Reaction system for detecting drug resistance genes of mycobacterium tuberculosis and rifampicin thereof

Abstract

The invention provides a reaction system for detecting mycobacterium tuberculosis and rifampicin drug resistance genes thereof. The reaction system comprises DNA polymerase, dNTPs, a reaction buffer solution and a primer for amplifying a template, the reaction system comprises magnesium ions, disodium hydrogen phosphate and a double-strand displacement probe composition, the reaction system can be combined with the double-strand displacement probe composition through synergy of the magnesium ions and the disodium hydrogen phosphate, and the defects that according to a conventional melting curve method, the number of impure peaks is large, the peak heights of melting curves are inconsistent under the same condition, and the sensitivity of the melting curves is low can be overcome; the method has the advantages of low fluorescence background, high detection sensitivity, good specificity and good stability, the purpose of multiple analysis of rifampicin sensitivity and drug resistance in the same optical channel can be achieved by combining melting curve analysis, and when the reaction system is applied to detection, the obtained melting curve peak shape is more stable than that of a conventional asymmetric PCR system, and the method is suitable for large-scale popularization and application. And the experiment repeatability and consistency are good.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and specifically to a reaction system for detecting Mycobacterium tuberculosis and its rifampicin resistance genes. Background Technology

[0002] Tuberculosis is a chronic infectious disease caused by the Mycobacterium tuberculosis complex. It can affect all organs of the body, with pulmonary tuberculosis being the most common. Common first-line drugs for the treatment of tuberculosis include rifampin, isoniazid, quinolones, ethambutol, etc.

[0003] Rifampin primarily binds to the β subunit of RNA polymerase in Mycobacterium tuberculosis (MTB), inhibiting mRNA transcription and thus exerting bacteriostatic and bactericidal effects against the bacillus. It has been established that rifampin resistance in Mycobacterium tuberculosis is related to mutations in the β subunit of RNA polymerase (rpoB gene), preventing rifampin from binding and resulting in resistance. Currently, over 70 gene mutation sites in the rpoB gene have been identified in rifampin-resistant clinical isolates, with approximately 96% of rifampin resistance mutations concentrated within the 81 bases of the rifampin resistance-determining region of the rpoB gene.

[0004] Traditional laboratory diagnostic methods for tuberculosis (sputum smear, sputum culture, traditional nucleic acid amplification) are time-consuming and cannot simultaneously achieve both sensitivity and specificity. The resistance mechanism of rifampin is particularly suitable for rapid molecular detection. Fluorescent PCR melting curve technology detects the presence of mutations in the target sequence by analyzing changes in the melting point of the target sequence on a real-time quantitative PCR instrument, thereby obtaining resistance information. Currently, molecular beacons combined with asymmetric amplification are commonly used for melting curve analysis, but molecular beacon design requires sophisticated techniques and a relatively complex optimization process. A molecular beacon is a fluorescently labeled oligonucleotide chain. It generally consists of three parts: ① a circular region: composed of 15–30 nucleotides that can specifically bind to the target molecule; ② a stem region: generally composed of 5–8 base pairs that can undergo reversible dissociation; ③ a fluorescent group and a quencher group: the two ends of the molecular beacon are labeled with a fluorescent group and a quencher group, respectively. In the absence of a target molecule, the fluorescence and quencher groups of the molecular beacon are very close, and the fluorescence is quenched. After binding to the target molecule, the spatial configuration of the molecular beacon changes, leading to the recovery of fluorescence. Due to its structure and binding properties, it may exhibit poor non-specific binding, resulting in a high fluorescence background.

[0005] In addition, the melting curve method obtained from conventional PCR reaction systems has many impurity peaks, which affects the appearance of the target peak, and the peak heights of the melting curves are inconsistent under the same conditions.

[0006] Therefore, there is an urgent need to develop a PCR reaction system that can achieve multiplex analysis of rifampicin sensitivity and resistance in the same optical channel by combining melting curve analysis with high detection sensitivity, good specificity, and high stability, and with stable melting curve peak shape. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a reaction system for detecting Mycobacterium tuberculosis and its rifampicin resistance gene. This invention can be used for the detection of Mycobacterium tuberculosis and its rifampicin resistance gene based on a PCR probe melting curve method using a double-stranded displacement probe combined with a dynamic magnesium ion system.

[0008] Specifically, on the one hand, the present invention provides a reaction system for detecting Mycobacterium tuberculosis and its rifampicin resistance gene, wherein the reaction system comprises: DNA polymerase, dNTPs, reaction buffer, and primers for amplifying the template; and

[0009] A composition of magnesium salt, disodium hydrogen phosphate, and double-chain displacement probe;

[0010] The nucleotide sequences of the primers used to amplify the template include SEQ ID NO.1 and SEQ ID NO.2;

[0011] The double-stranded substitution probe composition includes four types of double-stranded substitution probes;

[0012] The nucleotide sequences of the forward probes of the four double-stranded substitution probes include SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, and SEQ ID NO.13, respectively;

[0013] The nucleotide sequences of the reverse probes of the four double-stranded substitution probes include SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12, and SEQ ID NO.14, respectively.

[0014] The 5' end of the forward probe is labeled with a fluorescent group, and the 3' end of the reverse probe, which forms a complementary double strand with the forward probe, is labeled with a quenching group.

[0015] In the reaction system of this invention, DNA polymerase is used to polymerize new DNA chains, and the commonly used DNA polymerase is Taq polymerase; "dNTPs" can be used interchangeably with "deoxynucleotide triphosphate" or "nucleotide containing triphosphate group". DNA polymerase uses dNTPs to synthesize new DNA chains. dNTPs usually include dATP, dGTP, dTTP, and dCTP; "reaction buffer" provides a suitable chemical environment for DNA polymerase, such as Champagne Taq buffer.

[0016] The probes used in the reaction system of this invention are double-stranded substitution probes. These probes are formed by labeling two complementary oligonucleotide sequences with a fluorescent group and a quencher group, respectively. The fluorescent group is labeled at the 5' end of the forward probe, and the quencher group is labeled at the 3' end of the reverse probe, which forms a complementary double strand. In the absence of template DNA, the two complementary probes form a double strand, and because the fluorescent group is adjacent to the quencher group, quenching is more thorough, significantly reducing fluorescence background. Compared to commonly used molecular beacon probe systems in the prior art, during the melting experiment to form a melting curve, the fluorescence intensity change of the double-stranded substitution probe system used in this invention is greater and more pronounced within the same temperature range, thus improving detection sensitivity.

[0017] In PCR reaction systems, magnesium ions can bind to polymerase and activate its activity; however, excessive magnesium ions can also affect polymerase efficiency. As the number of cycles increases during PCR amplification, the enzyme activity, dNTPs, and primer concentrations in the system decrease, resulting in a difference in the system's magnesium ion requirement compared to the initial amplification reaction. The inventors of this invention have discovered through experimental research that adding disodium hydrogen phosphate (Na₂HPO₄) to the PCR reaction system allows hydrogen phosphate ions to dynamically bind with magnesium ions, forming a dynamic equilibrium. This ensures stable environmental conditions at any stage of the PCR amplification reaction, improving reaction stability and detection sensitivity.

[0018] According to a specific embodiment of the present invention, preferably, the reaction system further includes ammonium sulfite. Adding sodium sulfite to the PCR reaction system, which already contains magnesium ions and disodium hydrogen phosphate, can improve the stability of the reaction system and enhance the consistency of parallel experimental results.

[0019] According to a specific embodiment of the present invention, preferably, the magnesium salt includes magnesium chloride.

[0020] According to a specific embodiment of the present invention, preferably, the working concentration of the magnesium salt is 2-5 mM, more preferably 4 mM.

[0021] According to a specific embodiment of the present invention, preferably, the working concentration of the disodium hydrogen phosphate is 0.5-2 mM, more preferably 2 mM.

[0022] According to a specific embodiment of the present invention, preferably, the working concentration of the ammonium sulfite is 0.5-2 mM, more preferably 1 mM.

[0023] According to a specific embodiment of the present invention, preferably, the four types of double-stranded displacement probes are prepared by mixing the forward probe and its corresponding reverse probe in TE buffer.

[0024] According to a specific embodiment of the present invention, preferably, the working concentration of the four double-stranded substitution probes is independently 0.03-0.05 μM, more preferably 0.04 μM.

[0025] On the other hand, the present invention also provides a detection kit, wherein the detection kit includes the above-described reaction system.

[0026] The components of the kit of the present invention may be present in individually packaged form or in a mixture of one or more components. The components of the kit of the present invention may be present in a concentrated form. For example, the reaction buffer may be present in a concentrated form (e.g., 10×).

[0027] On the other hand, the present invention also provides the application of the above-described reaction system in a kit for preparing Mycobacterium tuberculosis and detecting its rifampicin resistance gene.

[0028] On the other hand, the present invention also provides a detection method for Mycobacterium tuberculosis and its rifampicin resistance gene, wherein the detection method includes:

[0029] Extract nucleic acid fragments from the sample to be tested;

[0030] The above-described reaction system or detection kit is used to perform PCR melting detection on the nucleic acid fragments of the sample to be tested. The nucleic acid fragments can be DNA fragments.

[0031] The detection method described above in this invention is not intended for the direct diagnosis or treatment of diseases.

[0032] In the above detection method, preferably, when performing PCR detection, the magnesium salt concentration is 2-5 mM and the disodium hydrogen phosphate concentration is 0.5-2 mM; more preferably, when performing PCR detection, the magnesium salt concentration is 4 mM and the disodium hydrogen phosphate concentration is 2 mM.

[0033] In the above detection method, preferably, when performing PCR detection, the sodium sulfite concentration is 0.5-2mM, more preferably 1mM.

[0034] In the above detection method, preferably, when performing PCR detection, the forward probe and its corresponding reverse probe are mixed in TE buffer to prepare the four double-stranded displacement probes.

[0035] In the above detection method, preferably, when performing PCR detection, the working concentration of the four double-stranded displacement probes is independently 0.03-0.05 μM, more preferably 0.04 μM.

[0036] This invention provides a PCR reaction system, a detection kit, and their applications. The reaction system, based on a double-stranded displacement probe combined with a dynamic magnesium ion system, enables PCR melting detection of test samples using the PCR probe melting curve method, achieving the detection of Mycobacterium tuberculosis and / or rifampicin resistance genes. The PCR reaction system of this invention, through the synergistic effect of magnesium ions and disodium hydrogen phosphate, and further by adding ammonium sulfite, combined with the double-stranded displacement probe composition of this invention, offers advantages such as low fluorescence background, high detection sensitivity, good specificity, and good stability when applied for detection. Furthermore, by combining melting curve analysis, it can achieve multiplex analysis of rifampicin sensitivity and resistance within the same optical channel, overcoming the disadvantages of conventional melting curve methods, such as numerous impurities, inconsistent peak heights under the same conditions, and low melting curve sensitivity. Moreover, when applied to detection, the melting curve peak shape obtained by the reaction system of this invention is more stable than that of conventional asymmetric PCR systems, exhibiting better experimental repeatability and consistency. Attached Figure Description

[0037] Figure 1 The results show the comparison between the double-stranded substitution probe in Example 1 and the conventional molecular beacon probe.

[0038] Figure 2 The result is the optimized magnesium ion concentration in Example 2.

[0039] Figure 3 The results show the optimized concentration of disodium hydrogen phosphate in Example 3.

[0040] Figure 4 The results show the optimized concentration of ammonium sulfite in Example 4.

[0041] Figure 5 The results of the Mycobacterium tuberculosis sensitivity detection in Example 5 are shown.

[0042] Figure 6 The results of rifampicin-resistant tuberculosis detection in Example 6 are shown.

[0043] Figure 7 This is the specific detection result in Example 7. Detailed Implementation

[0044] The content of this invention can be more readily understood by referring to the following embodiments, which are provided by way of illustration and not intended to limit the invention, unless otherwise specified.

[0045] Unless otherwise specifically defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods not specifically described in the embodiments shall be performed in accordance with conventional practice in the art or as advised by the manufacturer's instructions.

[0046] In the following examples, only the sample used in the verification of the rifampicin resistance mutation site specifically indicated in Example 6 was a Mycobacterium tuberculosis mutant sample; the samples used in the other examples were Mycobacterium tuberculosis (sensitive) samples.

[0047] Example 1: Comparative Detection of Double-Stranded Substitution Probe and Conventional Molecular Beacon Probe

[0048] This study selected the Mycobacterium tuberculosis reference sequence (Genebank number CP016888.1), designed primers and probes using the Mycobacterium tuberculosis rpoB gene, and compared and validated the double-stranded substitution probe and the molecular beacon probe at a sample concentration of 10 CFU / ml.

[0049] 1.1 Experimental Materials

[0050] 1.1.1 Instruments: Archimed medical fluorescence quantitative PCR instrument, Qubit nucleic acid quantitative instrument

[0051] 1.1.2 Reagents: Extraction reagent: Bacterial genomic DNA extraction kit (Tiangen)

[0052] 1.1.3 Primers: Synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0053] 1.1.4 Sample: The national reference standard for Mycobacterium tuberculosis was purchased from the China National Institute for Food and Drug Control.

[0054] 1.2 Primer and probe design

[0055] The primer and probe design for detecting rifampicin resistance in Mycobacterium tuberculosis in this study used the Mycobacterium tuberculosis rpoB gene, with the reference sequence number CP016888.1 in Genebank. The primer and probe sequences are shown in Table 1 below.

[0056] Table 1 Primer and Probe Table

[0057]

[0058]

[0059] Note: The underlined sequence is the stem sequence in the molecular beacon probe.

[0060] 1.3 Experimental Design

[0061] 1.3.1 Template Preparation

[0062] I purchased Mycobacterium tuberculosis samples (batch number 230033-202304) from the China National Institute for Food and Drug Control and extracted them using a bacterial genomic DNA extraction kit (Tiangen). The extraction method was performed entirely in accordance with the kit's instructions. The extracted genomic DNA samples were stored at -20°C.

[0063] 1.3.2 Preparation of double-stranded substitution probes

[0064] 5.0 nmol of forward probe (fluorescent probe) and 6.0 nmol of reverse probe (quencher probe) were mixed and dissolved in 50 μL of TE buffer. The concentration of the prepared probe was based on the concentration of the forward probe (fluorescent probe), as shown in Table 2.

[0065] Table 2. Preparation details of the double-stranded substitution probe

[0066]

[0067]

[0068] 1.3.3 Reaction System

[0069] (1) Molecular beacon probe system, as shown in Table 3 below.

[0070] Table 3

[0071]

[0072] (2) The double-stranded displacement probe system is shown in Table 4 below.

[0073] Table 4

[0074]

[0075] 1.3.4 The reaction procedure is shown in Table 5 below.

[0076] Table 5

[0077]

[0078] Note: "*" indicates that fluorescence signals were collected at this stage.

[0079] 1.4 Experimental Results

[0080] Molecular beacon probe detection system and double-strand substitution probe system were prepared according to the reaction system in 1.3.3, and the two systems were compared for detection. The detection results are as follows: Figure 1 As shown. By Figure 1 It can be seen that the peak heights of the melting curves of each optical channel in the double-stranded substitution probe system are all higher than those in the molecular beacon probe system, indicating higher sensitivity. Based on this experiment, the double-stranded substitution probe system is selected for subsequent system optimization experiments.

[0081] Optimization of magnesium ion concentration in the system of Example 2

[0082] Using a sample (Mycobacterium tuberculosis DNA 20 CFU / ml), the magnesium ion concentration in the system was optimized to verify the optimal magnesium ion concentration.

[0083] 2.1 Experimental Materials

[0084] 2.1.1 Instruments: Archimed medical fluorescence quantitative PCR instrument, Qubit nucleic acid quantitative instrument

[0085] 2.1.2 Reagents: Extraction reagent: Bacterial genomic DNA extraction kit (Tiangen)

[0086] 2.1.3 Primers: Synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0087] 2.1.4 Sample: The national reference standard for Mycobacterium tuberculosis was purchased from the China National Institute for Food and Drug Control.

[0088] 2.2 Experimental Design

[0089] 2.2.1 Template Preparation

[0090] A Mycobacterium tuberculosis sample (batch number 230033-202304) was purchased from the China National Institute for Food and Drug Control. The sample was extracted using a bacterial genomic DNA extraction kit (Tiangen). The extraction method was performed entirely in accordance with the kit's instructions. The extracted genomic DNA was stored at -20°C.

[0091] 2.2.2 The reaction system is shown in Table 6 below.

[0092] Table 6

[0093]

[0094] 2.2.4 The reaction procedure is shown in Table 7 below.

[0095] Table 7

[0096]

[0097] Note: "*" indicates that fluorescence signals were collected at this stage.

[0098] 2.3 Experimental Results

[0099] Double-stranded displacement probe systems with magnesium ion concentrations of 2 mM, 3 mM, 4 mM, and 5 mM in the reaction system described in section 2.2.2 were prepared respectively to detect samples and screen for the optimal magnesium ion concentration. The detection results are as follows: Figure 2 As shown, the peak height of the melting curve and the absence of non-specific peaks were used as indicators for optimizing the concentration.

[0100] Optimization results: Based on the melting curves of the four optical channels, the optimal magnesium ion concentration is 2 μL with a concentration of 4 mM.

[0101] Optimization of disodium hydrogen phosphate concentration in the system of Example 3

[0102] As the number of cycles increases during PCR amplification, the activity of enzymes, the concentration of dNTPs, and the concentration of primers in the PCR reaction system decrease. Compared to the early stage of the experiment, the system requires less magnesium ions in the later stage. At this time, excessive magnesium ions will interact with other components in the PCR reaction (such as primers) to form magnesium ion-primer complexes, which reduces the binding of primers to template DNA and thus affects amplification efficiency.

[0103] Similarly, the above-mentioned problems still exist when using the double-stranded displacement probe system of the present invention. In order to address this problem, the present invention further improves the formulation of the double-stranded displacement probe system by adding disodium hydrogen phosphate (Na2HPO4) to the double-stranded displacement probe system and further optimizing its concentration.

[0104] To simplify the description, this embodiment integrates the reaction stability experiment after adding disodium hydrogen phosphate and the disodium hydrogen phosphate concentration optimization experiment directly into the disodium hydrogen phosphate concentration optimization experiment. This can not only prove that adding disodium hydrogen phosphate to the double-stranded substitution probe system is beneficial to the reaction stability, but also further verify the optimal concentration of disodium hydrogen phosphate in the system.

[0105] Compared to the initial stage of the experiment, the system's demand for magnesium ions decreased in the later stages, leading to a magnesium ion surplus. This invention addresses this by adding disodium hydrogen phosphate to the PCR reaction system. In the later stages, the hydrogen phosphate ions in the system bind with the excess magnesium ions, forming a dynamic equilibrium that promotes reaction stability. Using 20 CFU / ml of Mycobacterium tuberculosis DNA, the concentration of disodium hydrogen phosphate in the system was optimized to verify the optimal concentration.

[0106] 3.1 Experimental Materials

[0107] 3.1.1 Instruments: Archimed medical fluorescence quantitative PCR instrument, Qubit nucleic acid quantitative instrument

[0108] 3.1.2 Reagents: Extraction reagent: Bacterial genomic DNA extraction kit (Tiangen)

[0109] 3.1.3 Primers: Synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0110] 3.1.4 Sample: The national reference standard for Mycobacterium tuberculosis was purchased from the China National Institute for Food and Drug Control.

[0111] 3.2 Experimental Design

[0112] 3.2.1 Template Preparation

[0113] A Mycobacterium tuberculosis sample (batch number 230033-202304) was purchased from the China National Institute for Food and Drug Control. The sample was extracted using a bacterial genomic DNA extraction kit (Tiangen). The extraction method was performed entirely in accordance with the kit's instructions. The extracted genomic DNA was stored at -20°C.

[0114] 3.2.2 The reaction system is shown in Table 8 below.

[0115] Table 8

[0116]

[0117]

[0118] 3.2.4 The reaction procedure is shown in Table 9 below.

[0119] Table 9

[0120]

[0121] Note: "*" indicates that fluorescence signals were collected at this stage.

[0122] 3.3 Experimental Results

[0123] According to the reaction system in 3.2.2, double-stranded substitution probe systems with disodium hydrogen phosphate concentrations of 0 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM were prepared and used to detect the samples. The detection results are as follows: Figure 3 The peak height and absence of nonspecific peaks in the melting curves were used as optimization indicators for concentration. Without Na₂HPO₄, the peak consistency of the melting curves in the three parallel experiments was poor, and the peak height was low, resulting in poor sensitivity. Adding Na₂HPO₄ improved the peak consistency and peak height, thus enhancing sensitivity. Adding 2 μL of Na₂HPO₄ resulted in higher melting curve peaks across all four optical channels and the best consistency among the three parallel experiments. Optimization results: Based on the melting curve peaks of the four optical channels, the optimal addition amount of Na₂HPO₄ was 2 μL, and the concentration was 2 mM.

[0124] Optimization of ammonium sulfite concentration in the system of Example 4

[0125] Compared to the system with disodium hydrogen phosphate added in Example 3, Example 4 adds ammonium sulfite to the system determined in Example 3. In the later stages of the experiment, hydrogen phosphate and sulfite ions in the system combine with excess magnesium ions to form a dynamic equilibrium, which is beneficial to the stability of the reaction. Using 20 CFU / ml of Mycobacterium tuberculosis DNA from the sample, the concentration of ammonium sulfite in the system was optimized to verify the optimal concentration.

[0126] 4.1 Experimental Materials

[0127] 4.1.1 Instruments: Archimed medical fluorescence quantitative PCR instrument, Qubit nucleic acid quantitative instrument

[0128] 4.1.2 Reagents: Extraction reagent: Bacterial genomic DNA extraction kit (Tiangen)

[0129] 4.1.3 Primers: Synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0130] 4.1.4 Sample: The national reference standard for Mycobacterium tuberculosis was purchased from the China National Institute for Food and Drug Control.

[0131] 4.2 Experimental Design

[0132] 4.2.1 Template Preparation

[0133] A Mycobacterium tuberculosis sample (batch number 230033-202304) was purchased from the China National Institute for Food and Drug Control. The sample was extracted using a bacterial genomic DNA extraction kit (Tiangen). The extraction method was performed entirely in accordance with the kit's instructions. The extracted genomic DNA was stored at -20°C.

[0134] 4.2.2 The reaction system is shown in Table 10 below.

[0135] Table 10

[0136]

[0137] 4.2.4 The reaction procedure is shown in Table 11 below.

[0138] Table 11

[0139]

[0140] Note: "*" indicates that fluorescence signals were collected at this stage.

[0141] 4.3 Experimental Results

[0142] According to the reaction system in 4.2.2, double-stranded substitution probe systems with ammonium sulfite concentrations of 0 mM, 0.5 mM, 1 mM, 1.5 mM, and 2 mM were prepared and used to detect the samples. The detection results are as follows: Figure 4The melting curve peak height and the absence of non-specific peaks were used as optimization indicators for concentration. Adding 1 μL of ammonium sulfite resulted in higher melting curve peaks across all four optical channels and showed the best consistency among the three parallel experiments. Adding 2 μL of ammonium sulfite led to the appearance of non-specific melting curve peaks. Furthermore, compared to the system with only disodium hydrogen phosphate, the addition of ammonium sulfite resulted in better consistency among parallel experiments. Optimization results: Based on the melting curve peaks of the four optical channels, the optimal addition amount of ammonium sulfite was selected as 1 μL, with a concentration of 1 mM.

[0143] Example 5: Sensitivity Verification of the Detection System

[0144] The sensitivity of the system was verified by detecting Mycobacterium tuberculosis DNA at concentrations of 200 CFU / mL, 100 CFU / mL, 50 CFU / mL, 10 CFU / mL, and 5 CFU / mL.

[0145] 5.1 Experimental Materials

[0146] 5.1.1 Instruments: Archimed medical fluorescence quantitative PCR instrument, Qubit nucleic acid quantitative instrument

[0147] 5.1.2 Reagents: Extraction reagent: Bacterial genomic DNA extraction kit (Tiangen)

[0148] 5.1.3 Primers: Synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0149] 5.1.4 Sample: Mycobacterium tuberculosis was purchased from the China National Institute for Food and Drug Control.

[0150] 5.2 Experimental Design

[0151] 5.2.1 Template Preparation

[0152] A Mycobacterium tuberculosis sample (batch number 230033-202304) was purchased from the China National Institute for Food and Drug Control. The sample was extracted using a bacterial genomic DNA extraction kit (Tiangen). The extraction method was performed entirely in accordance with the kit's instructions. The extracted genomic DNA was stored at -20°C.

[0153] 5.2.2 The reaction system is shown in Table 12 below.

[0154] Table 12

[0155]

[0156]

[0157] 5.2.3 The reaction procedure is shown in Table 13 below.

[0158] Table 13

[0159]

[0160] Note: "*" indicates that fluorescence signals were collected at this stage.

[0161] 5.3 Experimental Results

[0162] Using the reaction system shown in Table 12, Mycobacterium tuberculosis samples at concentrations of 200 CFU / mL, 100 CFU / mL, 50 CFU / mL, 10 CFU / mL, and 5 CFU / mL were tested. The results are shown in [Table 12]. Figure 5 The results showed that, for the reaction system shown in Table 12, the sensitivity of the reaction system against Mycobacterium tuberculosis was 10 CFU / mL, and the lower limit was 5 CFU / mL (e.g., ...). Figure 5 The ATTO, CY5, and CY55 channels in the reaction system (5.2.2) still allow for the detection of Mycobacterium tuberculosis when the sample concentration is 5 CFU / mL.

[0163] Example 6: Validation of Rifampicin Resistance Mutation Site Detection

[0164] DNA from four common Mycobacterium tuberculosis rifampicin resistance mutation sites (L511P, D516V, H526D, S531L) and Mycobacterium tuberculosis (sensitive) were used to detect and verify the ability of the reaction system to distinguish rifampicin-resistant tuberculosis.

[0165] 6.1 Experimental Materials

[0166] 6.1.1 Instruments: Archimed medical fluorescence quantitative PCR instrument and Qubit nucleic acid quantitative instrument;

[0167] 6.1.2 Reagents: Extraction reagent: Bacterial genomic DNA extraction kit (Tiangen);

[0168] 6.1.3 Primers: synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0169] 6.1.4 Sample: Mycobacterium tuberculosis was purchased from the China National Institute for Food and Drug Control.

[0170] 6.2 Experimental Design

[0171] 6.2.1 Template Preparation

[0172] Mycobacterium tuberculosis and rifampicin-resistant Mycobacterium tuberculosis (L511P, D516V, H526D, S531L) (batch number 230033-202304) were purchased from the China National Institute for Food and Drug Control. The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit (Tiangen), and the extraction method was carried out in strict accordance with the kit instructions. The extracted genomic DNA was stored at -20℃.

[0173] 6.2.2 The reaction system is shown in Table 14 below.

[0174] Table 14

[0175]

[0176] 6.2.3 The reaction procedure is shown in Table 15 below.

[0177] Table 15

[0178]

[0179] Note: "*" indicates that fluorescence signals were collected at this stage.

[0180] 6.3 Experimental Results

[0181] Using the reaction system shown in Table 14, rifampicin-resistant Mycobacterium tuberculosis L511P, D516V, H526D, S531L, and tuberculosis-sensitive samples were detected. The results are shown in [Table 14]. Figure 6 (The specific experiment involved adding the five samples to five separate reaction systems, followed by amplification and melting experiments. During this process, four optical channels were used for detection, and the final product was generated as shown in the image.) Figure 6 The melting curve is shown below. When detecting rifampicin resistance mutation sites (L511P, D516V, H526D, S531L) in DNA from four Mycobacterium tuberculosis strains, L511P showed resistance in the ATTO channel, while the other samples were sensitive (from...). Figure 6 As can be seen, only the Tm value of L511P is inconsistent with the Tm value of the tuberculosis-sensitive sample, while the rest are consistent; D516V is drug-resistant in the CY5 channel, while the other samples are sensitive; S531L is drug-resistant in the VIC channel, while the other samples are sensitive; H526D and S531L are drug-resistant in the CY55 channel, while the other samples are sensitive. It can be seen that each drug-resistant site can be distinguished from the sensitive site.

[0182] Example 7: Validation of the Detection System Specificity

[0183] Using an optimized system, 1000 CFU / mL of Mycobacterium tuberculosis, Mycobacterium guildrums, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium abscessis were used to detect and verify the system's specificity.

[0184] 7.1 Experimental Materials

[0185] 7.1.1 Instruments: Archimed medical fluorescence quantitative PCR instrument and Qubit nucleic acid quantitative instrument;

[0186] 7.1.2 Reagents: Extraction reagent: Bacterial genomic DNA extraction kit (Tiangen);

[0187] 7.1.3 Primers: synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0188] 7.1.4 Samples: Mycobacterium guildense, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasense, and Mycobacterium abscessus were all purchased from the Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains.

[0189] 7.2 Experimental Design

[0190] 7.2.1 Template Preparation

[0191] Mycobacterium tuberculosis (purchased from China National Institute for Food and Drug Control, batch number 230033-202304), Mycobacterium guilloché (purchased from Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, catalog number BNCC364361), Mycobacterium avium (purchased from Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, catalog number BNCC380501), Mycobacterium intracellulare (purchased from Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, catalog number BNCC382227), Mycobacterium kansasii (purchased from Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, catalog number BNCC379558), and Mycobacterium abscessii (purchased from Henan Provincial Engineering Technology Research Center for Industrial Microbial Strains, catalog number BNCC364507) were extracted using a bacterial genomic DNA extraction kit (Tiangen). The extraction method was performed strictly in accordance with the kit instructions. The extracted genomic DNA was stored at -20℃.

[0192] 7.2.2 The reaction system is shown in Table 16 below.

[0193] Table 16

[0194]

[0195] 7.2.3 The reaction procedure is shown in Table 17 below.

[0196] Table 17

[0197]

[0198] Note: "*" indicates that fluorescence signals were collected at this stage.

[0199] 7.3 Experimental Results

[0200] Prepare the corresponding double-stranded displacement probe systems as shown in Table 16, and use samples of Mycobacterium tuberculosis, Mycobacterium guildrums, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium abscessis for detection. The detection results are shown in [Table 16]. Figure 7 .like Figure 7 As shown, only Mycobacterium tuberculosis was detected (blue melting curve), while other strains were not detected (no melting curve). The results indicate that the reaction system in 7.2.2 showed no cross-reactivity with other common nontuberculous mycobacteria.

[0201] In summary, this invention provides a PCR reaction system that combines high detection sensitivity, good specificity, and high stability, while also enabling multiplex analysis of rifampicin sensitivity and resistance in the same optical channel by combining melting curve analysis, and with a stable melting curve peak shape.

Claims

1. A reaction system for detecting Mycobacterium tuberculosis and its rifampicin-resistant gene, wherein, The reaction system comprises: a DNA polymerase, dNTPs, a reaction buffer, primers for amplifying a template, and a magnesium salt, disodium hydrogen phosphate, a double-stranded displacement probe composition; the nucleotide sequence of the primers for amplifying the template comprises SEQ ID NO. 1 and SEQ ID NO. 2; the double-stranded displacement probe composition comprises four double-stranded displacement probes; the nucleotide sequence of the forward probe of the four double-stranded displacement probes comprises SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, and SEQ ID NO. 13, respectively; the nucleotide sequence of the reverse probe of the four double-stranded displacement probes comprises SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, and SEQ ID NO. 14, respectively; the 5' end of the forward probe is labeled with a fluorescent group, and the 3' end of the reverse probe that forms a complementary double strand with the forward probe is labeled with a quenching group.

2. The reaction system of claim 1, wherein, The reaction system further comprises ammonium sulfite.

3. The reaction system of claim 1, wherein, The working concentration of the magnesium salt is 2-5 mM.

4. The reaction system of claim 1, wherein, The working concentration of the disodium hydrogen phosphate is 0.5-2 mM.

5. The reaction system of claim 2, wherein, The working concentration of the ammonium sulfite is 0.5-2 mM.

6. The reaction system of claim 1, wherein, The forward probe and its corresponding reverse probe are mixed in a TE buffer to prepare the four double-stranded displacement probes.

7. The reaction system of claim 6, wherein, The working concentration of the four double-stranded displacement probes is independently 0.03-0.05 μM.

8. A test kit, wherein, The detection kit comprises the reaction system of any one of claims 1-7.

9. Use of the reaction system of any one of claims 1-7 in a kit for detecting Mycobacterium tuberculosis and its rifampicin-resistant gene.

10. A detection method for detecting Mycobacterium tuberculosis and its rifampin-resistant gene, wherein, The detection method comprises: extracting nucleic acid fragments from a sample to be tested; performing PCR melting detection on the nucleic acid fragments of the sample to be tested using the reaction system of any one of claims 1-7 or the detection kit of claim 8.

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