CN109112226B - Method and kit for simultaneously detecting mycobacterium tuberculosis rifampicin and isoniazid drug-resistant gene point mutation - Google Patents
Method and kit for simultaneously detecting mycobacterium tuberculosis rifampicin and isoniazid drug-resistant gene point mutation Download PDFInfo
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Abstract
The invention provides a method for simultaneously detecting mycobacterium tuberculosis rifampicin and isoniazid drug-resistant gene point mutation and a reagent thereof, wherein the method is based on an ARMS fluorescent quantitative PCR method, in the same reaction system, an ARMS primer aiming at a mutant type template of a drug-resistant gene mutation site, an ARMS primer aiming at a wild type template and an upstream or downstream primer shared by the two primers are respectively used, the mutant type reaction tube and the wild type reaction tube share two reaction tubes to detect the same sample to be detected, and the genotype of the sample is judged according to the Ct value and delta Ct value of the fluorescent PCR of each point mutation in the two reaction tubes. The method is simple and convenient to operate, and can detect the main drug-resistant sites of the first-line tuberculosis drug through multiple detection in a short time.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a method and a kit for simultaneously detecting the point mutation of drug-resistant genes of mycobacterium tuberculosis rifampicin and isoniazid.
Background
Mycobacterium Tuberculosis (TB), commonly known as tubercle bacillus, is a pathogenic bacterium causing tuberculosis, which can invade various organs of the body, but is the most common in clinical practice as pulmonary tuberculosis. Tuberculosis has now become a very important infectious disease worldwide, with the world population 1/3 infected with mycobacterium tuberculosis. China is one of 22 countries with serious tuberculosis prevalence in the world, the number of the disease patients is 2 in the world, and drug-resistant tuberculosis patients account for 46% of Chinese tuberculosis patients, and the generation of drug resistance greatly increases the difficulty for treating tuberculosis, so that the detection of tuberculosis drug-resistant mutation sites of the patients before tuberculosis treatment is very necessary, and the drugs are reasonably used.
According to the fifth national tuberculosis flow investigation report in China, the first-line drug resistance type of the drug-resistant tuberculosis patients in China is shown as follows: the two highest resistance rates of single and any drug resistance in naive patients were isoniazid (28.2%) and streptomycin (20.7%), while the highest several combinations of multiple and multi-drug resistance were: isoniazid + streptomycin (13.7%), isoniazid + rifampin (5.4%), rifampin + streptomycin (3.7%), isoniazid + rifampin + streptomycin (2.9%), isoniazid + ethambutol (5.8%); one drug that significantly increases the rate of resistance in retreated patients is rifampicin. At present, streptomycin is rarely or even no longer used clinically, so that the drug-resistant mutation sites of rifampicin and isoniazid two first-line drugs for treating tuberculosis are the drug-resistant mutation sites which are mainly detected by patients aiming at the drug-resistant condition of tuberculosis patients in China.
The main gene related to rifampicin resistance is rpoB, 80% of rifampicin resistant strains are due to mutations generated in rpoB gene, and more than 95% of rpoB mutations are generated in a highly conserved 81bp core region, wherein mutations at codons 531, 526 and 516 are commonly generated, and the main mutation forms are S531L, H526Y and D516V.
The main genes related to isoniazid drug resistance are katG and inhA, the 315 site mutation frequency of the katG gene is the highest, the 315 codon mutation can occur in 30-60% of isoniazid drug-resistant strains, and the mutation of S315T leads the enzyme to lose the capability of activating isoniazid; the mutation of the C base of-15 site of the promoter region of the inhA gene into T base is also an important point mutation for the generation of drug resistance of isoniazid drug-resistant plants.
The existing method for detecting the drug-resistant mutation site of the tubercle bacillus comprises the following steps: chip methods, MGB probe methods, fluorescence quantitative PCR melting curve methods, etc., for which the detection rate is high but the cost is high; the MGB probe method only depends on one probe for distinguishing, and sometimes results are not accurate enough; the result interpretation of the fluorescence quantitative PCR melting curve method needs to refer to the peak type and number of the melting peak and the Tm value, thereby improving the requirements on basic laboratory personnel.
The ARMS system is a commonly used analytical technique for gene mutations, and its genotyping detection usually involves two complementary PCR reactions, using the same DNA template and one and the same consensus primer and reaction conditions, differing only in the ARMS primer paired with the consensus primer, thereby allowing the two reactions to selectively amplify a specific DNA template. The method can be used for quickly and economically analyzing the genotype and has extremely high accuracy and cost performance. Various improvements to the ARMS system have also led to a number of highly accurate methods for detecting point mutations.
Disclosure of Invention
The invention provides a method for simultaneously detecting mycobacterium tuberculosis rifampicin and isoniazid drug-resistant gene point mutation, which is based on an ARMS fluorescent quantitative PCR method, wherein in the same reaction system, an ARMS primer aiming at a mutant type template of a drug-resistant gene mutation site, an ARMS primer aiming at a wild type template and an upstream or downstream primer shared by the two primers are respectively used, the mutant type reaction tube and the wild type reaction tube are used for detecting the same sample to be detected, the genotype of the sample is judged according to the Ct value and the delta Ct value of the fluorescent PCR of each point mutation in the two reaction tubes, and the delta Ct value is the difference between the Ct value of the mutant type reaction tube of the mutation site and the Ct value of the wild type reaction tube.
In one embodiment, the method uses a Taqman probe designed to detect the amplification efficiency of the ARMS primer against the template within the amplified fragment of the ARMS primer.
In one embodiment, the drug resistant gene point mutations are rifampicin resistant point mutations rpoB 516A > T, rpoB 526C > T \ G, rpoB 531C > G \ T, isoniazid resistant point mutations katG 315G > C \ A and InhA-15C > T.
In one embodiment, one multiplex reaction combination is a reaction solution of two mutation sites rpoB 526C > T \ G and katG 315G > C \ A and an internal reference gene; the other multiplex reaction combination is reaction liquid of three mutation sites of rpoB 516A > T, rpoB 531C > G \ T and InhA-15C > T, and two sites of rpoB 516A > T and 531C > G \ T share one downstream primer.
In one embodiment, the reference gene IS 6110.
In one embodiment, the primer sequences for detecting the point mutation and the reference gene are as follows:
in one embodiment, the probe sequence for detecting the point mutation and the reference gene is:
in one embodiment, rpoB 526C > T \ G uses FAM detection channel, katG 315G > C \ A uses Cy5 detection channel, and reference gene uses ROX channel; the FAM detection channel is used for rpoB 516A > T, the VIC detection channel is used for rpoB 531C > G \ T, and the ROX channel is used for InhA-15C > T.
In one embodiment, if the internal reference has no amplification signal, the sample is re-extracted for detection; if the internal reference gene has an amplification signal, 1) aiming at two sites of 516A > T and 531C > G \ T of rpoB gene, only judging whether the wild type and the two sites have mutation or not, but not specifically judging which site has mutation:
the mutations in the two sites 516 and 531 are interpreted in the following six cases: i: amplification signals are arranged in both FAM and VIC channels of the mutant reaction tube, and amplification signals are not arranged in both FAM and VIC channels of the wild reaction tube; II: amplification signals are respectively arranged in FAM and VIC channels of the mutant reaction tube and the wild reaction tube, and the respective delta Ct values in the two channels are delta Ct < -6; III: the FAM channel of the mutant reaction tube has no amplification signal, the FAM channel of the wild reaction tube has an amplification signal, and the delta Ct value in the channels of the mutant reaction tube and the wild reaction tube VIC is delta Ct < 16; IV: delta Ct values in FAM channels of the mutant reaction tubes and the wild reaction tubes are delta Ct >11, and delta Ct values in VIC channels of the mutant reaction tubes and the wild reaction tubes are delta Ct < 16; v: amplification signals exist in FAM channels of the mutant reaction tubes, no amplification signals exist in FAM channels of the wild reaction tubes, and delta Ct values in the channels of the mutant reaction tubes and the wild reaction tubes VIC are delta Ct < 16; VI: delta Ct values in FAM channels in the mutant reaction tubes and the wild type reaction tubes are delta Ct <11, and delta Ct values in VIC channels of the mutant reaction tubes and the wild type reaction tubes are delta Ct < 16;
(ii) interpretation of both sites 516 and 531 in two cases: i: if the mutant reaction tube FAM and the VIC channel have no amplification signals, and the wild reaction tube FAM and the VIC channel have amplification signals; II: if the mutant reaction tube and the wild reaction tube have amplification signals, the delta Ct value in the FAM channel is more than or equal to 11, and the delta Ct value in the VIC channel is more than or equal to 16;
2) the-15C > T three sites of 315G > C \ A, InhA gene aiming at 526C > T \ G, katG gene of rpoB gene:
judging that the mutant reaction tube is homozygous for the mutation if the mutant reaction tube has an amplification signal and the wild reaction tube has no amplification signal; if the mutant reaction tube and the wild reaction tube have amplification signals and the delta Ct value is delta Ct < -6, the mutant reaction tube and the wild reaction tube are also judged to be homozygous;
judging the wild type reaction tube to be the wild type if the mutant type reaction tube has no amplification signal and the wild type reaction tube has an amplification signal; if the mutant reaction tube and the wild reaction tube have amplification signals: for rpoB 526C > T \ G, if the delta Ct values of the two tubes are that the delta Ct is not less than 12, the wild type is also determined to be rpoB 526C > T \ G; for katG 315G > C \ A, if the delta Ct value of the two tubes is that the delta Ct is more than or equal to 18, the wild type of katG 315G > C \ A is also determined; for inhA-15C > T, if the delta Ct value of the two tubes is that delta Ct is not less than 15, the wild type inhA-15C > T is also determined;
and thirdly, if the mutant reaction tube and the wild reaction tube have amplification signals: aiming at rpoB 526C > T \ G, if the delta Ct values of the two reaction tubes are more than or equal to 0 and less than 12, determining that rpoB 526C > T \ G heterozygous mutation; for katG 315G > C \ A, if the delta Ct value of the two reaction tubes is more than or equal to 0 and less than 18, the result is judged as katG 315G > C \ A heterozygous mutation; and if the delta Ct value of the two reaction tubes is 0-15C > T and is more than or equal to 0 and less than 15, determining that the hybrid mutation type is inhA-15C > T.
In one embodiment, the invention provides a kit for detecting the point mutation of the rifampicin and isoniazid resistant genes of mycobacterium tuberculosis based on ARMS fluorescent quantitative PCR, wherein the kit is used in any one of the methods.
The ARMS method has the advantages of strong primer specificity, high sensitivity and low reagent cost, and the primers have small interference with each other in the multiple PCR reaction, so that the sensitivity of the ARMS method is greatly improved; the classical detection gene IS6110 of tuberculosis IS introduced as an internal reference group in the invention, which can ensure the high reliability of the detection result. The method is simple and convenient to operate, can detect the main drug-resistant sites of the first-line tuberculosis drug through multiple detection in a short time, and greatly reduces the detection cost and expense and the time required by detection. In addition, the method of the invention has direct judgment, greatly reduces the requirements for technical personnel using the method and is convenient for popularization and application.
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In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 shows the amplification plot of rpoB 516A > T, rpoB 531C > G \ T, InhA-15C > T three drug resistance sites of No. 1 clinical sample; wherein A1: rpoB 516A > T mutant primer amplification curve, A2: rpoB 516A > T wild type primer amplification curve; b1: rpoB 531C > G \ T mutant primer amplification curve, B2: the curve for rpoB 531C > G \ T wild-type primer amplification; c1: curve for InhA-15C > T mutant primer amplification, C2: InhA-15C > T wild type primer amplification curve.
FIG. 2 shows the amplification plot of rpoB 526C > T \ G and katG 315G > C \ A two drug resistant sites and internal reference gene IS6110 of No. 1 clinical sample, wherein A1: rpoB 526C > T \ G mutant primer amplification curve, A2: rpoB 526C > T \ G wild-type primer amplification curve; b1: katG 315G > curve for C \ A mutant primer amplification, B2: curve of katG 315G > C \ A wild type primer amplification; c1: and (3) amplification curve of the internal reference gene.
FIG. 3 shows the amplification profile of rpoB 516A > T, rpoB 531C > G \ T, InhA-15C > T three mutation sites of 2# clinical sample, wherein A1: rpoB 516A > T mutant primer amplification curve, A2: rpoB 516A > T wild type primer amplification curve; b1: rpoB 531C > G \ T mutant primer amplification curve, B2: the curve for rpoB 531C > G \ T wild-type primer amplification; c1: curve for InhA-15C > T mutant primer amplification, C2: InhA-15C > T wild type primer amplification curve.
FIG. 4 shows the amplification plot of rpoB 526C > T \ G and katG 315G > C \ A two drug resistant sites and internal reference gene IS6110 of No. 2 clinical sample, wherein A1: rpoB 526C > T \ G mutant primer amplification curve, A2: rpoB 526C > T \ G wild-type primer amplification curve; b1: katG 315G > curve for C \ A mutant primer amplification, B2: curve of katG 315G > C \ A wild type primer amplification; c1: and (3) amplification curve of the internal reference gene.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the present invention will be further described with reference to the following examples, and it is obvious that the described examples are only a part of the examples of the present application, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
In the following examples, unless otherwise specified, all methods are conventional in the art. In the following examples, PCR buffer, dNTP, dUTP, UDG and DNA polymerase were purchased from Shenzhen Fenpeng Biotech, Inc., the primers and probes used were synthesized by Shanghai Biotech service, Inc., and the standard plasmid extraction kit was purchased from Beijing Tiangen Biotech, Inc. The clinical sample is DNA extracted from sputum provided by a hospital.
Example A construction of the reaction System of the present invention
1. Design of primer probes
Downloading standard sequences near five mutation sites and standard sequences of internal reference genes from NCBI, and designing ARMS primers for detecting the five point mutation sites, primers for detecting the internal reference genes and probes for detecting the five point mutation sites and the internal reference genes. After a large number of and repeated screening optimization, the primers and probes used for detecting each site are respectively shown in tables 1 and 2.
Table 1: primer sequence information for detecting five point mutations and reference genes
Table 2: probe sequence information for detecting five point mutation and internal reference gene
2. The amplification procedure was:
example two screening experiments for the respective primers of the present invention
(I) screening experiment of mutant ARMS preferred primer of katG 315G > C \ A site in the invention
1. The probes and common downstream primers used in the screening procedure are described in tables 1 and 2, and the ARMS primers used are shown in the following tables:
2. two mutant standard plasmids and wild standard plasmids of which the samples are katG 315G > C \ A, and mixed plasmids of the mutant standard plasmids and the wild standard plasmids which are mixed according to a certain proportion: 50% mutation, 10% mutation, 1% mutation, 0.1% mutation, the specific mixing mode is shown in the following table:
3. the fluorescent PCR amplification system is as follows:
4. the amplification procedure was as described above;
5. and (4) screening results:
the ARMS primers were evaluated from the difference in amplification efficiency of the mutant template and the wild-type template in the primer pair and the sensitivity of the primers in amplifying the corresponding type of template: when the mutant ARMS primer K315-F1\ K315-F2\ K315-F3 \ K315-F4 is used for amplifying the mutant template and the wild template, the Ct difference value of the two types of templates is delta Ct<8, the amplification efficiency difference is small, which is not beneficial to the type discrimination and does not meet the requirement of the type discrimination. K315-F5\ K315-F6\ K315-F8 \ K315-F9\ K315-F10\ K315-F11 when amplifying low-concentration templates (10 copies/reaction and 10)2copies/reaction), the mutant template was amplified, but the Ct value was higher, the Ct value was higher>33(10 copies/reaction), no amplification of wild type template; when the high concentration template is amplified (not less than 10)3copies/reaction), the difference in Ct for the two types of template was 8<ΔCt<12, the difference of the amplification efficiency is obvious; 0.1% mutant molds when amplifying mixed templatesThe plate did not amplify the signal and could not distinguish between 0.1% of the mutations and wild type, so these mutant ARMS primers were also unsatisfactory. K315-F7 (which is K315-F in the preferred primer) at a template concentration of 10 copies/reaction, 102copies/reactions with 103Amplification mutant template amplified and Ct's in copies/reactions<32, no amplification is carried out on the amplified wild type template; the concentration of the template is more than or equal to 104copies/reaction, difference in Ct between mutant and wild type template is Δ Ct>12, amplification efficiency is obviously different, and when a mixed template is amplified, 0.1 percent of mutant templates also have amplification signals; when triple amplification IS carried out together with detection of the 526 site of the rpoB gene and the internal reference gene IS6110, the primer can still realize the differentiation of the mutant type, the wild type and the 0.1 percent mutant type; that is, the K315-F mutant ARMS primer has a good discrimination effect, has high sensitivity in amplifying the mutant template, and is not interfered in the multiplex reaction, so that it is preferably katG 315G in the present invention>Mutant ARMS primer of C \ A.
Wild type ARMS primers W-K315-F1\ W-K315-F2\ W-K315-F3 when amplifying low concentration template (10 copies/reaction and 10)2copies/reaction), no amplification of mutant template, amplification of wild-type template and Ct<30, of a nitrogen-containing gas; when the high concentration template is amplified (not less than 10)3copies/reaction), the difference in amplification efficiency is also evident, and the difference in Ct between the two types of templates is Δ Ct>17; however, when triple amplification IS carried out together with the detection of the 526 site of rpoB gene and the IS6110 reference gene, the primer cannot distinguish between wild type and 0.1% mutant type, which IS not satisfactory. When the W-K315-F7 is used for amplifying 10 copies/reaction template, the Ct difference between the mutant template and the wild template is delta Ct<6, the difference in amplification efficiency was small, and the types could not be distinguished effectively. When W-K315-F5 and W-K315-F6 amplify the mutant template and the wild template, the mutant template has no amplification signal, the wild template has amplification signal and Ct<31, the amplification efficiency is obviously different; however, when triple amplification was carried out together with detection of the 526 site of rpoB gene and IS6110, the primer binding efficiency was affected, and when a low-concentration template was amplified, the amplification efficiency of the mutant template and the wild-type template was not greatly different from that of the wild-type template, and none of them was observedThe methods are distinguished. W-K315-F4 (which is W-K315-F in the preferred primer), when amplifying the mutant template and the wild type template, the mutant template has no amplification signal, the wild type template has amplification signal and Ct<31(10 copies/reaction), the amplification efficiency difference is obvious; when triple amplification was carried out together with detection of the 526 site of rpoB gene and IS6110 reference gene, primer binding efficiency was not affected, at low template concentrations (10 copies/reaction, 10)2copies/reactions with 103copies/reaction), no amplification of mutant template, amplification of wild-type template and Ct<32, in the case of high concentration template (not less than 10)4copies/reaction), the difference in Ct between the mutant and wild type template is Δ Ct>16, the effective discrimination of the type can be made, so that it is preferable to katG 315G in the present invention>C \ A wild type ARMS primer.
(II) screening experiment of mutant ARMS preferred primers for rpoB 516A > T, 531C > G \ T, 526C > T \ G and InhA-15C > T sites in the present invention
The ARMS primers at other four sites except for katG 315G > C \ A are screened by adopting the primer screening principle and method of katG 315G > C \ A. The preferred mutant ARMS primer for rpoB 516A > T is 516-AF13, the preferred wild type ARMS primer is W-516-AF 8; the preferred mutant ARMS primer of rpoB 531C > G \ T is 531-F5, and the preferred wild type ARMS primer is W-531-F1; the preferred mutant ARMS primer of rpoB 526C > T \ G is 526-F1, and the preferred wild type ARMS primer is W-526-F2; a preferred mutant ARMS primer for InhA-15C > T is Ih-R16, and a preferred wild type ARMS primer is W-Ih-R5. The primers screened are shown in the following table:
example III verification of reaction solution consisting of two mutation sites rpoB 526C > T \ G and katG 315G > C \ A and internal reference gene IS6110
1. The primer probes are described in tables 1 and 2 as preferred primer probes;
2. the sample IS a mixed plasmid obtained by mixing mutant standard plasmids of rpoB 526C > T \ G, katG 315G > C \ A two sites and three sites of internal reference gene IS6110 with wild type standard plasmids in a corresponding mode, wherein the mutant plasmids of 526 sites comprise two types 526-T1(C > T) and 526-T2(C > G), and the wild type plasmid of 526 IS rpoB-W; the mutant plasmids of katG 315G \ C \ A are K315-T1(G > C) and K315-T2(G > A), and the wild type plasmid is K315-W; the standard plasmid of the internal reference gene IS6110 IS IS; and the plasmid obtained by mixing the mutant plasmid and the wild plasmid according to a certain proportion: 50% mutation, 10% mutation, 1% mutation, 0.1% mutation, specific mixture patterns are shown in the following table:
2. the fluorescent PCR amplification system is as follows:
3. the amplification procedure was as described above;
4. and (4) verification result:
in rpoB 526C>T\G、katG 315G>In the verification result of triple reaction liquid of C \ A and internal reference gene IS6110, in FAM channel (rpoB 526C)>T \ G detection channel): when the homozygous mutant plasmids at three sites were amplified and the templates-1 and-3 were mixed, the template was 10 copies/reaction, 102copies/reactions, 103copies/reaction mutant TransThe corresponding hole has amplification, the wild type reaction hole has no amplification, when the template is 104copies/reactions, 105copies/reactions, 106In copes/reaction, the Δ Ct values of the mutant reaction wells and the wild-type reaction wells were Δ Ct<-6, determination of rpoB 526C>T is homozygous mutation, and the detection result is consistent with the actual genotype condition of the template; when the homozygous mutant plasmids at three sites were amplified to mix templates-2 and-4, the template was 10 copies/reaction, 102copies/reactions, 103Amplification in the mutant reaction well and no amplification in the wild type reaction well in the copies/reaction where the template is 104copies/reactions, 105copies/reactions, 106In copes/reaction, the Δ Ct values of the mutant reaction wells and the wild-type reaction wells were Δ Ct<-6, determination of rpoB 526C>G is homozygous mutation, and the detection result is consistent with the actual genotype condition of the template. When the wild type plasmid mixed template at three sites is amplified, the template is 10 copies/reaction, 102copies/reactions, 103copies/reactions, 104Amplification in wild-type reaction well and no amplification in mutant reaction well in copies/reactions when template is 105copies/reactions, 106In copes/reaction, the Δ Ct values of the mutant reaction wells and the wild-type reaction wells were Δ Ct>12, can judge rpoB 526C>T \ G is wild type, and the detection result is consistent with the actual genotype condition of the template. When the wild type and mutant plasmid mixed templates-1, -2, -3 and-4 of the three sites are amplified, the delta Ct values of the mutant reaction holes and the wild type reaction holes are 0<ΔCt<12, can judge rpoB 526C>T \ G is heterozygous mutation, and the detection result is consistent with the actual genotype condition of the template.
Cy5 in-channel (katG 315G)>Detection channel of C \ A site): when the homozygous mutant plasmid mixed template-1 and the mutant plasmid mixed template-2 at three sites are amplified, the template is 10 copies/reaction and 102copies/reactions, 103Amplification in the mutant reaction well and no amplification in the wild type reaction well in the copies/reaction where the template is 104copies/reactions, 105copies/reactions, 106In copes/reaction, the Δ Ct values of the mutant reaction wells and the wild-type reaction wells were Δ Ct<-6, judged katG 315G>C is homozygous mutation, and the detection result is consistent with the actual genotype condition of the template. When the homozygous mutant plasmid mixed template-3 and the mutant plasmid mixed template-4 at three sites are amplified, the template is 10 copies/reaction and 102copies/reactions, 103Amplification in the mutant reaction well and no amplification in the wild type reaction well in the copies/reaction where the template is 104copies/reactions, 105copies/reactions, 106In copes/reaction, the Δ Ct values of the mutant reaction wells and the wild-type reaction wells were Δ Ct<-6, adjudicable katG 315G>A is homozygous mutation, and the detection result is consistent with the actual genotype condition of the template. When the wild type plasmid mixed template at three sites is amplified, the template is 10 copies/reaction, 102copies/reactions, 103copies/reactions, 104copies/reactions, 105Amplification in wild-type reaction well and no amplification in mutant reaction well in copies/reactions when template is 106In copes/reaction, the Δ Ct values of the mutant reaction wells and the wild-type reaction wells were Δ Ct>19, can judge katG 315G>C \ A is wild type, and the detection result is consistent with the actual genotype condition of the template. When the wild type and mutant plasmid mixed templates-1, -2, -3 and-4 of the three sites are amplified, the delta Ct values of the mutant reaction holes and the wild type reaction holes are 0<ΔCt<18, can judge katG 315G>C \ A is heterozygous mutation, and the detection result is consistent with the actual genotype condition of the template.
ROX channel (detection channel of reference gene IS 6110): in amplifying the mixed templates-1, -2, -3 and-4 of the mutant plasmids at three sites, the ROX channels of the mutant reaction wells are as follows: when the template is 10 copies/reaction, 102copies/reactions, 103copies/trans, 104copies/reactions, 105copies/reactions, 106Amplification signals exist in the copies/reaction, and the sensitivity can reach 10 copies/reaction.
Example IV verification of reaction solutions of the present invention comprising three mutation sites, rpoB 516A > T, rpoB 531C > G \ T and InhA-15C > T
1. The primer probes are described in tables 1 and 2 as preferred primer probes;
2. the sample is a mixed plasmid obtained by mixing mutant standard plasmids of rpoB 516A > T, rpoB 531C > G \ T and InhA-15C > T at three sites and wild-type standard plasmids in a corresponding mode, wherein the 516 site and the 531 site are constructed on one plasmid, so that the wild-type plasmids of the two plasmids are rpoB-W, the double-mutant plasmids of the two plasmids are rpoB-T1 (516A > T and 531C > G) and rpoB-T2(516A > T and 531C > T), the 516 mutant plasmids are 516-T, the 531 wild-type plasmids are 516-T, and the 531 plasmids are 531-T1(531C > G) and 531-T2(531C > T); the mutant plasmid of InhA-15C > T is InhA-T, and the wild type plasmid is InhA-W; and the plasmid obtained by mixing the mutant plasmid and the wild plasmid according to a certain proportion: 50% mutation, 10% mutation, 1% mutation, 0.1% mutation, specific mixture patterns are shown in the following table:
3. the fluorescent PCR amplification system is as follows:
4. the amplification procedure was as described above;
5. and (4) verification result:
in the verification results of triple reaction liquid of rpoB 516A > T, 531C > G \ T and InhA-15C > T, in FAM channel (detection channel of rpoB 516A > T) and VIC channel (detection channel of rpoB 531C > G \ T):
when the homozygous mutant plasmid mixed template-1 at three sites is amplified, the template is 10 copies/reaction and 102copies/reaction, 103copies/reactions, 104In the copes/reaction, amplification exists in two channels of the mutant reaction hole, and amplification does not exist in two channels of the wild type reaction hole; when the form is 105copies/reactions, 106In copies/reactions, the Δ Ct values in FAM channels in the mutant and wild type reaction wells are Δ Ct<6, the delta Ct value in the VIC channel in the mutant reaction well and the wild-type reaction well is delta Ct<8; therefore, the mutation in the 516 and 531 sites can be judged, and the detection result is consistent with the actual genotype condition of the template. When the homozygous mutant plasmids at three sites were amplified to mix templates-2 and-3, the template was 10 copies/reaction, 102copies/reactions, 103copies/reactions, 104When copies/reaction, the FAM channel of the mutant reaction hole has no amplification, the FAM channel of the wild type reaction hole has amplification, the VIC channels of the mutant reaction hole and the wild type reaction hole have amplification, and the delta Ct values of the two reaction holes in the VIC channels are delta Ct<8; when the form is 105copies/reactions, 106When copies/reaction are carried out, amplification is carried out in FAM channels of the mutant reaction wells and the wild reaction wells, and the delta Ct value in the FAM channels of the two reaction wells is delta Ct>11, the mutant reaction wells and the wild reaction wells have amplification in the VIC channels, and the delta Ct values in the VIC channels of the two reaction wells are delta Ct<8; therefore, the mutation in the 516 and 531 sites can be judged, and the detection result is consistent with the actual genotype condition of the template. When the homozygous mutant plasmid mixed template-4 at three sites is amplified, the template is 10 copies/reaction and 102copies/reactions, 103copies/reactions, 104In the copes/reaction, amplification exists in two channels of the mutant reaction hole, and amplification does not exist in two channels of the wild type reaction hole; when the form is 105copies/reactions, 106When copies/reaction are carried out, amplification is carried out in FAM channels of the mutant reaction wells and the wild reaction wells, and the delta Ct value in the FAM channels of the two reaction wells is delta Ct<6, amplifying the VIC channels of the mutant reaction wells and the wild reaction wells, wherein the delta Ct values in the VIC channels of the two reaction wells are delta Ct<-6; therefore, the mutation in the 516 and 531 sites can be judged, and the detection result and the actual genotype of the template can be judgedThe situation is the same.
When the mixed template-1 of the wild type and the mutant type plasmids of three sites is amplified, the delta Ct value in FAM channels in the mutant type reaction holes and the wild type reaction holes is delta Ct <10, and the delta Ct value in VIC channels in the mutant type reaction holes and the wild type reaction holes is delta Ct < 8; therefore, the mutation in the 516 and 531 sites can be judged, and the detection result is consistent with the actual genotype condition of the template. When the wild type and mutant type plasmids of three sites are amplified to mix the templates-2 and-3, when the template is 0.1 percent of mutation, 1 percent of mutation and 10 percent of mutation, the FAM channel of the mutant type reaction hole is not amplified, the FAM channel of the wild type reaction hole is amplified, the VIC channels of the mutant type reaction hole and the wild type reaction hole are amplified, and the delta Ct value in the VIC channels of the two reaction holes is delta Ct < 5; when the template is 50% mutated, the FAM channels of the mutant reaction holes and the wild type reaction holes are amplified, the delta Ct values in the FAM channels of the two reaction holes are more than 11, the VIC channels of the mutant reaction holes and the wild type reaction holes are amplified, and the delta CT values in the VIC channels of the two reaction holes are less than 5; therefore, the mutation in the 516 and 531 sites can be judged, and the detection result is consistent with the actual genotype condition of the template. When a wild type and mutant plasmid mixed template-4 of three sites is amplified, the FAM channels of the mutant reaction holes and the wild type reaction holes are amplified, the delta Ct values in the FAM channels of the two reaction holes are delta Ct <10, the VIC channels of the mutant reaction holes and the wild type reaction holes are amplified, and the delta Ct values in the VIC channels of the two reaction holes are delta Ct < 8; therefore, the mutation in the 516 and 531 sites can be judged, and the detection result is consistent with the actual genotype condition of the template.
When the wild type plasmid mixed template at three sites is amplified, the template is 10 copies/reaction, 102copies/reactions, 103copies/reactions, 104When in copies/reaction, FAM and VIC channels of the mutant reaction wells are not amplified, and FAM and VIC channels of the wild reaction wells are amplified; when the form is 105copies/reactions, 106When copies/reaction is carried out, amplification is carried out in FAM channels of the mutant reaction wells and the wild reaction wells, and the delta Ct values of the two reaction wells are delta Ct>11, both the mutant reaction wells and the wild-type reaction wells have amplification in the VIC channel, and the delta Ct values of the two reaction wells are delta Ct>16; from this, rpoB 516A can be judged>T and 531C>And the two G \ T loci are wild type, and the detection result is consistent with the actual genotype condition of the template.
ROX in-channel (InhA-15C)>T detection channel): when the homozygous mutant plasmids at three sites are amplified and the templates are mixed with the-1, -2 and-3, the template is 10 copies/reaction and 102copies/reactions, 103copies/reactions, 104copies/reactions, 105Amplification is present in the mutant reaction wells and not in the wild-type reaction wells; when the form is 106In copes/reaction, the mutant and wild type reaction wells are amplified and the Δ Ct value is Δ Ct>15; thus, it was determined that InhA-15C is present>T is homozygous mutation, and the detection result is consistent with the actual genotype condition of the template. When the wild type plasmid mixed template at three sites is amplified, the template is 10 copies/reaction, 102copies/reactions, 103copies/reactions, 104Amplification in wild-type reaction well and no amplification in mutant reaction well in copies/reactions when template is 105copies/reactions, 106In copes/reaction, the Δ Ct values of the mutant reaction wells and the wild-type reaction wells were Δ Ct>15, can determine InhA-15C>T is wild type, and the detection result is consistent with the actual genotype condition of the template. When the mixed templates-1, -2 and-3 of the wild type plasmid and the mutant type plasmid of the three sites are amplified, 0 of the mutant type reaction hole and the wild type reaction hole<ΔCt<15, can determine InhA-15C>T is heterozygous mutation, and the detection result is consistent with the actual genotype condition of the template.
Example five clinical samples of two known genotypes were examined using the system of the present invention for detecting five drug resistant mutation sites of rifampicin and isoniazid
1. The actual genotype of clinical samples # 1 and # 2 is: 1# is rifampicin resistant composite isoniazid katG 315 mutation, 2# is rifampicin resistant composite isoniazid InhA mutation;
2. primer probes the optimized primer probes described above were designed;
3. the fluorescent PCR amplification system is as follows:
4. the amplification procedure was as described above;
5. and (4) verification result:
the detection results of three mutation sites of clinical sample No. 1, rpoB 516A > T, rpoB 531C > G \ T, InhA-15C > T are shown in FIG. 1: the FAM channel of the mutant reaction hole has no amplification signal, the FAM channel of the wild reaction hole has amplification signal, and the delta Ct in the VIC channel of the mutant reaction hole and the wild reaction hole is less than 8, so that mutations in two sites of rpoB 516A T and 531C G \ T can be judged; in the ROX channel, the mutant reaction hole is not amplified, the wild type reaction hole is amplified, the InhA-15C > T can be judged to be the wild type, and the judgment results of the three sites are consistent with the actual genotype condition of the sample. The detection results of two drug resistant sites rpoB 526C > T \ G and katG 315G > C \ A and the internal reference gene IS6110 are shown in FIG. 2: in the FAM channel, the mutant reaction hole is not amplified, the wild type reaction hole is amplified, and rpoB 526C > T \ G can be judged to be the wild type; in the Cy5 channel, the mutant reaction hole has amplification, the wild type reaction hole has no amplification, and katG 315G > C \ A can be judged to be homozygous mutant; the judgment results of the two sites are consistent with the actual genotype of the sample; the Ct value in the ROX channel of the mutant reaction well was 30.3, indicating that this assay was effective.
The detection results of three mutation sites of clinical sample No. 2, rpoB 516A > T, rpoB 531C > G \ T, InhA-15C > T are shown in FIG. 3: the FAM channel and the VIC channel of the mutant reaction hole have no amplification signals, and the FAM channel and the VIC channel of the wild-type reaction hole have amplification signals, so that two sites rpoB 516A > T and 531C > G \ T can be judged to be wild-type; in the ROX channel, the mutant reaction hole is amplified, the wild type reaction hole is not amplified, the InhA-15C > T can be judged to be the homozygous mutant, and the judgment results of the three sites are consistent with the actual genotype of the sample. The detection results of two drug resistant sites rpoB 526C > T \ G and katG 315G > C \ A and the internal reference gene IS6110 are shown in FIG. 4: in the FAM channel, the mutant reaction hole is amplified, the wild reaction hole is not amplified, and rpoB 526C > T \ G can be judged to be the homozygous mutant; in the Cy5 channel, the mutant reaction hole has no amplification, the wild type reaction hole has amplification, and katG 315G > C \ A can be judged to be wild type; the judgment results of the two sites are consistent with the actual genotype of the sample; the Ct value in the ROX channel of the mutant reaction well was 24.11, indicating that this assay was effective.
It is to be understood that the invention disclosed is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.
Sequence listing
<110> Jiangsu Hongmite medicine science and technology Co., Ltd
BEIJING MACRO & MICRO TEST BIO TECH Co.,Ltd.
<120> method and kit for simultaneously detecting mycobacterium tuberculosis rifampicin and isoniazid drug-resistant gene point mutation
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Claims (5)
1. A method for simultaneously detecting drug-resistant gene point mutation of mycobacterium tuberculosis rifampicin and isoniazid with a non-diagnosis purpose is characterized in that the method is based on an ARMS fluorescent quantitative PCR method, in the same reaction system, an ARMS primer aiming at a mutant type template of a drug-resistant gene site, an ARMS primer aiming at a wild type template and an upstream primer or a downstream primer shared by the ARMS primer and the ARMS primer are respectively used, two reaction tubes of the mutant type reaction tube and the wild type reaction tube are used for detecting the same sample to be detected, the genotype of the sample is judged according to the Ct value and the delta Ct value of the fluorescent PCR of each point mutation in the two reaction tubes, and the delta Ct value is the difference between the Ct value of the mutant type reaction tube of the mutation site and the Ct value of the wild type reaction tube;
the method uses a Taqman probe which is designed in an ARMS primer amplification fragment and is used for detecting the amplification efficiency of the ARMS primer to a template;
the drug-resistant gene point mutation is rifampicin drug-resistant point mutation rpoB 516A > T, rpoB 526C > T \ G, rpoB 531C > G \ T, isoniazid drug-resistant point mutation katG 315G > C \ A and InhA-15C > T;
the internal reference gene IS gene IS 6110;
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 as follows:
2. the method of claim 1, wherein one multiplex reaction combination is a reaction solution of two mutation sites rpoB 526C > T \ G and katG 315G > C \ A and an internal reference gene; the other multiplex reaction combination is reaction liquid of three mutation sites of rpoB 516A > T, rpoB 531C > G \ T and InhA-15C > T, and two sites of rpoB 516A > T and 531C > G \ T share one downstream primer.
3. The method of claim 2, wherein rpoB 526C > T \ G uses FAM detection channel, katG 315G > C \ A uses Cy5 detection channel, and reference gene uses ROX channel; the FAM detection channel is used for rpoB 516A > T, the VIC detection channel is used for rpoB 531C > G \ T, and the ROX channel is used for InhA-15C > T.
4. The method of claim 3,
if the internal reference has no amplification signal, the sample needs to be extracted again for detection; if the internal reference gene has an amplification signal,
1) aiming at two sites of 516A > T and 531C > G \ T of rpoB gene, only the wild type and the two sites are judged whether to have mutation, but not specifically which site is mutated:
the mutations in the two sites 516 and 531 are interpreted in the following six cases: i: amplification signals are arranged in both FAM and VIC channels of the mutant reaction tube, and amplification signals are not arranged in both FAM and VIC channels of the wild reaction tube; II: amplification signals are respectively arranged in FAM and VIC channels of the mutant reaction tube and the wild reaction tube, and the respective delta Ct values in the two channels are delta Ct < -6; III: the FAM channel of the mutant reaction tube has no amplification signal, the FAM channel of the wild reaction tube has an amplification signal, and the delta Ct value in the channels of the mutant reaction tube and the wild reaction tube VIC is delta Ct < 16; IV: delta Ct values in FAM channels of the mutant reaction tubes and the wild reaction tubes are delta Ct >11, and delta Ct values in VIC channels of the mutant reaction tubes and the wild reaction tubes are delta Ct < 16; v: amplification signals exist in FAM channels of the mutant reaction tubes, no amplification signals exist in FAM channels of the wild reaction tubes, and delta Ct values in the channels of the mutant reaction tubes and the wild reaction tubes VIC are delta Ct < 16; VI: delta Ct values in FAM channels in the mutant reaction tubes and the wild type reaction tubes are delta Ct <11, and delta Ct values in VIC channels of the mutant reaction tubes and the wild type reaction tubes are delta Ct < 16;
(ii) interpretation of both sites 516 and 531 in two cases: i: if the mutant reaction tube FAM and the VIC channel have no amplification signals, and the wild reaction tube FAM and the VIC channel have amplification signals; II: if the mutant reaction tube and the wild reaction tube have amplification signals, the delta Ct value in the FAM channel is more than or equal to 11, and the delta Ct value in the VIC channel is more than or equal to 16;
2) the-15C > T three sites of 315G > C \ A, InhA gene aiming at 526C > T \ G, katG gene of rpoB gene:
judging that the mutant reaction tube is homozygous for the mutation if the mutant reaction tube has an amplification signal and the wild reaction tube has no amplification signal; if the mutant reaction tube and the wild reaction tube have amplification signals and the delta Ct value is delta Ct < -6, the mutant reaction tube and the wild reaction tube are also judged to be homozygous;
judging the wild type reaction tube to be the wild type if the mutant type reaction tube has no amplification signal and the wild type reaction tube has an amplification signal; if the mutant reaction tube and the wild reaction tube have amplification signals: for rpoB 526C > T \ G, if the delta Ct values of the two tubes are that the delta Ct is not less than 12, the wild type is also determined to be rpoB 526C > T \ G; for katG 315G > C \ A, if the delta Ct value of the two tubes is that the delta Ct is more than or equal to 18, the wild type of katG 315G > C \ A is also determined; for inhA-15C > T, if the delta Ct value of the two tubes is that delta Ct is not less than 15, the wild type inhA-15C > T is also determined;
and thirdly, if the mutant reaction tube and the wild reaction tube have amplification signals: aiming at rpoB 526C > T \ G, if the delta Ct values of the two reaction tubes are more than or equal to 0 and less than 12, determining that rpoB 526C > T \ G heterozygous mutation; for katG 315G > C \ A, if the delta Ct value of the two reaction tubes is more than or equal to 0 and less than 18, the result is judged as katG 315G > C \ A heterozygous mutation; and if the delta Ct value of the two reaction tubes is 0-15C > T and is more than or equal to 0 and less than 15, determining that the hybrid mutation type is inhA-15C > T.
5. A kit for detecting mycobacterium tuberculosis rifampicin and isoniazid drug-resistant gene point mutation based on ARMS fluorescent quantitative PCR, which is characterized in that the kit is used in the method of any one of claims 1-4.
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| PCT/CN2019/098074 WO2020052356A1 (en) | 2018-09-12 | 2019-07-28 | Method for simultaneously detecting point mutations of rifampicin- and isoniazid-resistant genes for mycobacterium tuberculosis, and kit therefor |
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| CN105603084A (en) * | 2016-02-01 | 2016-05-25 | 海南医学院 | Primers, probes and method for detecting Mycobacterium tuberculosis drug-resistant gene mutation sites |
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| TaqMan实时荧光定量PCR检测动物组织中结核分枝杆菌的研究;孟凤珍等;《中国病原生物学杂志》;20160930;第11卷(第9期);第769-774页 * |
| 利福平耐药基因突变位点的分析及M-ARMS-PCR检测方法的建立;石晓丹;《中国优秀硕士学位论文全文数据库（电子期刊）》;20140215;E061-57 * |
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