Composition and kit for detecting tuberculosis drug-resistant gene and application of composition and kit

Abstract

The invention belongs to the field of molecular biology detection, particularly relates to detection of tuberculosis drug-resistant genes, and more particularly relates to detection of at least four genes in inhA, KatG, ahpC-2, rpoB-3, rpoB-4 and FabG1 genes. According to the combined detection composition provided by the invention, a multicolor melting curve analysis technology is mainly utilized, different target spots are detected, and drug resistance target spots of pathogens are detected, so that drug resistance detection of isoniazide and rifampicin is realized, and a targeted strategy is provided for subsequent treatment. The composition disclosed by the invention is higher in detection sensitivity (up to 800 bacteria / mL), good in specificity and more accurate in detection, provides more accurate and comprehensive tuberculosis drug-resistant medication guidance for clinicians, and accelerates the implementation of treatment measures on patients.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biological detection, and specifically, relates to the detection of tuberculosis resistance genes, and more specifically, to the detection of isoniazid resistance, rifampicin resistance, and mycobacterium tuberculosis and non-tuberculous mycobacteria. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease that endangers human health and is mainly caused by infection with Mycobacterium tuberculosis (MTB). It is one of the important public health problems facing my country. TB is transmitted through the air, and symptoms include persistent cough, fever, night sweats, weight loss, chest pain, and hemoptysis. The treatment of TB usually requires long-term use of multiple antibiotics, including isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA), and ethambutol (EMB). TB drug resistance has always been a major problem in clinical treatment due to its long course, complex treatment, and high cost. In view of the needs of monitoring and treatment, the World Health Organization divides drug-resistant TB into five categories: isoniazid-resistant TB (HR-TB), rifampicin-resistant TB (RR-TB), multidrug-resistant TB (MDR-TB), quasi-extensively drug-resistant TB (Pre-XDR TB), and extensively drug-resistant TB (XDR-TB). MDR-TB is defined as resistance to at least INH and RIF, two of the most effective first-line anti-TB drugs, and resistance to these two is the most worrying problem in current TB treatment.

[0003] Early diagnosis of pathogens and timely and effective anti-infection treatment are the key to improving prognosis and can greatly reduce morbidity and mortality. However, it is difficult to quickly and accurately distinguish MTB and NTM through clinical symptoms and traditional drug resistance sensitivity testing, and it is impossible to quickly judge drug resistance, which brings great difficulties to clinical diagnosis, medication and cure of the disease.

[0004] Therefore, there is a need in this field for a product that can simply and quickly detect the above targets, and has good sensitivity and specificity, can accurately and efficiently distinguish between MTB and NTM infections in the early stages of the disease, and provide guidance for the subsequent use of rifampicin and isoniazid. Summary of the invention

[0005] In view of this, in a first aspect, the present invention provides a composition for detecting tuberculosis drug-resistant genes, comprising:

[0006] A first nucleic acid composition, comprising an upstream primer, a downstream primer and a probe for detecting at least 4 of the following 5 targets:

[0007] Upstream primers, downstream primers and probes for detecting inhA as shown in SEQ ID NOs: 1 to 3;

[0008] Upstream primers, downstream primers and probes for detecting KatG as shown in SEQ ID NOs: 4 to 6;

[0009] Upstream primers, downstream primers and probes for detecting aphC-2 as shown in SEQ ID NOs: 7 to 9;

[0010] The upstream primers, downstream primers and probes for detecting rpoB-3 as shown in SEQ ID NOs: 10 to 12; or

[0011] The upstream primers, downstream primers and probe for detecting rpoB-4 are shown in SEQ ID NOs: 10-11 and 13.

[0012] Optionally, the first nucleic acid composition may further include:

[0013] The upstream primers, downstream primers and probe for detecting fabG1 are shown in SEQ ID NOs: 14-16.

[0014] The combined detection composition provided by the present invention mainly utilizes multicolor melting curve analysis technology to detect the drug resistance targets of pathogens by detecting different targets, thereby realizing the detection of isoniazid and rifampicin resistance, so as to provide targeted strategies for subsequent treatment. The composition of the present invention has a higher detection sensitivity of 800 bacteria / mL, good specificity, and more accurate detection, providing more accurate and comprehensive guidance on tuberculosis resistance medication for clinicians, and accelerating the implementation of treatment measures for patients.

[0015] Furthermore, the first nucleic acid composition comprises:

[0016] Upstream primers, downstream primers and probes for detecting inhA as shown in SEQ ID NOs: 1 to 3;

[0017] Upstream primers, downstream primers and probes for detecting KatG as shown in SEQ ID NOs: 4 to 6;

[0018] Upstream primers, downstream primers and probes for detecting aphC-2 as shown in SEQ ID NOs: 7 to 9;

[0019] Upstream primers, downstream primers and probes for detecting rpoB-3 as shown in SEQ ID NOs: 10 to 12;

[0020] Upstream primers, downstream primers and probes for detecting rpoB-4 as shown in SEQ ID NOs: 10-11, 13; and

[0021] The upstream primers, downstream primers and probe for detecting fabG1 are shown in SEQ ID NOs: 14-16.

[0022] Furthermore, the composition also includes a second nucleic acid composition for more comprehensive detection of tuberculosis drug resistance.

[0023] Furthermore, the second nucleic acid composition comprises:

[0024] Upstream primers, downstream primers and probes for detecting ahpC-1 as shown in SEQ ID NOs: 17 to 19; and / or

[0025] Upstream primers, downstream primers and probes for detecting rpoB-1 as shown in SEQ ID NOs: 10 to 11, 20; and / or

[0026] The upstream primers, downstream primers and probe for detecting rpoB-2 are shown in SEQ ID NOs: 10-11, 21.

[0027] Optionally, the second nucleic acid composition may further include:

[0028] The upstream primers, downstream primers and probe for detecting fabG1 are shown in SEQ ID NOs: 14-16.

[0029] Through the additional primer and probe set included in the above nucleic acid composition, the joint detection composition provided by the present invention can also provide more comprehensive and accurate detection of tuberculosis drug-resistant genes.

[0030] Furthermore, the composition of the present invention also includes a third nucleic acid composition for distinguishing Mycobacterium tuberculosis from non-tuberculous mycobacteria.

[0031] Furthermore, the third nucleic acid composition comprises:

[0032] Upstream primers, downstream primers and probes for detecting IS6110 as shown in SEQ ID NOs: 22 to 24;

[0033] Upstream primers, downstream primers and probes for detecting IS1081 as shown in SEQ ID NOs: 25 to 27; and

[0034] The upstream primers, downstream primers and probe for detecting NTM are shown in SEQ ID NOs: 28-30.

[0035] The composition of the present invention can simultaneously detect 58 drug-resistant mutations, specifically, rpoB 510-512del; 511 site CTG-CCG; 513 site CAA-CTA / CCA / AAA / CGA; 516 site GAC-TAC / ACC / GCC / GTC / GGC; 517-518del; 522 site TCG-TGG / CAG; 526 site CAC-TAC / TGC / GAC / GGC / TGC / TCC / CGC / AAC / CTC / CAG / CTG / CAA / AGC; 531 site TCG-TGG / TTG / GGG / TTC / CCG; fabG1 G609A, G603A; ahpC G-48A, T-44A, T-42C, C-39T, T-34C, G-32A, C-30T, C-15T, C-12T, C-10T, C-10A, C-10G, G-9 A, G-6A; inhAG-17T, A-16G, C-15T, A-11T, T-8C, T-8A; katG315 codon AGC-ACC / AAC / ATC / ACA.

[0036] Furthermore, the composition includes an upstream primer, a downstream primer and a probe for detecting an internal standard.

[0037] In some specific embodiments, an exogenous internal standard is added to the sample, and further, the internal standard is an internal standard gene of Bacillus subtilis.

[0038] Furthermore, the fluorescent groups of the probes in each nucleic acid composition of the composition of the present invention are different from each other and do not interfere with each other.

[0039] In this article, "different from each other and not interfering with each other" means that the fluorescent groups used in each probe in the composition are different and will not affect each other's detection, that is, different channels can be used for detection. For example, ATTO425, Quasar705, FAM, HEX, ROX and CY5 can be used. The absorbance values ​​of these groups are not close, and different channels can be selected, so they will not interfere with each other.

[0040] In some specific embodiments, in the first nucleic acid composition, the fluorescent reporter group of the probe for detecting inhA is FAM; the fluorescent reporter group of the probe for detecting KatG is HEX; the fluorescent reporter group of the probe for detecting rpoB-3 is ROX; the fluorescent reporter group of the probe for detecting rpoB-4 is CY5; the fluorescent reporter group of the probe for detecting fabG1 is ATTO425; and the fluorescent reporter group of the probe for detecting ahpC-2 is QUASAR 705.

[0041] In some specific embodiments, in the second nucleic acid composition, the fluorescent reporter group of the probe for detecting RIF-1 is HEX; the fluorescent reporter group of the probe for detecting ahpC-1 is ROX; and the fluorescent reporter group of the probe for detecting RIF-2 is CY5.

[0042] In some specific embodiments, in the third nucleic acid composition, the fluorescent reporter group of the probe for detecting IS6110 is FAM; the fluorescent reporter group of the probe for detecting IS1081 is CY5; and the fluorescent reporter group of the probe for detecting NTM is ROX.

[0043] Further, in some embodiments, the composition of the present invention may include one or more of the above primer and probe pairs. In the present invention, "set" refers to upstream and downstream primers and probes that match each other for detecting a target.

[0044] The composition of the present invention can be arbitrarily combined into any combination of detection corresponding to 12 targets. Those skilled in the art can combine as needed to detect which targets, that is, to combine the primers and probe pairs corresponding to the targets. These combinations are all included in the present invention, for example, any 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 of the 12 targets mentioned in the present invention can be detected. These are all expected by those skilled in the art.

[0045] For example, it may include any 11 of the 12 sets of primers and probes, it may include any 10 of the 12 sets of primers and probes, it may include any 9 of the 12 sets of primers and probes, and it may include any 8 of the 12 sets of primers and probes.

[0046] In some specific embodiments, the compositions of the present invention are used for fluorescent PCR.

[0047] Furthermore, the 3' end of the probe also has a non-fluorescent quencher.

[0048] Furthermore, the 3' end of the probe also has a quencher group, such as BHQ1 or BHQ2.

[0049] In a specific embodiment, the 3' end of the probe is BHQ1.

[0050] In a specific embodiment, the 3' end of the probe is BHQ2.

[0051] In a specific embodiment, each component of the composition of the present invention is present in a separate package.

[0052] In a particular embodiment, the components of the composition of the present invention are present in the same package.

[0053] Furthermore, the components of the nucleic acid composition of the present invention are present in a mixed form.

[0054] Furthermore, different nucleic acid compositions of the present invention exist in separate forms.

[0055] In a second aspect, the present invention provides use of the above-mentioned composition of the present invention in preparing a kit for detecting tuberculosis drug-resistant genes.

[0056] In a third aspect, the present invention provides a kit for detecting tuberculosis drug-resistant genes, wherein the kit comprises the composition of the present invention as described above.

[0057] Furthermore, the kit also includes a negative quality control product and / or a positive quality control product.

[0058] In a specific embodiment, the negative control product is at least one of DEPC H2O and normal saline; the positive control product is a fragment plasmid mixture of fabG1, inhA, KatG, ahpC, rpoB, IS6110, IS1081, and NTM genes.

[0059] Furthermore, the kit also includes dNTP(U)s, PCR buffer and Mg 2+ At least one of .

[0060] Furthermore, the kit also includes at least one of DNA polymerase and uracil glycosylase (UNG).

[0061] Furthermore, the kit also includes at least one of a nucleic acid releasing reagent and a nucleic acid extracting reagent.

[0062] Furthermore, the kit also includes a nucleic acid release reagent, a nucleic acid extraction reagent, dNTP, dUTP, uracil glycosylase (UNG), DNA polymerase, PCR buffer and Mg 2+ At least one of .

[0063] Furthermore, the concentration of the DNA polymerase is 5 U / reaction to 15 U / reaction, for example, the DNA polymerase may be Taq enzyme.

[0064] In a specific embodiment, the kit of the present invention comprises Taq enzyme, UDG enzyme, Mg 2+ , dNTP(U)s, primers, probes and PCR buffer.

[0065] The main components of PCR buffer include Tris-HCl, NaCl, NH4Cl, and Tween 20.

[0066] In some specific embodiments, the total volume of the reactants in a single PCR reaction tube is 20 μL to 60 μL.

[0067] In a specific embodiment, the kit of the present invention is compatible with a digital PCR amplification system, that is, it can be directly used for amplification on a digital PCR instrument.

[0068] In a fourth aspect, a method for detecting tuberculosis drug-resistant genes is provided, the method comprising the following steps:

[0069] 1) Extracting nucleic acid from the sample to be tested;

[0070] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0071] 3) Obtain and analyze the results.

[0072] In the present invention, the sample used for detection can be sputum, alveolar lavage fluid, respiratory secretions, blood, etc., but is not limited thereto.

[0073] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0074] UDG enzyme reaction, temperature is 30-50℃, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, extension, 60-80℃, time is 10-30 seconds, 30-50 cycles; denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; melting analysis.

[0075] Furthermore, the method for detecting tuberculosis drug resistance is for non-diagnostic purposes.

[0076] In a specific embodiment, a composition is provided for preparing a reagent for detecting tuberculosis drug-resistant genes, wherein the detection comprises the following steps:

[0077] 1) Extracting nucleic acid from the sample to be tested;

[0078] 2) using the composition of the present invention or the kit of the present invention as described above to perform fluorescent PCR detection on the nucleic acid obtained in step 1);

[0079] 3) Obtain and analyze the results.

[0080] Furthermore, the reaction conditions of the fluorescent quantitative PCR are:

[0081] UDG enzyme reaction, temperature is 30-50℃, time is 1-5 minutes, 1 cycle; pre-denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; denaturation, temperature is 90-99℃, time is 5-20 seconds, annealing, temperature is 55℃-60℃, time is 10-60 seconds, extension, 60-80℃, time is 10-30 seconds, 30-50 cycles; denaturation, temperature is 90-99℃, time is 0.5-6min, 1 cycle; melting analysis.

[0082] In this context, the term "non-diagnostic purpose" means that the purpose is not to obtain information on whether an individual is infected with the above-mentioned pathogens and suffers from the infection. For example, the method can detect whether the environment, culture and drinking water sources have the above-mentioned resistant pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 This is a result diagram of the first nucleic acid composition of the present invention detecting 6 targets;

[0084] Figure 2 This is a result diagram of the second nucleic acid composition of the present invention detecting three targets (ahpC-1, rpoB-1, and rpoB-2);

[0085] Figure 3 This is a result diagram of the third nucleic acid composition of the present invention detecting three targets;

[0086] Figure 4 This is a result diagram of the first nucleic acid composition of the present invention detecting five targets (inhA, KatG, ahpC-2, rpoB-3, and rpoB-4);

[0087] Figure 5 This is a result diagram of the first nucleic acid composition of the present invention detecting four targets (inhA, KatG, ahpC-2, and rpoB-3);

[0088] Figure 6 The result graph of the second nucleic acid composition of the composition of the present invention detecting four targets (ahpC-1, rpoB-1, rpoB-2, fabG1);

[0089] Figures 7 to 15 Specific result diagrams of the composition of the present invention (respectively, the detection results of Mycobacterium tuberculosis quality control product, Mycobacterium avium quality control product, Mycobacterium schrenckii quality control product, Mycobacterium kansasii quality control product, rpoB gene 526 codon CAC>TAC, katG gene 315 codon AGC>ACC, inhA gene C-15T, ahpC gene C-12T, fabG1 gene G 609A);

[0090] Figures 16 to 18This is a graph showing the test results of different compositions of the comparative examples of the present invention;

[0091] Fig.19 This is the result of rifampicin rpoB testing in the same tube. DETAILED DESCRIPTION

[0092] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0093] Example 1: Primers and probes used in the present invention

[0094] The primers and probes used in the present invention are shown in Table 1 below:

[0095] Table 1

[0096]

[0097]

[0098] In the first nucleic acid composition, the fluorescent reporter group of the probe for detecting inhA is FAM; the fluorescent reporter group of the probe for detecting KatG is HEX; the fluorescent reporter group of the probe for detecting rpoB-3 is ROX; the fluorescent reporter group of the probe for detecting rpoB-4 is CY5; the fluorescent reporter group of the probe for detecting fabG1 is ATTO425; and the fluorescent reporter group of the probe for detecting ahpC-2 is QUASAR 705.

[0099] In the second nucleic acid composition, the fluorescent reporter group of the probe for detecting rpoB-1 is HEX; the fluorescent reporter group of the probe for detecting ahpC-1 is ROX; and the fluorescent reporter group of the probe for detecting rpoB-2 is CY5.

[0100] In the third nucleic acid composition, the fluorescent reporter group of the probe for detecting IS6110 is FAM; the fluorescent reporter group of the probe for detecting IS1081 is CY5; and the fluorescent reporter group of the probe for detecting NTM is ROX.

[0101] Among them, "+" indicates that the base behind it is modified by locked nucleic acid; lowercase indicates non-target base.

[0102] Example 2: Method for detecting pathogens

[0103] Reagent preparation

[0104] Take out each component in the box, place it at room temperature, wait for the temperature to equilibrate to room temperature, mix well and centrifuge for a short time before use.

[0105] According to the number of samples to be tested, negative controls, and positive controls, take the corresponding amount of components in proportion (26 μL / person of PCR reaction solution + 4 μL / person of enzyme mixture), mix them thoroughly to form a PCR mixture, and centrifuge it for standby use.

[0106] Transfer the above prepared reagents to the sample processing area for later use.

[0107] Sample handling and loading

[0108] The sputum samples were liquefied with 2 to 3 times the volume of sample diluent, 1.5 mL of the liquefied sample was concentrated and sonicated, 200 μL of the pre-treated sample and quality control product were simultaneously extracted using extraction reagents or purification reagents for fully automatic nucleic acid extraction, and 20 μL of the extracted nucleic acid or quality control product was added to the reaction solution for machine testing. The reaction solution is shown in Table 2.

[0109] Table 2

[0110] Components Volume / concentration in each reaction <![CDATA[Mg 2+ ]]> 2mM dNTPs(100mM) 0.3mM Taq enzyme (5U / μl) 10U Primers 400nM / strip Probe 200nM / strip Template DNA 20μl PCR buffer (1x) Make up to 50 μl

[0111] PCR amplification

[0112] PCR amplification was performed on PCR instruments such as ABI 7500 fluorescence quantitative PCR instrument and SLAN-96P fully automatic medical PCR analysis system according to certain temperature and time setting programs. PCR amplification was performed according to the temperature and time setting programs shown in Table 3.

[0113] Table 3

[0114]

[0115] Result analysis and judgment

[0116] In the first nucleic acid composition, the ATTO 425 channel detects a characteristic peak of the melting curve and the Tm value is between 67.10±2℃, the FAM channel detects a characteristic peak of the melting curve and the Tm value is between 65.29±2℃, the HEX channel detects a characteristic peak of the melting curve and the Tm value is between 66.91±2℃, and the QUASAR 705 channel detects a characteristic peak of the melting curve and the Tm value is between 68.51±2℃, indicating that the tested sample is an isoniazid sensitive sample; if the characteristic peak of the melting curve is detected in any of the above four channels and the Tm value is not between the positive judgment values, it indicates that the tested sample is an isoniazid resistant sample; the ROX channel detects a characteristic peak of the melting curve and the Tm value is between 72.69±2℃, and the CY5 channel detects a characteristic peak of the melting curve and the Tm value is between 72.87±2℃, indicating that the tested sample is a rifampicin sensitive sample; if the characteristic peak of the melting curve is detected in any of the above two channels and the Tm value is not between the positive judgment values.

[0117] In the second nucleic acid composition, the HEX channel detects a characteristic peak of the melting curve and the Tm value is between 76.07±2°C, and the reaction tube B CY5 channel detects a characteristic peak of the melting curve and the Tm value is between 65.56±2°C, indicating that the sample under test is a rifampicin sensitive sample; if a characteristic peak of the melting curve is detected in any of the above two channels and the Tm value is not between the positive judgment values, it indicates that the sample under test is a rifampicin resistant sample; the ROX channel detects a characteristic peak of the melting curve and the Tm value is between 61.55±2°C, indicating that the sample under test is an isoniazid sensitive sample. If the channel detects a characteristic peak of the melting curve and the Tm value is not between the positive judgment values, it indicates that the sample under test is an isoniazid resistant sample;

[0118] In the third nucleic acid composition, if the FAM channel detects a characteristic peak of the melting curve and the Tm value is between 64.96±2°C, or the CY5 channel detects a characteristic peak of the melting curve and the Tm value is between 68.29±2°C, it indicates that the tested sample is a positive sample for Mycobacterium tuberculosis, otherwise it is negative; if the ROX channel detects a characteristic peak of the melting curve, it indicates that the tested sample is a positive sample for non-tuberculosis Mycobacterium, otherwise it is negative.

[0119] The details are shown in Table 4 below.

[0120] Table 4

[0121]

[0122]

[0123] In addition, each Tm value in the present invention is a common value obtained on a specific sample diluent, nucleic acid extraction or purification reagent and SLAN-96P instrument, which is used as a reference. When other sample diluents, nucleic acid extraction or purification reagents and instruments are used, the Tm value may vary slightly, and the Tm value obtained by the positive control (wild type) of the test is used as a reference.

[0124] The Tm value is based on the automatic interpretation of the instrument. When the instrument gives more than one Tm value, please refer to the peak type of the positive control to select a valid Tm value. When the instrument cannot automatically give a Tm value, the Tm value can be obtained by adjusting the baseline or directly interpreting it manually.

[0125] Interpretation of heterozygous sample results: When double peaks or fusion peaks appear in the reaction tube, it is judged as a mutant type and the strain to be tested is a heterogeneous drug-resistant strain.

[0126] If some channels of the target channel have no characteristic peaks of the melting curve, it indicates that the content of Mycobacterium tuberculosis or non-tuberculous mycobacteria in the sample to be tested is lower than the detection sensitivity of this product, or there is an experimental operation error; the kit is invalid, etc.

[0127] Example 3: Test results of the test samples of the composition of the present invention

[0128] The primers and probes shown in Example 1 were used according to the method of Example 2 to select a mixed sample of Mycobacterium tuberculosis IS6110, IS1081 gene sequences, non-tuberculous mycobacterium ku gene sequences, rifampicin rpoB resistance determining region sequences, isoniazid resistance genes katG, ahpC, fabG1, inhA gene sequences, and PCR detection was performed on a SLAN-96P real-time fluorescence quantitative PCR instrument. The results showed that the composition of the present invention can detect and distinguish the combination of the above targets, specifically, for example:

[0129] The first nucleic acid composition: inhA, KatG, ahpC-2, rpoB-3, rpoB-4, fabG1 ( Figure 1 );

[0130] Second nucleic acid composition: ahpC-1, rpoB-1, rpoB-2 ( Figure 2 );

[0131] The third nucleic acid composition: IS6110, IS1081, NTM ( Figure 3 );

[0132] First nucleic acid composition: inhA, KatG, ahpC-2, rpoB-3, rpoB-4 ( Figure 4 );

[0133] First nucleic acid composition: inhA, KatG, ahpC-2, rpoB-3 ( Figure 5 );

[0134] Second nucleic acid composition: ahpC-1, rpoB-1, rpoB-2, fabG1 ( Figure 6 )

[0135] These figures can prove that the present invention is a multiplex PCR that can amplify the above-mentioned targets separately at one time, for example, the highest can be 6-plex PCR of the first nucleic acid composition, and can be distinguished using a 6-channel PCR instrument. It also proves that any corresponding pair of combinations of the compositions of the present invention can detect corresponding combinations of any targets.

[0136] Example 4: Sensitivity of the composition of the present invention

[0137] Using the composition in Example 1 of the present invention, the gradient dilution of the plasmid has a high sensitivity. The sensitivity of Mycobacterium tuberculosis detection reaches 10 bacteria / mL, and the sensitivity of non-tuberculous mycobacteria detection, rifampicin, and isoniazid resistance detection reaches 800 bacteria / mL. The detection results of different concentrations of each detection target are shown in the following table (the concentration with a detection rate of ≥95% is used as the detection limit concentration). The results are shown in Table 5.

[0138] Table 5

[0139]

[0140] Example 5. Specificity of the composition of the present invention

[0141] The quality control products of Mycobacterium tuberculosis and non-tuberculous mycobacteria were tested on the fluorescence quantitative PCR instrument; plasmids with different mutation types of rifampicin and isoniazid were added to sputum negative samples to simulate MTB tuberculosis-resistant samples, and the results showed that the third nucleic acid composition can accurately detect Mycobacterium tuberculosis and non-tuberculous mycobacteria, and the first and second nucleic acid compositions both showed specific Tm melting peaks in specific channels according to the design of the kit, and can accurately detect drug-resistant mutations. Figures 7 to 15 shown.

[0142] Example 6. Accuracy of the composition of the present invention

[0143] Clinical samples were used for real testing, and the consistency of the reagent of the present invention was compared with the gold standard Sanger sequencing. Through the study of 284 clinical samples, the method of the present invention tested a total of 156 MTB positive, 38 NTM positive, and 90 negative samples, of which 58 MTB positive samples contained rifampicin resistance and 82 isoniazid mutation samples. The overall consistency rate between the Sanger sequencing method and the method of the present invention was 98%, with good consistency, indicating that the accuracy of the method of the present invention is superior.

[0144] Comparative Example 1: Other primers and probes designed by the present invention that have poor effects

[0145] Due to the principle of complementary base pairing, dimers will form between primers and (or) probes, but this probability is very small and can be ruled out at the beginning of the design. However, when multiple pathogens are detected together, there are many primers and probes, and dimers are likely to occur between primers and primers, probes and probes, or primers and probes. In order to ensure the conservatism of the design (conservatism is crucial to the accuracy of the detection), and to consider the mutual interference between different primers and probes, the primers and probes need to be carefully designed.

[0146] Therefore, the inventors also designed some other primers and probes to form different detection systems (sequences not shown), which are also used to detect the above pathogens. Figures 16 to 18 As shown in the figure, it can be seen that after amplification, the melting curve peak is low and other non-target peaks appear, so the overall detection effect is poor.

[0147] Comparative Example 2: Detection of rpoB target of rifampicin

[0148] The present invention attempts to use 4 probes to fully cover the 81bp resistance-determining region of the rpoB gene of rifampicin and place them in the same tube. In order to prevent the situation where the two adjacent probes cannot excite fluorescence and an abnormal melting curve occurs due to the probes being modified with a fluorescent group at the 5' end and a quenching group at the 3' end, two probes are used with adjacent fluorescent groups or adjacent quenching groups. However, since the adjacent probes are too close, fluorescence resonance energy transfer will still occur. At this time, the high Tm value probe will first form an inverted peak and then a positive peak. The temperature corresponding to the trough of the inverted peak is exactly the Tm value of the low Tm value probe. The low Tm value probe will form a normal melting peak, but the peak value is low, and the detection ability is affected. Fig.19 Therefore, the probe of the 81bp resistance-determining region of the rpoB gene of rifampicin was set in two tubes for detection.

Claims

1. A composition for detecting tuberculosis drug-resistant genes, comprising: A first nucleic acid composition, comprising an upstream primer, a downstream primer and a probe for detecting at least 4 of the following 5 targets: Upstream primers, downstream primers and probes for detecting inhA as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting KatG as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting aphC-2 as shown in SEQ ID NOs: 7 to 9; The upstream primers, downstream primers and probes for detecting rpoB-3 as shown in SEQ ID NOs: 10 to 12; or The upstream primers, downstream primers and probe for detecting rpoB-4 are shown in SEQ ID NOs: 10-11 and 13.

2. The composition according to claim 1, characterized in that The composition further comprises a second nucleic acid composition, wherein the second nucleic acid composition comprises: Upstream primers, downstream primers and probes for detecting ahpC-1 as shown in SEQ ID NOs: 17 to 19; Upstream primers, downstream primers and probes for detecting rpoB-1 as shown in SEQ ID NOs: 10-11, 20; and The upstream primers, downstream primers and probe for detecting rpoB-2 are shown in SEQ ID NOs: 10-11, 21.

3. The composition according to claim 2, characterized in that The first nucleic acid composition and / or the second nucleic acid composition further comprises: The upstream primers, downstream primers and probe for detecting fabG1 are shown in SEQ ID NOs: 14-16.

4. The composition according to claim 1, characterized in that The first nucleic acid composition comprises: Upstream primers, downstream primers and probes for detecting inhA as shown in SEQ ID NOs: 1 to 3; Upstream primers, downstream primers and probes for detecting KatG as shown in SEQ ID NOs: 4 to 6; Upstream primers, downstream primers and probes for detecting aphC-2 as shown in SEQ ID NOs: 7 to 9; Upstream primers, downstream primers and probes for detecting rpoB-3 as shown in SEQ ID NOs: 10 to 12; Upstream primers, downstream primers and probes for detecting rpoB-4 as shown in SEQ ID NOs: 10-11, 13; and The upstream primers, downstream primers and probe for detecting fabG1 are shown in SEQ ID NOs: 14-16.

5. The composition according to any one of claims 1 to 4, characterized in that The composition further comprises a third nucleic acid composition, wherein the third nucleic acid composition comprises: Upstream primers, downstream primers and probes for detecting IS6110 as shown in SEQ ID NOs: 22 to 24; Upstream primers, downstream primers and probes for detecting IS1081 as shown in SEQ ID NOs: 25 to 27; and The upstream primers, downstream primers and probe for detecting NTM are shown in SEQ ID NOs: 28-30.

6. The composition according to claim 5, characterized in that The different nucleic acid compositions of the composition are present in separate forms.

7. Use of the composition according to any one of claims 1 to 6 in the preparation of a kit for detecting drug resistance of tuberculosis.

8. A kit for detecting tuberculosis drug-resistant genes, the kit comprising the composition according to any one of claims 1 to 6.

9. The kit according to claim 8, characterized in that The kit also includes a negative quality control product and / or a positive quality control product.

10. Use of a composition for preparing a reagent for detecting tuberculosis drug-resistant genes, the detection comprising the following steps: 1) Extracting nucleic acid from the sample to be tested; 2) performing fluorescent PCR detection on the nucleic acid obtained in step 1) using the composition according to any one of claims 1 to 6 or the kit according to any one of claims 8 to 9; 3) Obtain and analyze the results.

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