110, as shown in Figure 4: In the FAM channel, the mutant reaction wells are amplified, and the wild-type reaction There is no amplification in the well, and it can be judged that rpoB 526 C> T ⁇ G is a homozygous mutant; in the Cy5 channel, there is no amplification in the mutant reaction well and there is amplification in the wild-type reaction well, it can be judged that katG 315 G> C ⁇ A is Wild type; the determination results of these two loci are consistent with the actual genotype of the sample; the Ct value in the ROX channel of the mutant reaction well is 24.11, which indicates that the detection is valid.

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Abstract

一种同时检测结核分枝杆菌利福平与异烟肼耐药基因点突变的方法及其试剂，所述方法基于ARMS荧光定量PCR方法，在相同的反应体系中，分别使用针对所述耐药基因突变位点的突变型模板的ARMS引物和针对野生型模板的ARMS引物及它们共用的上游或下游引物，用突变型反应管与野生型反应管共两个反应管对同一份待测样本进行检测，通过各个点突变在两个反应管中的荧光PCR的Ct值和ΔCt值的大小判定样本的基因型。

Description

一种同时检测结核分枝杆菌利福平与异烟肼耐药基因点突变的方法及其试剂盒 技术领域
本发明涉及分子生物学技术领域，特别涉及一种同时检测结核分枝杆菌利福平与异烟肼耐药基因点突变的方法及其试剂盒。
背景技术
结核分枝杆菌(M.tuberculosis，TB)，俗称结核杆菌，是引起结核病的病原菌，其可侵犯全身各器官，但在临床上以肺结核最为多见。现如今结核病已经成为全球非常重要的传染病，世界1/3的人口感染结核分枝杆菌。我国是全球22个结核病流行严重的国家之一，发病人数位居全球第2位，且我国结核病人中耐药结核病人占46％，耐药性的产生给结核的治疗大大地增加了难度，这就使得非常有必要在结核治疗前对病人进行结核耐药突变位点的检测，从而进行合理用药。
根据我国第五次全国结核病流调调查报告，国内耐药结核病人一线药物的耐药类型表现为：初治患者中单耐药与任何耐药的耐药率最高的两种药都是异烟肼(28.2％)与链霉素(20.7％)，而耐多药和多耐药最高的几种组合是：异烟肼+链霉素(13.7％)，异烟肼+利福平(5.4％)，利福平+链霉素(3.7％)，异烟肼+利福平+链霉素(2.9％)，异烟肼+乙胺丁醇(5.8％)；复治患者中耐药率显著提升的一个药是利福平。现今，临床上已经很少甚至不再使用链霉素这项药物，所以针对我国结核病人的耐药情况，利福平与异烟肼两种治疗结核病的一线药物的耐药突变位点，应为病人首要检测的耐药突变位点。
与利福平耐药相关的主要基因为rpoB，80％的利福平耐药株都是因为rpoB基因产生了突变，而超过95％的rpoB突变都是发生在一个高度保守的81bp的核心区域内，其中通常发生在第531、526与516位密码子的突变，主要的突变形式为S531L、H526Y、D516V这三种形式。
与异烟肼耐药相关的主要基因为katG与inhA，katG基因的315位点的突变频率最高，30％-60％的异烟肼耐药株会发生315位密码子的突变，S315T这一突变导致酶失去活化异烟肼的能力；inhA基因启动子区的-15位点的C碱基突变成T碱基也是异烟肼耐药株产生耐药的一个重要点突变。
现有的检测结核杆菌耐药突变位点的方法中有：芯片法、MGB探针法、荧光定量PCR熔解曲线法等，对于芯片法，其检出率虽高但是其成本也高；MGB探针法只依靠一条探 针进行区别，有时造成结果不够准确；荧光定量PCR熔解曲线法其结果判读需要参看熔解峰的峰型与个数及Tm值，这就提高了对基层实验室人员的要求。
ARMS系统是基因突变常用的分析技术，其基因分型检测通常包括两个互补的PCR反应，使用相同的DNA模板和一条相同的共有引物和反应条件，区别仅在于与共有引物配对的ARMS引物不同，从而使两个反应选择性扩增特定的DNA模板。其可以快速经济的进行基因型分析，且具有极高的准确率及性价比。对ARMS系统进行各方面的改进，也催生出了多种检测点突变的高精确度的方法。
发明内容
本发明提供一种同时检测结核分枝杆菌利福平与异烟肼耐药基因点突变的方法，所述方法基于ARMS荧光定量PCR方法，在相同的反应体系中，分别使用针对所述耐药基因突变位点的突变型模板的ARMS引物和针对野生型模板的ARMS引物及它们共用的上游或下游引物，用突变型反应管与野生型反应管共两个反应管对同一份待测样本进行检测，通过各个点突变在两个反应管中的荧光PCR的Ct值和ΔCt值的大小判定样本的基因型，所述ΔCt值为突变位点的突变型反应管Ct值与野生型反应管Ct值之差。
在一种实施方式中，所述方法使用Taqman探针，所述探针为在ARMS引物扩增片段内设计用于检测ARMS引物对模板的扩增效率。
在一种实施方式中，所述耐药基因点突变是利福平耐药点突变rpoB 516 A>T、rpoB 526 C>T\G、rpoB 531 C>G\T，异烟肼耐药点突变katG 315 G>C\A和InhA-15 C>T。
在一种实施方式中，一个多重反应组合是rpoB 526 C>T\G与katG 315 G>C\A两个突变位点以及内参基因的反应液；另一个多重反应组合是rpoB 516 A>T、rpoB 531 C>G\T和InhA-15 C>T三个突变位点的反应液，rpoB 516 A>T与531 C>G\T两个位点共用一条下游引物。
在一种实施方式中，所述内参基因为基因IS6110。
在一种实施方式中，检测所述点突变与内参基因的引物序列如下：
Figure PCTCN2019098074-appb-000001
Figure PCTCN2019098074-appb-000002
在一种实施方式中，检测所述点突变与内参基因的探针序列是：
Figure PCTCN2019098074-appb-000003
在一种实施方式中，rpoB 526 C>T\G使用FAM检测通道，katG 315 G>C\A使用Cy5检测通道，内参基因使用ROX通道；rpoB 516 A>T使用FAM检测通道，rpoB 531 C>G\T使用VIC检测通道，InhA-15 C>T使用ROX通道。
在一种实施方式中，若内参无扩增信号，需重新提取样本进行检测；若内参基因有扩增信号下，1)针对rpoB基因的516 A>T与531 C>G\T两个位点，只判读野生型与这两个位点中是否有突变，而不判读具体是哪个位点的突变：
①以下六种情况判读516与531这两个位点中有突变：Ⅰ：突变型反应管FAM与VIC通道内都有扩增信号，而野生型反应管FAM与VIC通道内都没有扩增信号；Ⅱ：突变型反应管与野生型反应管FAM与VIC通道内都有扩增信号，且两个通道内各自的ΔCt值都为ΔCt<-6；Ⅲ：突变型反应管FAM通道内没有扩增信号，野生型反应管FAM通道内有扩增信号，且突变型反应管与野生型反应管VIC通道内ΔCt值为ΔCt<16；Ⅳ：突变型反应管与野生型反应管FAM通道内的ΔCt值为ΔCt>11,且同时突变型反应管与野生型反应管VIC通道内的ΔCt值为ΔCt<16；Ⅴ：突变型反应管FAM通道内有扩增信号，野生型反应管内FAM通道内无扩增信号，且突变型反应管与野生型反应管VIC通道内ΔCt值为ΔCt<16；Ⅵ：突变型反应管与野生型反应管内FAM通道内ΔCt值为ΔCt<11，且突变型反应管与野生型反应管VIC通道内ΔCt<16；
②以下两种情况判读516与531这两个位点均为野生型：Ⅰ：若突变型反应管FAM与VIC通道内都无扩增信号，同时野生型反应管FAM与VIC通道内都有扩增信号；Ⅱ：若突变型反应管与野生型反应管都有扩增信号，且FAM通道内的ΔCt值为ΔCt≥11，VIC通道内的ΔCt值为ΔCt≥16；
2)针对rpoB基因的526 C>T\G、katG基因的315 G>C\A、InhA基因的-15 C>T三 个位点：
①若突变型反应管有扩增信号，而野生型反应管无扩增信号，则判定为突变纯合型；若突变型反应管与野生型反应管均有扩增信号，且ΔCt值为ΔCt<-6，也判定为突变纯合型；
②若突变型反应管无扩增信号，而野生型反应管有扩增信号，则判定为野生型；若突变型反应管与野生型反应管均有扩增信号：针对rpoB 526 C>T\G，若两管的ΔCt值为ΔCt≥12，也判定为rpoB 526 C>T\G野生型；针对katG 315 G>C\A，若两管的ΔCt值为ΔCt≥18，也判定为katG 315 G>C\A野生型；针对inhA-15 C>T，若两管的ΔCt值为ΔCt≥15，也判定为inhA-15 C>T野生型；
③若突变型反应管与野生型反应管均有扩增信号：针对rpoB 526 C>T\G，若两反应管的ΔCt值为0≤ΔCt＜12，则判定为rpoB 526 C>T\G杂合突变；针对katG 315 G>C\A，若两反应管的ΔCt值为0≤ΔCt＜18，则判定为katG 315 G>C\A杂合突变；针对inhA-15 C>T，若两反应管的ΔCt值为0≤ΔCt＜15，则判定为inhA-15 C>T杂合突变型。
在一种实施方式中，本发明提供一种基于ARMS荧光定量PCR检测结核分枝杆菌利福平与异烟肼耐药基因点突变的试剂盒，所述试剂盒在上述任一所述的方法中使用。
本发明ARMS方法的引物特异性强、敏感性高，试剂成本低廉，各引物在多重PCR反应中彼此之间干扰小，使得本发明方法的灵敏度大大提高；本发明中引入结核的经典检出基因IS6110作为内参基团，可保证检测结果高度可靠。其操作简便，可在短时间内通过多重检测，检出结核一线药物的主要耐药位点，大大降低了检测成本和费用，以及检测所需要的时间。另外，本发明方法判断直接，对于使用本方法的技术人员要求大大降低，便于本发明方法的推广和应用。
附图说明
为了更清楚地说明本申请实施例中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请中记载的一些实施例，对于本领域普通技术人员来说，在不付出创造性劳动的前提下，还可以根据这些附图获得其它的附图。
图1显示1#临床样本的rpoB 516 A>T、rpoB 531 C>G\T、InhA-15 C>T三个耐药位点的扩增曲线图；其中A1：rpoB 516 A>T突变型引物扩增的曲线，A2：rpoB 516 A>T野生型引物扩增的曲线；B1：rpoB 531 C>G\T突变型引物扩增的曲线，B2：为rpoB 531 C>G\T野生型引物扩增的曲线；C1：InhA-15 C>T突变型引物扩增的曲线，C2：InhA-15 C>T野生型引物扩增的曲线。
图2显示1#临床样本的rpoB 526 C>T\G与katG 315 G>C\A两个耐药位点以及内参基因IS6110的扩增曲线图，其中A1：rpoB 526 C>T\G突变型引物扩增的曲线，A2：rpoB 526 C>T\G野生型引物扩增的曲线；B1：katG 315 G>C\A突变型引物扩增的曲线，B2：katG 315 G>C\A野生型引物扩增的曲线；C1：内参基因的扩增曲线。
图3显示2#临床样本的rpoB 516 A>T、rpoB 531 C>G\T、InhA-15 C>T三个突变位 点的扩增曲线图，其中A1：rpoB 516 A>T突变型引物扩增的曲线，A2：rpoB 516 A>T野生型引物扩增的曲线；B1：rpoB 531 C>G\T突变型引物扩增的曲线，B2：为rpoB 531 C>G\T野生型引物扩增的曲线；C1：InhA-15 C>T突变型引物扩增的曲线，C2：InhA-15 C>T野生型引物扩增的曲线。
图4显示2#临床样本的rpoB 526 C>T\G与katG 315 G>C\A两个耐药位点以及内参基因IS6110的扩增曲线图，其中A1：rpoB 526 C>T\G突变型引物扩增的曲线，A2：rpoB 526 C>T\G野生型引物扩增的曲线；B1：katG 315 G>C\A突变型引物扩增的曲线，B2：katG 315 G>C\A野生型引物扩增的曲线；C1：内参基因的扩增曲线。
具体实施方式
为了使本领域技术领域人员更好地理解本申请中的技术方案，下面将结合实施例对本发明作进一步说明，显然，所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其它实施例，都应当属于本申请保护的范围。
下述实施例中，如无特殊说明，均为本领域常规方法。以下实施例中，PCR缓冲液、dNTP、dUTP、UDG、DNA聚合酶购于深圳菲鹏生物股份有限公司，所用引物及探针均由上海生物工程技术服务有限公司合成，标准质粒的提取试剂盒购自北京天根生物科技有限公司。临床样本为医院提供的痰液提取的DNA。
实施例一 本发明反应体系的构建
1.引物探针的设计
从NCBI上下载五个突变位点附近的标准序列及内参基因的标准序列，设计了检测五个点突变位点的ARMS引物和检测内参基因的引物，以及检测五个点突变位点与内参基因的探针。经过大量和反复筛选优化，本发明对各个位点的检测所使用引物和探针分别见表1和表2。
表1：检测五个点突变与内参基因的引物序列信息
Figure PCTCN2019098074-appb-000004
表2：检测五个点突变与内参基因的探针序列信息
Figure PCTCN2019098074-appb-000005
2.扩增程序为：
Figure PCTCN2019098074-appb-000006
实施例二 本发明中的各个引物的筛选实验
(一)本发明中的katG 315 G>C\A位点的突变型ARMS优选引物的筛选实验
1.筛选过程中用到探针及共用下游引物如表1与表2中所述，用到的ARMS引物如下表所示：
Figure PCTCN2019098074-appb-000007
2.样本为katG 315 G>C\A的两种突变型标准质粒与野生型标准质粒，以及用突变型标准质粒与野生型标准质粒按一定比例混合的混合质粒：50％突变、10％突变、1％突变、0.1％突变，具体混合方式如下表所示：
Figure PCTCN2019098074-appb-000008
3.荧光PCR扩增体系为：
Figure PCTCN2019098074-appb-000009
4.扩增程序如上所述；
5.筛选结果：
从引物对突变型模板与野生型模板的扩增效率的差别与引物扩增相应型别的模板时的灵敏度两方面对ARMS引物进行评价：突变型ARMS引物K315-F1\K315-F2\K315-F3\K315-F4在扩增突变型模板与野生型模板时,两种类型的模板的Ct差值为ΔCt<8，扩增效率差别较小，不利于型别的区分，不符合型别区分的要求。K315-F5\K315-F6\K315-F8\K315-F9\K315-F10\K315-F11在扩增低浓度模板时(10copies/反应与10 2copies/反应)， 突变型模板有扩增，但是Ct值较高，Ct>33(10copies/反应)，野生型模板无扩增；在扩增高浓度模板时(≥10 3copies/反应)，两种类型的模板的Ct差值为8<ΔCt<12，扩增效率的差别明显；扩增混合模板时，0.1％突变型模板没有扩增信号，没法区分0.1％突变与野生型，所以这几条突变型ARMS引物也不符合要求。K315-F7(其为优选引物中的K315-F)在模板浓度为10copies/反应、10 2copies/反应与10 3copies/反应时，扩增突变型模板有扩增且Ct<32，扩增野生型模板无扩增；在模板浓度为≥10 4copies/反应，突变型模板与野生型模板的Ct差值为ΔCt>12，扩增效率有明显差别，在扩增混合模板时，0.1％突变模板也有扩增信号；当与rpoB基因的526位点及内参基因IS6110的检测一起进行三重扩增时，这条引物仍可以实现突变型、野生型、0.1％突变型别的区分；即K315-F这条突变型ARMS引物，既有很好的区分效果，其扩增突变型模板的灵敏度又很高，且在多重反应里未受干扰，所以将其优选为本发明中katG 315 G>C\A的突变型ARMS引物。
野生型ARMS引物W-K315-F1\W-K315-F2\W-K315-F3在扩增低浓度模板时(10copies/反应与10 2copies/反应)，突变型模板无扩增，野生型模板有扩增且Ct<30；在扩增高浓度模板时(≥10 3copies/反应)，扩增效率差别也很明显，两种类型的模板的Ct差值为ΔCt>17；但当与rpoB基因的526位点及内参基因IS6110的检测一起进行三重扩增时，这条引物无法实现野生型与0.1％突变型别的区分，不符合要求。W-K315-F7在扩增10copies/反应模板时，突变型模板与野生型模板的Ct差值为ΔCt<6,扩增效率的差别较小，无法进行型别的有效区分。W-K315-F5与W-K315-F6在扩增突变型模板与野生型模板时，突变型模板没有扩增信号，野生型模板有扩增信号且Ct<31，扩增效率差别明显；但当与rpoB基因的526位点及内参基因IS6110的检测一起进行三重扩增时，引物的结合效率受到影响，在扩增低浓度模板时，突变型模板与野生型模板的扩增效率差别不大，无法进行区分。W-K315-F4(其为优选引物中的W-K315-F)，在扩增突变型模板与野生型模板时，突变型模板没有扩增信号，野生型模板有扩增信号且Ct<31(10copies/反应)，扩增效率差别明显；当与rpoB基因的526位点及内参基因IS6110的检测一起进行三重扩增时，引物结合效率不受影响，在低浓度模板时(10copies/反应、10 2copies/反应与10 3copies/反应)，突变型模板无扩增，野生型模板有扩增且Ct<32，在高浓度模板时(≥10 4copies/反应)，突变型模板与野生型模板的Ct差值为ΔCt>16，仍可以进行型别的有效区分，所以将其优选为本发明中katG 315 G>C\A的野生型ARMS引物。
(二)本发明中的rpoB 516 A>T、531 C>G\T、526 C>T\G以及InhA-15 C>T位点的突变型ARMS优选引物的筛选实验
除katG 315 G>C\A外的其它四个位点的ARMS引物的筛选也采用上述katG 315 G>C\A的引物筛选原则与方法。rpoB 516 A>T的优选突变型ARMS引物为516-AF13，优选野生型ARMS引物为W-516-AF8；rpoB 531 C>G\T的优选突变型ARMS引物为531-F5，优选野生型ARMS引物为W-531-F1；rpoB 526 C>T\G的优选突变型ARMS引物为526-F1，优选野生型ARMS引物为W-526-F2；InhA-15 C>T的优选突变型ARMS引物为Ih-R16，优选野生型ARMS引物为W-Ih-R5。被筛选的引物如下表所示：
Figure PCTCN2019098074-appb-000010
Figure PCTCN2019098074-appb-000011
实施例三 本发明中的rpoB 526 C>T\G与katG 315 G>C\A两个突变位点以及内参基因IS6110组成的反应液的验证
1.引物探针如表1与表2中的优选引物探针所述；
2.样本为rpoB 526 C>T\G、katG 315 G>C\A两个位点以及内参基因IS6110共三个位点的突变型标准质粒与野生型标准质粒按相应的方式混合后的混合质粒，其中，526位点的突变型质粒有两种526-T1(C>T)与526-T2(C>G)，526的野生型质粒为rpoB-W；katG 315 G>C\A的突变型质粒为K315-T1(G>C)与K315-T2(G>A)，野生型质粒为K315-W；内参基因IS6110的标准质粒为IS；以及用突变型质粒与野生型质粒按一定比例混合后的质粒：50％突变，10％突变，1％突变，0.1％突变，具体混合模式如下表所示：
Figure PCTCN2019098074-appb-000012
Figure PCTCN2019098074-appb-000013
Figure PCTCN2019098074-appb-000014
2.荧光PCR扩增体系为：
Figure PCTCN2019098074-appb-000015
3.扩增程序如上所述；
4.验证结果：
在rpoB 526 C>T\G、katG 315 G>C\A与内参基因IS6110三重反应液的验证结果中，FAM通道内(rpoB 526 C>T\G的检测通道)：在扩增三个位点的纯合突变型质粒混合模板-1与-3时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应时突变型反应孔有扩增，野生型反应孔无扩增，当模板为10 4copies/反应、10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的ΔCt值为ΔCt<-6，可判断rpoB 526 C>T为纯合突变， 检测结果与模板的实际基因型情况相符；在扩增三个位点的纯合突变型质粒混合模板-2与-4时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应时突变型反应孔有扩增，野生型反应孔无扩增，当模板为10 4copies/反应、10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的ΔCt值为ΔCt<-6，可判断rpoB 526 C>G为纯合突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型质粒混合模板时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应时野生型反应孔有扩增，突变型反应孔无扩增，当模板为10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的ΔCt值为ΔCt>12，可判断rpoB 526 C>T\G为野生型，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型与突变型质粒混合模板-1、-2、-3与-4时，突变型反应孔与野生型反应孔的ΔCt值为0<ΔCt<12，可判断rpoB 526 C>T\G为杂合突变，检测结果与模板的实际基因型情况相符。
Cy5通道内(katG 315 G>C\A位点的检测通道)：在扩增三个位点的纯合突变型质粒混合模板-1与突变型质粒混合模板-2时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应时突变型反应孔有扩增，野生型反应孔无扩增，当模板为10 4copies/反应、10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的ΔCt值为ΔCt<-6，可判断katG 315 G>C为纯合突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的纯合突变型质粒混合模板-3与突变型质粒混合模板-4时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应时突变型反应孔有扩增，野生型反应孔无扩增，当模板为10 4copies/反应、10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的ΔCt值为ΔCt<-6，可判断katG 315 G>A为纯合突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型质粒混合模板时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应、10 5copies/反应时野生型反应孔有扩增，突变型反应孔无扩增，当模板为10 6copies/反应时，突变型反应孔与野生型反应孔的ΔCt值为ΔCt>19，可判断katG 315 G>C\A为野生型，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型与突变型质粒混合模板-1、-2、-3与-4时，突变型反应孔与野生型反应孔的ΔCt值为0<ΔCt<18，可判断katG 315 G>C\A为杂合突变，检测结果与模板的实际基因型情况相符。
ROX通道内(内参基因IS6110的检测通道)：在扩增三个位点的突变型质粒的混合模板-1、-2、-3与-4时，突变型反应孔的ROX通道内：当模板为10copies/反应、10 2copies/反应、10 3copies/反、10 4copies/反应、10 5copies/反应、10 6copies/反应时，都有扩增信号，灵敏度可以达到10copies/反应。
实施例四 本发明中的rpoB 516 A>T、rpoB 531 C>G\T以及InhA-15 C>T三个突变位点的组成的反应液的验证
1.引物探针如表1与表2中的优选引物探针所述；
2.样本为rpoB 516 A>T、rpoB 531 C>G\T以及InhA-15 C>T三个位点的突变型标准质粒与野生型标准质粒按相应的方式混合后的混合质粒，其中，516位点与531位点构 建在一个质粒上，所以二者的野生型质粒同为rpoB-W，二者的双突变型质粒为rpoB-T1(516 A>T、531 C>G)与rpoB-T2(516 A>T、531 C>T)，516突变型而531野生型质粒为516-T，516野生型而531突变型质粒为531-T1(531 C>G)与531-T2(531 C>T)；InhA-15 C>T的突变型质粒为InhA-T，野生型质粒为InhA-W；以及用突变型质粒与野生型质粒按一定比例混合后的质粒：50％突变，10％突变，1％突变，0.1％突变，具体混合模式如下表所示：
Figure PCTCN2019098074-appb-000016
Figure PCTCN2019098074-appb-000017
3.荧光PCR扩增体系为：
Figure PCTCN2019098074-appb-000018
Figure PCTCN2019098074-appb-000019
4.扩增程序如上所述；
5.验证结果：
在rpoB 516 A>T、531 C>G\T与InhA-15 C>T三重反应液的验证结果中，FAM通道内(rpoB 516 A>T的检测通道)与VIC通道内(rpoB 531 C>G\T的检测通道)：
在扩增三个位点的纯合突变型质粒混合模板-1时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应时，突变型反应孔的两个通道内有扩增，野生型反应孔的两个通道内无扩增；当模板为10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔内的FAM通道内的ΔCt值为ΔCt<-6，突变型反应孔与野生型反应孔内的VIC通道内的ΔCt值为ΔCt<8；由此可判断516与531两个位点中有突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的纯合突变型质粒混合模板-2与-3时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应时，突变型反应孔的FAM通道内无扩增，野生型反应孔的FAM通道内有扩增，突变型反应孔与野生型反应孔的VIC通道内均有扩增，且VIC通道内两反应孔的ΔCt值为ΔCt<8；当模板为10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的FAM通道内均有扩增，且两反应孔的FAM通道内ΔCt值为ΔCt>11，突变型反应孔与野生型反应孔的VIC通道内均有扩增，且两反应孔的VIC通道内ΔCt值为ΔCt<8；由此可判断516与531两个位点中有突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的纯合突变型质粒混合模板-4时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应时，突变型反应孔的两个通道内均有扩增，野生型反应孔的两个通道内均无扩增；当模板为10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的FAM通道内均有扩增，且两反应孔的FAM通道内ΔCt值为ΔCt<-6，突变型反应孔与野生型反应孔的VIC通道内均有扩增，且两反应孔的VIC通道内ΔCt值为ΔCt<-6；由此可判断516与531两个位点中有突变，检测结果与模板的实际基因型情况相符。
在扩增三个位点的野生型与突变型质粒混合模板-1时，突变型反应孔与野生型反应孔内的FAM通道内的ΔCt值为ΔCt<10，突变型反应孔与野生型反应孔内的VIC通道内的ΔCt值为ΔCt<8；由此可判断516与531两个位点中有突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型与突变型质粒混合模板-2与-3时，当模板为0.1％突变，1％突变、10％突变时，突变型反应孔的FAM通道内无扩增，野生型反应孔的FAM通道内有扩增，突变型反应孔与野生型反应孔的VIC通道内均有扩增，且两反应孔的VIC通道内的ΔCt值为ΔCt<5；当模板为50％突变时，突变型反应孔与野生型反应孔的FAM通道内均有扩增，且两反应孔的FAM通道内ΔCt值为ΔCt值>11，突变型反应 孔与野生型反应孔的VIC通道内均有扩增，且两反应孔的VIC通道内的ΔCT值为ΔCT<5；由此可判断516与531两个位点中有突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型与突变型质粒混合模板-4时，突变型反应孔与野生型反应孔的FAM通道内均有扩增，且两反应孔的FAM通道内ΔCt值为ΔCt<10，突变型反应孔与野生型反应孔的VIC通道内均有扩增，且两反应孔的VIC通道内ΔCt值为ΔCt<8；由此可判断516与531两个位点中有突变，检测结果与模板的实际基因型情况相符。
在扩增三个位点的野生型质粒混合模板时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应时，突变型反应孔的FAM与VIC通道内均无扩增，野生型反应孔的FAM与VIC通道内均有扩增；当模板为10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的FAM通道内均有扩增，且两反应孔的ΔCt值为ΔCt>11，突变型反应孔与野生型反应孔的VIC通道内也均有扩增，且两反应孔的ΔCt值为ΔCt>16；由此可判断rpoB 516 A>T与531 C>G\T两个位点均为野生型，检测结果与模板的实际基因型情况相符。
ROX通道内(InhA-15 C>T的检测通道)：在扩增三个位点的纯合突变型质粒混合模板-1、-2与-3时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应、10 5copies/反应时突变型反应孔有扩增，野生型反应孔无扩增；当模板为10 6copies/反应时，突变型与野生型反应孔均有扩增，且其ΔCt值为ΔCt>15；由此可判断InhA-15 C>T为纯合突变，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型质粒混合模板时，当模板为10copies/反应、10 2copies/反应、10 3copies/反应、10 4copies/反应时野生型反应孔有扩增，突变型反应孔无扩增，当模板为10 5copies/反应、10 6copies/反应时，突变型反应孔与野生型反应孔的ΔCt值为ΔCt>15，可判断InhA-15 C>T为野生型，检测结果与模板的实际基因型情况相符。在扩增三个位点的野生型与突变型质粒混合模板-1、-2与-3时，突变型反应孔与野生型反应孔的0<ΔCt<15，可判断InhA-15 C>T为杂合突变，检测结果与模板的实际基因型情况相符。
实施例五 利用本发明中的利福平与异烟肼五个耐药突变位点的检测体系，检测两例已知基因型的临床样本
1.临床样本1#与2#的实际基因型别为：1#是利福平耐药复合异烟肼katG 315突变，2#是利福平耐药复合异烟肼InhA突变；
2.引物探针设计如上所述的优化引物探针；
3.荧光PCR扩增体系为：
Figure PCTCN2019098074-appb-000020
Figure PCTCN2019098074-appb-000021
4.扩增程序如上所述；
5.验证结果：
临床1#样本，rpoB 516 A>T、rpoB 531 C>G\T、InhA-15 C>T三个突变位点的检测结果，如图1所示：突变型反应孔的FAM通道内无扩增信号，野生型反应孔的FAM通道内有扩增信号，突变型反应孔与野生型反应孔的VIC通道内的ΔCt<8，可判断rpoB 516 A>T与531 C>G\T两个位点中有突变；ROX通道内，突变型反应孔无扩增，野生型反应孔有扩增，可判断InhA-15 C>T为野生型，这三个位点的判定结果与样本的实际基因型别情况相符。rpoB 526 C>T\G与katG 315 G>C\A两个耐药位点以及内参基因IS6110的检测结果，如图2所示：FAM通道内，突变型反应孔无扩增，野生型反应孔有扩增，可判断rpoB 526 C>T\G为野生型；Cy5通道内，突变型反应孔有扩增，野生型反应孔无扩增，可判断katG 315 G>C\A为纯合突变型；这两个位点的判定结果与样本的实际基因型别情况相符；突变型反应孔的ROX通道内的Ct值为30.3，说明此次检测是有效的。
临床2#样本，rpoB 516 A>T、rpoB 531 C>G\T、InhA-15 C>T三个突变位点的检测结果，如图3所示：突变型反应孔的FAM通道与VIC通道内无扩增信号，野生型反应孔的FAM通道与VIC通道内有扩增信号，可判断rpoB 516 A>T与531 C>G\T两个位点均为野生型；ROX通道内，突变型反应孔有扩增，野生型反应孔无扩增，可判断InhA-15 C>T为纯合突变型，这三个位点的判定结果与样本的实际基因型别情况相符。rpoB 526 C>T\G与katG 315 G>C\A两个耐药位点以及内参基因IS6110的检测结果，如图4所示： FAM通道内，突变型反应孔有扩增，野生型反应孔无扩增，可判断rpoB 526 C>T\G为纯合突变型；Cy5通道内，突变型反应孔无扩增，野生型反应孔有扩增，可判断katG 315 G>C\A为野生型；这两个位点的判定结果与样本的实际基因型别情况相符；突变型反应孔的ROX通道内的Ct值为24.11，说明此次检测是有效的。
应该理解到披露的本发明不仅仅限于描述的特定的方法、方案和物质，因为这些均可变化。还应理解这里所用的术语仅仅是为了描述特定的实施方式方案的目的，而不是意欲限制本发明的范围，本发明的范围仅受限于所附的权利要求。
本领域的技术人员还将认识到，或者能够确认使用不超过常规实验，在本文中所述的本发明的具体的实施方案的许多等价物。这些等价物也包含在所附的权利要求中。

Claims (10)

  1. 一种同时检测结核分枝杆菌利福平与异烟肼耐药基因点突变的方法，其特征在于，所述方法基于ARMS荧光定量PCR方法，在相同的反应体系中，分别使用针对所述耐药基因位点的突变型模板的ARMS引物和针对野生型模板的ARMS引物及它们共用的上游或下游引物，用突变型反应管与野生型反应管共两个反应管对同一份待测样本进行检测，通过各个点突变在两个反应管中的荧光PCR的Ct值和ΔCt值的大小判定样本的基因型，所述ΔCt值为突变位点的突变型反应管Ct值与野生型反应管Ct值之差。
  2. 根据权利要求1所述的方法，其特征在于，所述方法使用Taqman探针，所述探针为在ARMS引物扩增片段内设计用于检测ARMS引物对模板的扩增效率。
  3. 根据权利要求1所述的方法，其特征在于，所述耐药基因点突变是利福平耐药点突变rpoB 516 A>T、rpoB 526 C>T\G、rpoB 531 C>G\T，异烟肼耐药点突变katG 315 G>C\A和InhA-15 C>T。
  4. 根据权利要求3所述的方法，其特征在于，一个多重反应组合是rpoB 526 C>T\G与katG 315 G>C\A两个突变位点以及内参基因的反应液；另一个多重反应组合是rpoB 516 A>T、rpoB 531 C>G\T和InhA-15 C>T三个突变位点的反应液，rpoB 516 A>T与531 C>G\T两个位点共用一条下游引物。
  5. 根据权利要求4所述的方法，所述内参基因为基因IS6110。
  6. 根据权利要求5所述的方法，其特征在于，检测所述点突变与内参基因的引物序列如下：
    Figure PCTCN2019098074-appb-100001
    Figure PCTCN2019098074-appb-100002
  7. 根据权利要求5所述的方法，其特征在于，检测所述点突变与内参基因的探针序列是：
    Figure PCTCN2019098074-appb-100003
  8. 根据权利要求5所述的方法，其特征在于，rpoB 526 C>T\G使用FAM检测通道，katG 315 G>C\A使用Cy5检测通道，内参基因使用ROX通道；rpoB 516 A>T使用FAM检测通道，rpoB 531 C>G\T使用VIC检测通道，InhA-15 C>T使用ROX通道。
  9. 根据权利要求8所述的方法，其特征在于，
    若内参无扩增信号，需重新提取样本进行检测；若内参基因有扩增信号下，
    1)针对rpoB基因的516 A>T与531 C>G\T两个位点，只判读野生型与这两个位点中是否有突变，而不判读具体是哪个位点的突变：
    ①以下六种情况判读516与531这两个位点中有突变：Ⅰ：突变型反应管FAM与VIC通道内都有扩增信号，而野生型反应管FAM与VIC通道内都没有扩增信号；Ⅱ：突变型反应管与野生型反应管FAM与VIC通道内都有扩增信号，且两个通道内各自的ΔCt值都为ΔCt<-6；Ⅲ：突变型反应管FAM通道内没有扩增信号，野生型反应管FAM通道内有扩增信号，且突变型反应管与野生型反应管VIC通道内ΔCt值为ΔCt<16；Ⅳ：突变型反应管与野生型反应管FAM通道内的ΔCt值为ΔCt>11,且同时突变型反应管与野生型反应管VIC通道内的ΔCt值为ΔCt<16；Ⅴ：突变型反应管FAM通道内有扩增信号，野生型反应管内FAM通道内无扩增信号，且突变型反应管与野生型反应管VIC通道内ΔCt值为ΔCt<16；Ⅵ：突变型反应管与野生型反应管内FAM通道内ΔCt值为ΔCt<11，且突变型反应管与野生型反应管VIC通道内ΔCt<16；
    ②以下两种情况判读516与531这两个位点均为野生型：Ⅰ：若突变型反应管FAM与VIC通道内都无扩增信号，同时野生型反应管FAM与VIC通道内都有扩增信号；Ⅱ： 若突变型反应管与野生型反应管都有扩增信号，且FAM通道内的ΔCt值为ΔCt≥11，VIC通道内的ΔCt值为ΔCt≥16；
    2)针对rpoB基因的526 C>T\G、katG基因的315 G>C\A、InhA基因的-15 C>T三个位点：
    ①若突变型反应管有扩增信号，而野生型反应管无扩增信号，则判定为突变纯合型；若突变型反应管与野生型反应管均有扩增信号，且ΔCt值为ΔCt<-6，也判定为突变纯合型；
    ②若突变型反应管无扩增信号，而野生型反应管有扩增信号，则判定为野生型；若突变型反应管与野生型反应管均有扩增信号：针对rpoB 526 C>T\G，若两管的ΔCt值为ΔCt≥12，也判定为rpoB 526 C>T\G野生型；针对katG 315 G>C\A，若两管的ΔCt值为ΔCt≥18，也判定为katG 315 G>C\A野生型；针对inhA-15 C>T，若两管的ΔCt值为ΔCt≥15，也判定为inhA-15 C>T野生型；
    ③若突变型反应管与野生型反应管均有扩增信号：针对rpoB 526 C>T\G，若两反应管的ΔCt值为0≤ΔCt＜12，则判定为rpoB 526 C>T\G杂合突变；针对katG 315 G>C\A，若两反应管的ΔCt值为0≤ΔCt＜18，则判定为katG 315 G>C\A杂合突变；针对inhA-15 C>T，若两反应管的ΔCt值为0≤ΔCt＜15，则判定为inhA-15 C>T杂合突变型。
  10. 一种基于ARMS荧光定量PCR检测结核分枝杆菌利福平与异烟肼耐药基因点突变的试剂盒，其特征在于，所述试剂盒在权利要求1-9任一所述的方法中使用。
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