Probe set, kit and method for detecting rifampicin resistance of mycobacterium tuberculosis

Abstract

The application provides a probe set, a kit and a method for detecting rifampicin resistance of mycobacterium tuberculosis, and specifically, the probe set comprises five Drop-off probes, target regions of the five Drop-off probes successively cover the rifampicin resistance determining region of the mycobacterium tuberculosis rpoB gene; and the rifampicin resistance determining region of the mycobacterium tuberculosis rpoB gene comprises codons 507-533. The probe set can realize high-sensitivity and high-precision detection of rifampicin heterogeneity resistance of mycobacterium tuberculosis and accurate quantification of the proportion of drug-resistant mutations, and the detection is rapid and simple; meanwhile, the probe set is suitable for various complex clinical few-bacterium samples, can effectively detect low-abundance drug-resistant mutations in the samples, and has wide sample applicability.

Description

Technical Field

[0001] This invention relates to the field of molecular detection, specifically to probe sets, kits, and methods for detecting rifampicin resistance in Mycobacterium tuberculosis, and more specifically to probe sets and compositions thereof, kits, and methods for detecting rifampicin resistance in Mycobacterium tuberculosis. Background Technology

[0002] Tuberculosis is caused by Mycobacterium tuberculosis (Mycobacterium tuberculosis) Mycobacterium tuberculosis Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB). According to the World Health Organization's Global Tuberculosis Report, TB remains a global public health problem. Rifampicin (RIF) is a core first-line drug for anti-TB treatment, possessing potent bactericidal activity and indispensable in short-course chemotherapy regimens for TB. The emergence of rifampicin resistance in Mycobacterium tuberculosis significantly impacts the success rate of TB treatment and the effectiveness of TB control. Therefore, rapid and accurate detection of rifampicin resistance in Mycobacterium tuberculosis is of significant clinical importance for guiding rational drug use and controlling the spread of drug-resistant strains.

[0003] Rifampicin resistance is mainly caused by Mycobacterium tuberculosis. rpoB This is caused by a single nucleotide mutation in the rifampicin resistance determining region (RRDR) of the rifampicin gene. Commonly used clinical methods for rifampicin resistance detection include phenotypic susceptibility culture and molecular detection. Phenotypic susceptibility culture is the traditional gold standard method, but the detection cycle is long (1-3 weeks) and it relies on live bacterial growth, making it unsuitable for inactivated or degraded samples. Molecular detection, represented by the GeneXpertMTB / RIF system, can provide resistance results within 2 hours, but its detection sensitivity and sample applicability have certain limitations. Furthermore, these methods are significantly inadequate in identifying heterogeneous resistance; when the proportion of resistant bacteria is below 10% or even 5%, these molecular detection methods are prone to false negatives. Patients may be misdiagnosed as rifampicin sensitive and receive ineffective traditional rifampicin-containing treatment regimens, which not only delays precise treatment but may also further screen and amplify resistant bacteria, increasing the risk of resistance transmission. In clinical practice, not all tuberculosis patients can provide qualified sputum samples for testing. Current molecular detection methods suffer from significantly reduced sensitivity and reliability in low-bacterial samples due to the sample's inherent characteristics (extremely low bacterial count, nucleic acid degradation, interference from PCR inhibitors, etc.).

[0004] Therefore, it is still necessary to further develop a precise detection method that can both detect low-frequency drug-resistant mutations with high sensitivity and stably detect drug-resistant Mycobacterium tuberculosis in samples with few bacteria, so as to provide new technical means for the precise detection of tuberculosis. Summary of the Invention

[0005] This application is based on the inventor's discoveries and understanding of the following facts and problems: Digital PCR (dPCR) is a technique for absolute quantification of nucleic acids, enabling single-molecule amplification and detection, and then achieving absolute quantification of target nucleic acids based on Poisson distribution statistics. This invention, based on digital PCR technology, employs a drop-off probe design strategy targeting Mycobacterium tuberculosis. rpoB A drop-off probe set designed to cover the entire RRDR region of rifampicin resistance was developed. This probe set effectively distinguished the RRDR region regardless of the mutation site's location. rpoB The probe set contains wild-type and mutant gene sequences, and the digital PCR detection method based on this probe set can detect low-frequency drug-resistant mutations with high sensitivity, and is also suitable for stable detection in samples with few bacteria.

[0006] Therefore, in a first aspect, the present invention provides a probe set. According to an embodiment of the invention, the probe set includes five drop-off probes, the target regions of which continuously cover Mycobacterium tuberculosis. rpoB The rifampicin resistance-determining region of the gene; the Mycobacterium tuberculosis rpoB The rifampicin resistance-determining region of the gene includes codons 507-533. The probe set described can be used to effectively distinguish... rpoB The probe set proposed in this invention contains both wild-type and mutant gene sequences and has broad-spectrum detection of RRDR region mutations in Mycobacterium tuberculosis. Regardless of the location of the mutation site, the probe set can cover the entire spectrum. It can be used for highly sensitive detection of heterogeneous drug resistance in Mycobacterium tuberculosis and accurate quantification of the proportion of drug resistance mutations. It is also suitable for various low-bacterial samples and has wide sample applicability.

[0007] According to an embodiment of the present invention, the probe set has a nucleic acid sequence as shown in any of SEQ ID NO:1 to 5.

[0008] According to an embodiment of the present invention, the reference probe for any one drop-off probe in the probe group is the other four drop-off probes.

[0009] According to an embodiment of the present invention, at least one drop-off probe in the probe set targets the Mycobacterium tuberculosis. rpoB The antisense strand of the rifampicin resistance-determining region of the gene.

[0010] According to an embodiment of the present invention, the probe set has locked nucleic acid modification.

[0011] According to an embodiment of the present invention, the probe group has fluorescent groups or quenching groups labeled at both ends.

[0012] According to embodiments of the present invention, the fluorescent group label includes at least one of the following: FAM, Q705, CY5, ROX, or VIC.

[0013] According to an embodiment of the present invention, the quenching group includes at least one of the following: BHQ1, BHQ2, or BHQ3.

[0014] In a second aspect, the present invention provides a probe composition. According to embodiments of the present invention, the probe composition comprises the probe set described above. The probe composition proposed in this invention can be used for highly sensitive detection of heterogeneous drug resistance in Mycobacterium tuberculosis and accurate quantification of the proportion of drug resistance mutations; it is also suitable for various low-bacterial samples, effectively detecting low-abundance drug resistance mutations, and has broad sample applicability.

[0015] According to an embodiment of the present invention, the probe composition further includes: a first primer, the first primer being used to amplify the Mycobacterium tuberculosis. rpoB Rifampicin resistance-determining regions in genes.

[0016] According to an embodiment of the present invention, the probe composition further includes: an exogenous internal reference plasmid template, a second primer and an exogenous internal reference probe, wherein the second primer is used to amplify the exogenous internal reference plasmid template, and the exogenous internal reference probe has fluorescent groups or quenching groups labeled at both ends.

[0017] According to an embodiment of the present invention, the first primer has a nucleic acid sequence as shown in any of SEQ ID NO:6~7.

[0018] According to an embodiment of the present invention, the second primer has a nucleic acid sequence as shown in any of SEQ ID NO:8~9.

[0019] According to an embodiment of the present invention, the exogenous internal reference plasmid template has a nucleic acid sequence as shown in SEQ ID NO:10.

[0020] According to an embodiment of the present invention, the exogenous internal reference probe has a nucleic acid sequence as shown in SEQ ID NO:11.

[0021] According to an embodiment of the present invention, the fluorescent group of the exogenous internal reference probe is labeled Q705.

[0022] In a third aspect, the present invention proposes the use of the aforementioned probe set or probe composition in the preparation of a kit for detecting rifampicin resistance in Mycobacterium tuberculosis. The kit prepared based on the aforementioned probe set or probe composition exhibits high sensitivity and accuracy in detecting rifampicin resistance in Mycobacterium tuberculosis, is rapid and simple to use, and is applicable to various low-bacterial samples. It can effectively detect low-abundance resistance mutations and has broad sample applicability.

[0023] In a fourth aspect, the present invention provides a reagent kit. According to embodiments of the present invention, the reagent kit comprises the probe set or probe composition described above. The reagent kit proposed in this invention exhibits high sensitivity and accuracy in detecting rifampicin resistance in Mycobacterium tuberculosis, is rapid and simple to use, and is applicable to various low-bacterial samples. It can effectively detect low-abundance drug-resistant mutations and has broad sample applicability.

[0024] In a fifth aspect, this invention provides a method for detecting rifampicin resistance in Mycobacterium tuberculosis. According to an embodiment of the invention, the method includes: generating a droplet from a nucleic acid to be tested, the droplet comprising the probe composition described above; performing PCR amplification on the droplet to obtain a fluorescence signal, the fluorescence signal comprising: a first fluorescence channel signal, the first fluorescence channel signal being the channel signal corresponding to the five drop-off probes in the probe set; a second fluorescence channel signal, the second fluorescence channel signal being the channel signal corresponding to the exogenous internal reference probe; and evaluating rifampicin resistance in Mycobacterium tuberculosis based on the fluorescence signal. The method proposed in this invention has high sensitivity and accuracy, is simple and rapid to operate, and provides reliable results. It is also applicable to various complex clinical samples with low bacterial counts, effectively detecting low-abundance drug-resistant mutations and exhibiting broad sample applicability.

[0025] According to an embodiment of the present invention, the nucleic acid to be tested is not less than 8 copies / reaction.

[0026] According to an embodiment of the present invention, the nucleic acid to be tested is derived from at least one of the following: sputum, culture, tissue, cerebrospinal fluid or bronchoalveolar lavage fluid.

[0027] According to an embodiment of the present invention, the tissue is formalin-fixed paraffin-embedded tissue.

[0028] According to an embodiment of the present invention, the annealing temperature of the method is 56°C to 63°C.

[0029] According to embodiments of the present invention, the drug resistance evaluation includes at least one of the following: rifampicin resistance heterogeneity rate of Mycobacterium tuberculosis, rifampicin resistance of Mycobacterium tuberculosis, or the effectiveness of detection.

[0030] According to an embodiment of the present invention, the determination rules for drug resistance evaluation include: if the second fluorescence channel signal is abnormal, the detection is deemed invalid; if the detection is valid, and the copy number corresponding to all five first fluorescence channel signals is <12 copies / reaction, Mycobacterium tuberculosis is deemed undetectable; if the detection is valid, and the copy number corresponding to all five first fluorescence channel signals is ≥12 copies / reaction, and the copy numbers corresponding to the five first fluorescence channel signals are equal after correction, then Mycobacterium tuberculosis is deemed to be present in the sample to be tested. Mycobacteria are sensitive to rifampicin; if the detection is valid, and at least one of the five first fluorescent channel signals has a corresponding copy number ≥ 12 copies / reaction, and at least one corresponding copy number < 12 copies / reaction, then the Mycobacterium tuberculosis in the test sample is determined to be resistant to rifampicin; if the detection is valid, and all the corresponding copy numbers of the five first fluorescent channel signals are ≥ 12 copies / reaction, and the corresponding copy numbers of the five first fluorescent channel signals are not completely consistent after correction, then the Mycobacterium tuberculosis in the test sample is determined to be resistant to rifampicin, and heterogeneous resistance exists.

[0031] According to an embodiment of the present invention, when the test sample exhibits heterogeneous drug resistance, and the highest copy number among the five corrected copy numbers of the first fluorescence channel signals is ≥100 copies / reaction, the five corrected copy numbers of the first fluorescence channel signals are sorted from high to low; channels with copy numbers lower than the highest copy number are identified as low copy number channels, and their corresponding copy numbers are denoted as A1, A2, ..., A1. N Where N is 1~4; let the highest copy number be denoted as B; then the heterogeneity corresponding to each low copy number channel is calculated by the following formula: Heterogeneity = (BA) N ) / B 100%.

[0032] According to an embodiment of the present invention, the correction includes: sorting the original copy numbers of the five first fluorescence channel signals from high to low; taking the arithmetic mean of the original copy numbers of the fluorescence channels whose relative deviation from the highest original copy number is not higher than a preset threshold, to obtain the corrected highest copy number.

[0033] According to an embodiment of the present invention, the preset threshold is 1%.

[0034] The beneficial effects of this invention are at least as follows: The probe set proposed in this invention can achieve high sensitivity and high accuracy in detecting rifampicin heterogeneous resistance in Mycobacterium tuberculosis and accurately quantify the proportion of drug resistance mutations. The detection is rapid and simple. At the same time, it is applicable to various complex clinical samples with few bacteria and can effectively detect low abundance of drug resistance mutations, thus having wide sample applicability. Attached Figure Description

[0035] Figure 1 This is a scatter plot of dPCR detection results for different mutation sites according to embodiments of the present invention, wherein, Figure 1 a is a scatter plot of the dPCR detection results of the L511P mutant droplet population; Figure 1 b is a scatter plot of the dPCR detection results of the D516V mutant droplet population; Figure 1 c is a scatter plot of the dPCR detection results of the H526D mutant droplet population; Figure 1 d is a scatter plot of the dPCR detection results of the S531L mutant droplet population; Figure 1 e is a scatter plot of dPCR detection results for the S522L mutant droplet population; the labeled droplet populations in the figure all correspond to the mutant type, and the marked droplet populations all correspond to the wild type.

[0036] Figure 2 This is a scatter plot of dPCR detection results for different mutation sites according to embodiments of the present invention, wherein, Figure 2 a is a scatter plot of the dPCR detection results of the L511R mutant droplet population; Figure 2 b is a scatter plot of the dPCR detection results of the Q513P mutant droplet population; Figure 2 c is a scatter plot of the dPCR detection results of the H526L mutant droplet population; Figure 2 d is a scatter plot of the dPCR detection results of the L533P mutant droplet population; Figure 2 e is a scatter plot of dPCR detection results for the N518D mutant droplet population; the labeled droplet populations in the figure all correspond to the mutant type, and the marked droplet populations all correspond to the wild type.

[0037] Figure 3 This is a scatter plot showing the effect of the exogenous internal control on target amplification according to an embodiment of the present invention, wherein... Figure 3 a is a scatter plot of the results for the experimental group (including the exogenous internal reference plasmid and its primers and probes). Figure 3 b is a scatter plot of the results for the control group (excluding the exogenous internal reference plasmid and its primers and probes).

[0038] Figure 4 This is a scatter plot showing the results of the exogenous internal control system according to an embodiment of the present invention dealing with interference from PCR inhibitors in the template, wherein... Figure 4 Group a is the experimental group (containing exogenous internal reference plasmid, primers and probes, and the template is a wild-type plasmid containing 1% formalin). There were no positive signals in the FAM channel and Q705 channel, and the exogenous internal reference failed to be amplified, indicating that there was interference from PCR inhibitors. Figure 4b is the control group (containing exogenous internal control plasmid, primers and probes, with an equivalent amount of DEPC water as the template). No positive signal was detected in the FAM channel, and a stable number of positive droplets were detected in the Q705 channel. The exogenous internal control was successfully amplified, indicating that the amplification system was normal.

[0039] Figure 5 This is a scatter plot of the results of using an exogenous internal reference as a signal reference according to an embodiment of the present invention. In the figure, all the labeled droplet groups are negative droplet groups, all the labeled droplet groups correspond to wild type, all the labeled droplet groups correspond to S522L mutant type, and all the labeled droplet groups correspond to exogenous internal reference plasmids.

[0040] Figure 6 This is a graph showing the effect of different annealing temperatures on amplification results according to an embodiment of the present invention. Figure 6 a is a graph showing the signal separation results of the S531L mutant plasmid in the FAM channel at different annealing temperatures; Figure 6 Figure b shows the signal separation results of the S531L mutant plasmid in the VIC channel at different annealing temperatures.

[0041] Figure 7 This is a graph showing the quantitative accuracy results of different mutant strains according to embodiments of the present invention.

[0042] Figure 8 This is a graph showing the quantitative accuracy results for different copy numbers according to an embodiment of the present invention.

[0043] Figure 9 This is a graph showing the linear relationship between the measured total copy number and the theoretical total copy number according to an embodiment of the present invention.

[0044] Figure 10 This is a Probit analysis result graph of low-concentration sample detection data according to an embodiment of the present invention.

[0045] Figure 11 This is a schematic diagram of the distribution of five drop-off probes according to an embodiment of the present invention. Detailed Implementation

[0046] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0047] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0048] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0049] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. In the description of this invention, the terms used herein have been explained and described; these explanations and descriptions are merely for the purpose of facilitating understanding and should not be construed as limiting the scope of protection of this invention.

[0050] In this article, the term "heterogeneous drug resistance" refers to the phenomenon that within the same bacterial population, there exist bacterial subgroups with different sensitivities to a certain drug, meaning that most bacteria are sensitive to the drug, while a few bacteria exhibit varying degrees of drug resistance.

[0051] In this article, "heterogeneity rate" is the proportion of drug-resistant subgroups in the total bacterial population, and is a quantitative indicator used to characterize the degree of heterogeneous drug resistance.

[0052] In this article, the term "drop-off probe" refers to a labeled probe used in digital PCR (dPCR) technology, specifically designed for the highly sensitive and specific detection and quantification of any type of sequence variation (such as point mutations, insertions, and deletions) on nucleic acids. Drop-off probe sequences perfectly match normal wild-type sequences but do not match any mutated sequences. It acts like a highly specific "lock" and "key," only a perfectly matching target can "unlock" and generate a signal. This design ensures that even a single base change is sufficient to prevent binding to the mutated template, causing it to "fall off." Drop-off probes are typically used in pairs with reference probes, where the reference probe binds to a conserved region of the target gene and is used solely to confirm the presence of target DNA (generating a basal fluorescent signal).

[0053] In this article, the term "rifampicin resistance determination zone" or "RRDR" refers to Mycobacterium tuberculosis (Mycobacterium tuberculosis). Mycobacterium tuberculosis ) rpoB A specific nucleotide sequence region on the gene, approximately 81 bp in length, covering positions 507 to 533 of the codon.

[0054] In this article, the terms "spectral crosstalk" or "fluorescent crosstalk" refer to the natural overlap of emission spectra of different fluorescent dyes when using multicolor fluorescent probes (such as FAM, VIC, ROX, CY5, etc.) for multiplex dPCR detection. The signal detected by one fluorescent channel is mixed with leakage signals from other fluorescent dyes, causing signal interference between channels.

[0055] In this paper, the term "boundary codon" refers to the codon spanned by the junction boundary between two adjacent drop-off probes, meaning that the 3' end of the preceding probe and the 5' start of the following probe are both located within the nucleotide sequence of that codon. The two adjacent probes each cover a portion of the nucleotides of the boundary codon, and their coverage areas are non-overlapping and seamlessly connected within the codon. For example, in the 517th codon of the RRDR resistance determination region, the target region of drop-off probe 2 covers one nucleotide at the 5' end of the codon, and the target region of drop-off probe 3 covers two nucleotides at the 3' end of the codon; together, they completely cover the boundary codon.

[0056] In this paper, the term "continuous coverage" refers to the sequential arrangement of the target regions of multiple drop-off probes on the target DNA sequence, with no gaps or overlaps between the target regions of adjacent probes, thus completely covering the target region. In this invention, it refers to the target regions of five drop-off probes covering Mycobacterium tuberculosis. rpoB The nucleotide sequences corresponding to codons 507-533 of the gene are arranged sequentially, with no gaps or overlaps in the target regions of adjacent probes. Depending on the position of the junction boundary between adjacent probes, this continuous coverage includes the following two forms: (1) The junction boundary of two adjacent drop-off probes is located inside the same codon. The 3' end of the first probe covers N nucleotides at the 5' end of the boundary codon, and the 5' end of the second probe begins to cover (3-N) nucleotides at the 3' end of the boundary codon, where N is 1 or 2. The coverage areas of the two probes are non-overlapping and gapless within the codon, and together they completely cover all 3 nucleotides of the boundary codon.

[0057] (2) The junction boundary between two adjacent drop-off probes is located between two adjacent codons. That is, the 3' end of the first probe terminates exactly at the last nucleotide of a codon, and the 5' end of the second probe begins at the first nucleotide of the next codon. The coverage of the two probes does not overlap or have gaps, and the junction is achieved at the junction of the two codons.

[0058] The technical solution of this application is described in detail below: probe sets and probe compositions In some embodiments of the present invention, a probe set is proposed. According to an embodiment of the present invention, the probe set includes five drop-off probes, the target regions of which continuously cover Mycobacterium tuberculosis. rpoB Rifampicin resistance-determining regions in genes.

[0059] According to embodiments of the present invention, the reference probe for any one drop-off probe in the probe set proposed in this invention is the other four. When a mutation occurs in a certain segment, the other four normal probes together form a reference system, achieving "self-calibrated" quantification without the need for additional reference probe design. For example: when there is one mutation site, the reference probe for any one of the five drop-off probes is the other four drop-off probes; when there are two mutation sites, the reference probe for any two of the five drop-off probes is the other three drop-off probes; when there are three mutation sites, the reference probe for any three of the five drop-off probes is the other two drop-off probes; when there are four mutation sites, the reference probe for any four of the five drop-off probes is the remaining one drop-off probe; the case of five mutation sites has not been reported to date.

[0060] The probe set can be used to effectively distinguish... rpoB The proposed probe set contains both wild-type and mutant gene sequences and possesses broad-spectrum detection capabilities for mutations in the RRDR region of Mycobacterium tuberculosis. Regardless of the location of the mutation site, the probe set can be covered, enabling highly sensitive detection of heterogeneous drug resistance in Mycobacterium tuberculosis and accurate quantification of the proportion of drug-resistant mutations. It is also applicable to various low-bacterial samples, exhibiting broad sample applicability. The drop-off probe proposed in this invention can detect any single nucleotide mutation in the RRDR resistance-determining region.

[0061] According to an embodiment of the present invention, the Mycobacterium tuberculosis rpoB The rifampicin resistance-determining region of the gene includes codons 507-533. The target regions of adjacent probes do not overlap, collectively covering the complete 81 bp RRDR region.

[0062] According to an embodiment of the present invention, at least one drop-off probe in the probe set targets the Mycobacterium tuberculosis. rpoB The antisense strand of the rifampicin resistance-determining region of the gene.

[0063] like Figure 11As shown, the target binding sequences of the five drop-off probes continuously cover the entire double-stranded region of the rifampicin resistance determination region. Drop-off probe 1 targets the antisense strand of the rifampicin resistance determination region, while drop-off probes 2-5 target the sense strand. Specifically, drop-off probes 2-4 are arranged sequentially from the 5' end to the 3' end, with adjacent probes covering a portion of the corresponding boundary codon. They are seamlessly connected within the codon without sequence overlap, collectively covering all three nucleotides of the boundary codon. The target regions of drop-off probes 4 and 5 do not overlap or have gaps, but are joined end-to-end by adjacent codons. The target region of drop-off probe 1 and the target region of probe 2 on the sense strand are complementary at the double-stranded level. Thus, the target regions of the five drop-off probes collectively cover the Mycobacterium tuberculosis. rpoB Rifampicin resistance-determining regions in genes.

[0064] Specifically, the target regions of the five drop-off probes correspond to the Mycobacterium tuberculosis, respectively. rpoB Codons 507-512, 512-517, 517-523, 523-528, and 529-533 of the rifampicin resistance-determining region of the gene.

[0065] According to embodiments of the present invention, the probe set has nucleic acid sequences as shown in any of SEQ ID NO: 1 to 5. According to embodiments of the present invention, each probe sequence is a perfect match for the wild-type corresponding region sequence and exhibits a base mismatch with the mutant (most known mutations are single-base mutations). During digital PCR amplification, perfectly matched probes can anneal normally with the template, resulting in high hydrolysis efficiency and producing a high-intensity fluorescence signal; while probes with single-base mismatches cannot anneal effectively with the template or have reduced annealing efficiency, therefore they cannot undergo hydrolysis or have low hydrolysis efficiency, and thus produce no fluorescence signal or a lower-intensity fluorescence signal.

[0066] According to an embodiment of the present invention, the probe set has locked nucleic acid (LNA) modification. The introduction of LNA modification into the probe can enhance the difference in Tm values ​​between the probe binding wild-type and common mutant types, thereby achieving distinguishable differences in fluorescence signals.

[0067] According to an embodiment of the present invention, the probe group has fluorescent groups or quenching groups labeled at both ends.

[0068] According to embodiments of the present invention, the fluorescent group label includes at least one of the following: FAM, Q705, CY5, ROX, or VIC.

[0069] It should be noted that HEX and VIC are defined as interchangeable groups with equivalent fluorescence spectral characteristics in the detection system described in this invention. They can be used interchangeably when labeling probes without affecting the multi-channel resolution of the detection results. Furthermore, Cy5.5 and Q705 are defined as interchangeable equivalent fluorescent labeling groups in the detection system described in this invention.

[0070] According to an embodiment of the present invention, the quenching group includes at least one of the following: BHQ1, BHQ2, or BHQ3.

[0071] In some embodiments of the present invention, a probe composition is proposed. According to embodiments of the present invention, the probe composition comprises the probe set described above. The probe composition proposed in this invention can be used for highly sensitive detection of heterogeneous drug resistance in Mycobacterium tuberculosis and accurate quantification of the proportion of drug resistance mutations; it is also suitable for various low-bacterial samples, effectively detecting low-abundance drug resistance mutations, and has broad sample applicability.

[0072] According to an embodiment of the present invention, the probe composition further includes: a first primer, the first primer being used to amplify the Mycobacterium tuberculosis. rpoB Rifampicin resistance-determining regions in genes.

[0073] According to embodiments of the present invention, the probe composition further includes: an exogenous internal reference plasmid template, a second primer, and an exogenous internal reference probe. The second primer is used to amplify the exogenous internal reference plasmid template, and the exogenous internal reference probe is labeled with fluorescent groups or quenching groups at both ends. The probe composition introduces an exogenous internal reference quality control system. The exogenous internal reference plasmid and its specific primers and probes are added to each reaction system, and the effectiveness of the PCR amplification process is monitored by the signal of the exogenous internal reference. When the presence of PCR inhibitors in the sample or abnormal amplification conditions lead to amplification failure, the exogenous internal reference signal is abnormal, thereby promptly determining that the experimental results are invalid and avoiding false negative results. This quality control mechanism significantly improves the reliability and reproducibility of the detection results, and is particularly suitable for processing complex clinical samples, such as FFPE tissue, cerebrospinal fluid, and other samples containing inhibitors.

[0074] According to an embodiment of the present invention, the first primer has a nucleic acid sequence as shown in any of SEQ ID NO:6~7.

[0075] According to an embodiment of the present invention, the second primer has a nucleic acid sequence as shown in any of SEQ ID NO:8~9.

[0076] According to an embodiment of the present invention, the exogenous internal reference plasmid template has a nucleic acid sequence as shown in SEQ ID NO:10. The exogenous internal reference plasmid template has no homology with the Mycobacterium tuberculosis genome, the human genome, and other common pathogens (such as bacteria, fungi, and viruses). The exogenous internal reference plasmid and its primer-probe system are independent of the target detection system, and its amplification is not affected by the presence or content of Mycobacterium tuberculosis DNA in the sample.

[0077] According to an embodiment of the present invention, the exogenous internal reference probe has a nucleic acid sequence as shown in SEQ ID NO:11.

[0078] According to an embodiment of the present invention, the exogenous internal reference probe is labeled with the fluorescent group Q705. The exogenous internal reference probe is used to determine detection effectiveness and calibrate spectral crosstalk. The second drop-off probe in the RRDR region shares the Q705 fluorescence channel with the exogenous internal reference probe, but the fluorescence intensity of the positive droplets produced by the two differs significantly: the second drop-off probe in the RRDR region has lower amplification efficiency, producing a low-amplitude positive signal; the exogenous internal reference probe has higher amplicon amplification efficiency, producing a high-amplitude positive signal. During data analysis, by setting two fluorescence intensity thresholds within the Q705 channel, low-amplitude positive droplet clusters and high-amplitude positive droplet clusters can be effectively distinguished, and the copy number of the target of the second drop-off probe in the RRDR region and the copy number of the exogenous internal reference plasmid can be quantitatively counted separately. The present invention labels the probe of the exogenous internal reference plasmid in the Q705 channel, which can serve as a reference for the positive signal in the Q705 channel, avoiding incorrect interpretation due to crosstalk between the CY5 and Q705 channels. In multiplex digital PCR, crosstalk can occur between different fluorescence channels, especially when the emission spectra of CY5 and Q705 are similar. A positive signal from one channel may be misinterpreted as a signal from another, affecting the accuracy of the results. This invention labels the exogenous internal reference plasmid probe onto the Q705 channel, which remains positive throughout the reaction system (as long as amplification is successful), providing a stable positive signal reference for the Q705 channel. During data interpretation, the fluorescence intensity and distribution of positive droplets from the exogenous internal reference Q705 channel can be used to calibrate and distinguish between the true positive signals and crosstalk signals of the CY5 and Q705 channels, thereby avoiding misinterpretations due to channel crosstalk.

[0079] Reagent kit and preparation purpose In some embodiments of the present invention, the use of the aforementioned probe sets or probe compositions in the preparation of kits for detecting rifampicin resistance in Mycobacterium tuberculosis is proposed. Kits prepared based on the aforementioned probe sets or probe compositions exhibit high sensitivity and accuracy in detecting rifampicin resistance in Mycobacterium tuberculosis, are rapid and simple to use, and are applicable to various low-bacterial samples. They can effectively detect low-abundance resistance mutations and have broad sample applicability.

[0080] In some embodiments of the present invention, a kit is provided. According to embodiments of the present invention, the kit comprises the probe set or probe composition described above. The kit proposed by the present invention has high sensitivity and accuracy in detecting rifampicin resistance in Mycobacterium tuberculosis, is rapid and simple to use, and is applicable to various low-bacterial samples. It can effectively detect low-abundance drug-resistant mutations and has broad sample applicability.

[0081] Detection methods In some embodiments of the present invention, a method for detecting rifampicin resistance in Mycobacterium tuberculosis is proposed. According to an embodiment of the present invention, the method includes: generating a droplet from the nucleic acid to be tested, the droplet comprising the probe composition described above; performing PCR amplification on the droplet to obtain a fluorescence signal, the fluorescence signal comprising: a first fluorescence channel signal, the first fluorescence channel signal being the channel signal corresponding to the five drop-off probes in the probe set; a second fluorescence channel signal, the second fluorescence channel signal being the channel signal corresponding to the exogenous internal reference probe; and evaluating the rifampicin resistance of Mycobacterium tuberculosis based on the fluorescence signal. The method proposed in this invention has high sensitivity and accuracy, and is applicable to various complex clinical samples with few bacteria, effectively detecting low-abundance drug-resistant mutations, and has broad sample applicability. The detection method proposed in this invention is simple, rapid, and reliable. From nucleic acid extraction to result reporting, the entire process can be completed within 4 to 6 hours, much faster than phenotypic drug sensitivity culture (1 to 3 weeks) and next-generation sequencing (1 to 2 days), and requires no complex bioinformatics analysis, making it suitable for widespread application in clinical testing laboratories.

[0082] According to an embodiment of the present invention, the nucleic acid to be tested is not less than 8 copies / reaction. According to a preferred embodiment of the present invention, the nucleic acid to be tested is not less than 12 copies / reaction.

[0083] According to an embodiment of the present invention, the nucleic acid to be tested is derived from at least one of the following: sputum, culture, tissue, cerebrospinal fluid or bronchoalveolar lavage fluid.

[0084] According to an embodiment of the present invention, the tissue is formalin-fixed paraffin-embedded tissue (FFPE).

[0085] According to embodiments of the present invention, current molecular detection methods are mainly applicable to sputum samples with high bacterial loads. Their effectiveness is significantly limited for low-bacterial samples such as biopsy tissues from extrapulmonary tuberculosis (especially FFPE samples), cerebrospinal fluid from tuberculous meningitis, bronchoalveolar lavage fluid, and sputum from patients with smear-negative pulmonary tuberculosis. In FFPE samples, nucleic acids undergo significant degradation and DNA fragmentation during fixation and embedding. Furthermore, residual formalin fixative, paraffin, and dyes in the samples inhibit PCR reactions, affecting amplification efficiency and detection sensitivity. Additionally, the presence of the blood-brain barrier typically results in extremely low Mycobacterium tuberculosis loads in cerebrospinal fluid samples. Low-bacterial respiratory samples such as bronchoalveolar lavage fluid also lead to a significant decrease in the sensitivity and reliability of current molecular detection methods. This invention utilizes the high sensitivity of digital PCR, making it equally applicable to the aforementioned low-bacterial samples. It can effectively detect low-abundance drug-resistant mutations, thereby expanding the application of rifampicin resistance detection from traditional sputum samples to a wider range of clinical sample types.

[0086] According to embodiments of the present invention, the annealing temperature of the method is 56°C to 63°C. Exemplarily, the annealing temperature of the method is 56°C, 56.1°C, 56.5°C, 57°C, 57.5°C, 58°C, 58.5°C, 59°C, 59.4°C, 59.5°C, 60°C, 61°C, 62.7°C, or 63°C, or a range between any two of the above values. According to some preferred embodiments of the present invention, the annealing temperature of the method is 59°C to 63°C; more preferably, it is 60°C.

[0087] According to embodiments of the present invention, the drug resistance evaluation includes at least one of the following: rifampicin resistance heterogeneity rate in Mycobacterium tuberculosis, rifampicin resistance in Mycobacterium tuberculosis, or the effectiveness of the detection. According to embodiments of the present invention, based on the absolute quantification principle of digital PCR technology, the present invention can stably detect drug resistance mutation rates as low as 1%, its detection sensitivity meets the requirements of the World Health Organization, and the detection cycle is shortened from several weeks to several hours, thereby effectively compensating for the shortcomings of existing rapid molecular detection technologies in identifying heterogeneous drug resistance.

[0088] According to an embodiment of the present invention, the determination rules for drug resistance evaluation include: if the second fluorescence channel signal is abnormal, the detection is deemed invalid; if the detection is valid, and the copy number corresponding to all five first fluorescence channel signals is <12 copies / reaction, Mycobacterium tuberculosis is deemed undetectable; if the detection is valid, and the copy number corresponding to all five first fluorescence channel signals is ≥12 copies / reaction, and the copy numbers corresponding to the five first fluorescence channel signals are equal after correction, then Mycobacterium tuberculosis is deemed to be present in the sample to be tested. Mycobacteria are sensitive to rifampicin; if the detection is valid, and at least one of the five first fluorescent channel signals has a corresponding copy number ≥ 12 copies / reaction, and at least one corresponding copy number < 12 copies / reaction, then the Mycobacterium tuberculosis in the test sample is determined to be resistant to rifampicin; if the detection is valid, and all the corresponding copy numbers of the five first fluorescent channel signals are ≥ 12 copies / reaction, and the corresponding copy numbers of the five first fluorescent channel signals are not completely consistent after correction, then the Mycobacterium tuberculosis in the test sample is determined to be resistant to rifampicin, and heterogeneous resistance exists.

[0089] According to an embodiment of the present invention, the correction is to eliminate background noise and experimental system bias.

[0090] According to an embodiment of the present invention, when the test sample exhibits heterogeneous drug resistance, and the highest copy number among the five corrected copy numbers of the first fluorescence channel signals is ≥100 copies / reaction, the five corrected copy numbers of the first fluorescence channel signals are sorted from high to low; channels with copy numbers lower than the highest copy number are identified as low copy number channels, and their corresponding copy numbers are denoted as A1, A2, ..., A1. N Where N is 1~4; let the highest copy number be denoted as B; then the heterogeneity corresponding to each low copy number channel is calculated by the following formula: Heterogeneity = (BA) N ) / B 100%.

[0091] According to an embodiment of the present invention, the correction includes: sorting the original copy numbers of the five first fluorescence channel signals from high to low; taking the arithmetic mean of the original copy numbers of the fluorescence channels whose relative deviation from the highest original copy number is not higher than a preset threshold, to obtain the corrected highest copy number.

[0092] According to an embodiment of the present invention, the preset threshold is 1%.

[0093] According to an embodiment of the present invention, the relative deviation is the percentage of the absolute value of the difference between the original copy number and the reference copy number (highest original copy number) of each channel relative to the reference copy number. The preset threshold is determined based on the signal-to-noise ratio of the first fluorescence channel signal and the dispersion of the copy number distribution. When the consistency of the original copy number of each channel is good, the threshold can be set to 1% to ensure that the number of channels participating in the mean calculation meets statistical requirements.

[0094] According to an embodiment of the present invention, when the test sample exhibits heterogeneous drug resistance, the heterogeneity rate is calculated based on the corrected copy number, where the highest copy number among the five first fluorescence channel signals is ≥100 copies / reaction, using the following formula: (1) Single low copy number channel: Sort the five signals of the first fluorescence channel according to copy number, determine the unique channel with the lowest copy number as A, and denote the arithmetic mean of the copy numbers of the remaining four channels as B. Heterogeneity = (BA) / B 100%.

[0095] (2) Multiple low copy number channels: Sort the five signals of the first fluorescence channel according to copy number, and determine the copy numbers of the N channels with the lowest copy numbers (2≤N≤4), denoted as A1, A2, ..., A N The arithmetic mean of the copy numbers of the remaining (5-N) channels is denoted as B. The heterogeneity of each low copy number channel is calculated using the following formula: Heterogeneity = (BA) i ) / B 100%, where i = 1, 2, ..., N.

[0096] It should be noted that the term "low copy number" here is a relative concept, referring to the copy number of other fluorescence channels, rather than a limitation on the absolute copy number. When the copy number of any fluorescence channel is significantly lower than the copy numbers of the other fluorescence channels, that fluorescence channel is determined to be a low copy number channel. According to an embodiment of the present invention, when the copy number of a certain fluorescence channel is significantly lower than the copy number of the other fluorescence channels, it suggests that heterogeneous drug resistance may exist at each point corresponding to that fluorescence channel.

[0097] The method proposed in this invention enables precise quantification of the proportion of drug-resistant mutations. Unlike existing related technologies (such as GeneXpert MTB / RIF) that only provide qualitative or semi-quantitative results, this invention directly calculates the absolute copy number of wild-type and mutant nucleic acids in the sample using Poisson distribution statistics, thereby obtaining an accurate mutation ratio. This quantitative information can not only be used to determine the existence of heterogeneous drug resistance but also to assess the development and changes of drug-resistant subgroups, providing data support for monitoring the efficacy of clinical anti-tuberculosis treatment and adjusting treatment plans.

[0098] The sequences involved in this invention are shown in Table 1 below.

[0099] Table 1. Sequences involved in this invention

[0100] Note: "+" in the table indicates locked nucleic acid modification.

[0101] Embodiments of the present invention will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0102] Example 1: Primer and probe design for the RRDR region 1.1 Primer and probe design for the RRDR region Obtain Mycobacterium tuberculosis from the NCBI database rpoB The nucleotide sequence of the rifampicin resistance-determining region (RRDR) of the gene is 81 bp in length, covering codons 507 to 533, and contains common rifampicin resistance mutation sites (such as codons 516, 526, and 531 mutations). A drop-off probe design strategy was employed, designing five probes that are contiguously linked to cover the entire RRDR region. Each probe sequence is a perfect match for the wild-type corresponding region sequence and exhibits a base mismatch with the mutant region (most known mutations are single-base mutations). During digital PCR amplification, perfectly matched probes anneal normally with the template, exhibiting high hydrolysis efficiency and producing a high-intensity fluorescence signal; while probes with single-base mismatches cannot anneal effectively with the template or have reduced annealing efficiency, thus failing to hydrolyze or exhibiting low hydrolysis efficiency, resulting in no fluorescence signal or a low-intensity fluorescence signal.

[0103] The primer design principles are as follows: (1) The upstream and downstream primer binding sites are located upstream and downstream of the RRDR region, respectively; (2) The amplicon should be as short as possible; (3) The primer length is generally 18-25 bp; (4) The difference in Tm values ​​between the upstream and downstream primers should be ≤2℃; (5) Dimers and self-complementarity should be avoided. The probe design principles are as follows: In order to ensure that the probe is effective for the target region, the target region should be designed for the target region. rpoB For effective detection of common mutations, priority should be given to ensuring that the difference in Tm values ​​between the probe binding wild-type and common mutants is at least 5°C to achieve a distinguishable difference in fluorescence signal. This method introduces locked nucleic acid (LNA) modification into the probe to enhance the difference in Tm values ​​between the probe binding wild-type and common mutants.

[0104] In this embodiment, for rpoB The primer and probe sequences designed for the RRDR region of the gene are shown in Table 2.

[0105] Table 2 Primer and probe sequences for the RRDR region

[0106] 1.2 RRDR region primers, probes, and verification experimental methods and materials To verify the effectiveness of the primers and probes, the following plasmids were constructed. These plasmids were purchased from General Biotechnology (Anhui) Co., Ltd.: a wild-type plasmid for the RRDR region, mutant plasmids for common sites in the RRDR region corresponding to different probes (including L511P, D516V, S522L, H526D, and S531L sites), and mutant plasmids for other sites in the RRDR region corresponding to different probes (including L511R, Q513P, N518D, H526L, and L533P sites). All purchased plasmids were dissolved and diluted to 1000 copies / µL with DEPC water. The mutant plasmids corresponding to different sites were then mixed 1:1 with the wild-type plasmid to obtain simulated samples corresponding to different mutation sites.

[0107] Wild-type and simulated samples with different mutation sites were used as templates to prepare digital PCR reaction systems, as shown in Table 3. Droplet generation was performed using the Drop Maker M1 sample preparation instrument manufactured by Xinyi Manufacturing Technology (Beijing) Co., Ltd. The chip and consumables used were provided by the Xinyi Manufacturing Technology (Beijing) Co., Ltd. general sample preparation kit (catalog number 10001, including the generation chip and generation oil). 30 µL of the prepared digital PCR reaction system was added to the sample wells of the droplet generation chip, along with 160 µL of generation oil. The chip was then covered with a rubber pad and placed in the chip loading area of ​​the Drop Maker M1. Water-in-oil droplets were generated in the instrument and stored in the reaction tube.

[0108] Table 3 Components and dosage of digital PCR reaction system

[0109] Place the reaction tube containing the water-in-oil droplets into a PCR amplification instrument for amplification. The reference amplification procedure is shown in Table 4.

[0110] Table 4 Digital PCR Amplification Program

[0111] After PCR amplification, microdroplet detection was performed using the Chip Reader R2 biochip analyzer manufactured by Xinyi Manufacturing Technology (Beijing) Co., Ltd. The chip and consumables used were provided by Xinyi Manufacturing Technology (Beijing) Co., Ltd.'s universal sample preparation kit (catalog number 10002, including the detection chip and detection oil). The reaction tube containing the droplets was placed in the Chip Reader R2, and 430 µL and 500 µL of detection oil were added to the wells of the detection chip, respectively. The analyzer sequentially read the fluorescence intensity of each droplet in the FAM, VIC, ROX, CY5, and Q705 channels. The detection results were displayed as a scatter plot or a one-dimensional plot. By setting the fluorescence amplitude threshold, droplets containing amplification products of different targets could be distinguished.

[0112] 1.3 Validation results of primers and probes for the RRDR region The raw experimental data corresponding to the simulated samples of common mutation sites are shown in Table 5, and the detection results are attached. Figure 1 As shown in Table 6. The original experimental data for other simulated mutation sites are shown in Table 6, and the detection results are attached. Figure 2 As shown, in droplets containing wild-type templates, all five fluorescence channels detected fluorescence signals significantly different from those in droplets without wild-type templates, appearing as double-positive droplets in the two-dimensional plots of pairwise fluorescence channels. In droplets containing mutant templates, the fluorescence channels corresponding to the probes at the mutation sites detected fluorescence signals comparable to those in droplets without wild-type templates, appearing as single-positive droplets in the two-dimensional plots of pairwise fluorescence channels. By setting corresponding fluorescence thresholds for each of the five channels, droplet clusters with different fluorescence signal amplitudes can be effectively distinguished, and the corresponding copy number can be calculated based on the number of droplets, followed by copy number correction (with a relative deviation not exceeding 1%, and subsequent corrections are all based on a relative deviation not exceeding 1%).

[0113] Based on the copy number measured for each channel, the heterogeneity can be calculated using the following formula: Heterogeneity = (BA) / B 100%; where A is the copy number of the target site corresponding to the probe that generates a low copy number signal, and B is the average copy number of the target site corresponding to the other probes that generate high copy number signals. Taking the L511P mutation site as an example: heterogeneity rate = (8157.7 - 4113.2) / 8157.7 100% = 49.58%. The heterogeneity calculation results for the remaining channels are listed in Tables 5 and 6, respectively. This result demonstrates that the primers and probes designed in this embodiment can effectively distinguish between different channels. rpoB The wild-type gene and any single nucleotide mutant sequence in the RRDR region can achieve the expected function.

[0114] Table 5. Digital PCR detection results of simulated samples with common mutation sites

[0115] Table 6. Digital PCR detection results of simulated samples from other mutation sites.

[0116] Example 2: Design of exogenous internal reference plasmids, primers, and probes 2.1 Design of exogenous internal reference plasmids, primers, and probes A synthetic DNA sequence was designed using a random algorithm to serve as an exogenous internal reference plasmid. The generated DNA sequence was then screened for homology and its effectiveness was verified. The sequence met the following conditions: (1) it had no homology with the Mycobacterium tuberculosis genome, the human genome, or other common pathogens (such as bacteria, fungi, and viruses); (2) the GC content was 40%-60%; and (3) the length was controlled between 100-200 bp to facilitate synthesis and amplification.

[0117] Using primer design software such as Primer3 and Oligo, specific primers and probes were designed based on the sequence. The design principles are as follows: (1) Amplicon ≤ 100 bp; (2) Primer length is generally 18-25 bp; (3) Primer GC content is generally 40-60%; (4) Tm value difference between upstream and downstream primers ≤ 2℃; (5) Avoid dimers and self-complementarity.

[0118] The sequences of the exogenous internal reference plasmid, primers, and probes involved in this embodiment are shown in Table 7.

[0119] Table 7. Sequences of exogenous internal reference plasmids, primers, and probes

[0120] 2.2 Validation of Exogenous Internal Reference Plasmids, Primers, and Probes: Experimental Materials and Methods To verify the effectiveness of the exogenous internal reference plasmid, the following three sets of experiments were set up: (1) Two experimental groups were set up: Based on the RRDR region system of Example 1, one group was supplemented with exogenous internal control plasmid (1000 copies / reaction) and its primers and probes (experimental group), and the other group was supplemented with an equivalent amount of DEPC water (control group). Both groups were supplemented with the same number of wild-type plasmids (10000 copies / reaction) as targets. The digital PCR reaction system was prepared as shown in Table 8, and the remaining digital PCR operation steps were the same as in Example 1. The copy number of RRDR and the copy number of exogenous internal control in the five channels were recorded, and the results were displayed as a two-dimensional scatter plot.

[0121] Table 8 Components and dosage of digital PCR reaction system (for group (1) experiment)

[0122] (2) Two experimental groups were set up: RRDR region systems containing exogenous internal control plasmids, primers, and probes were used. One group used wild-type plasmids containing 1% formalin (Beijing Solarbio Science & Technology Co., Ltd., Beijing) (10,000 copies / reaction) to prepare positive simulated samples containing PCR inhibitors (experimental group). The other group added an equivalent amount of DEPC water to prepare negative simulated samples (control group). The digital PCR reaction system was prepared as shown in Table 9. The remaining digital PCR operation steps were the same as in Example 1. The copy number of RRDR and the copy number of exogenous internal control in the five channels were recorded, and the results were displayed as a two-dimensional scatter plot.

[0123] Table 9. Components and dosages of digital PCR reaction system (including exogenous and internal controls)

[0124] (3) Four experimental groups were set up: Groups 1 and 2 used RRDR region systems containing exogenous internal control plasmids, primers, and probes, with templates of 5000 copies / reaction for wild-type plasmids and S522L mutant plasmids, respectively. Groups 3 and 4 used RRDR region systems without exogenous internal control plasmids, primers, and probes, with templates of 5000 copies / reaction for wild-type plasmids and S522L mutant plasmids, respectively. The digital PCR reaction systems were prepared according to Table 9 for Groups 1 and 2, and according to Table 3 for Groups 3 and 4. The remaining digital PCR operation steps were the same as in Example 1. The signals of the CY5 channel and Q705 channel were recorded, and the results were displayed as a two-dimensional scatter plot.

[0125] 2.3 Verification of experimental results using exogenous internal reference plasmids, primers, and probes (1) The experimental results are attached. Figure 3 As shown in Table 10, the original data were obtained. In normal samples, a stable number of positive droplets were detected in the Q705 channel of the experimental group, and the target copy number was not significantly different from that of the control group. This experimental result indicates that the exogenous internal control does not affect the target detection efficiency.

[0126] Table 10 Results of Digital PCR Copy Number in Group 1 Experiment

[0127] (2) The experimental results are attached. Figure 4 As shown in Table 11, the original data were as follows. Due to the influence of PCR inhibitors, neither the FAM nor Q705 channels in the experimental group showed positive signals, indicating that the exogenous internal control plasmid failed to amplify successfully, suggesting that the experiment was invalid and the detection result was a false negative. In the control group, the FAM channel showed no positive signal, but a stable number of positive droplets were detected in the Q705 channel, indicating that the exogenous internal control plasmid was successfully amplified, and the detection result was a true negative. These results indicate that the exogenous internal control quality control system can effectively monitor the PCR amplification process and improve the reliability of the detection results.

[0128] Table 11 Results of digital PCR copy number in Group 2

[0129] (3) The experimental results are attached. Figure 5 As shown, spectral crosstalk exists between the CY5 and Q705 channels, meaning their emission spectra (CY5 peak at approximately 670 nm, Q705 peak at approximately 705 nm) overlap in the long-wavelength direction, causing signal interference during detection. In systems without an exogenous internal reference plasmid, the lack of a Q705 monopositive droplet cluster as a reference leads to the wild-type plasmid's CY5 and Q705 bipositive droplet cluster signals being extremely similar to the S522L mutant plasmid's Q705 monopositive droplet cluster signal, severely interfering with accurate signal segmentation and interpretation. In systems with an exogenous internal reference plasmid, the presence of the Q705 monopositive droplet cluster as a reference prevents the wild-type plasmid's CY5 and Q705 bipositive droplet cluster from being misidentified as a Q705 monopositive droplet cluster, effectively avoiding signal interpretation obstacles caused by fluorescence channel crosstalk. Furthermore, the exogenous internal control generates a high-amplitude positive droplet cluster in the Q705 channel, while the RRDR probe 2 (if a mutant target is present) generates a low-amplitude positive droplet cluster in the Q705 channel. By setting two fluorescence intensity thresholds, the high-amplitude exogenous internal control droplet cluster and the low-amplitude RRDR probe 2 droplet cluster can be clearly distinguished, thus avoiding misjudgment due to channel crosstalk.

[0130] Example 3: Screening of annealing temperatures for digital PCR reaction systems To determine the optimal annealing temperature, this embodiment used the S531L mutant plasmid as a template (5000 copies / reaction) and set annealing temperature gradients (56.1℃, 57.2℃, 58.3℃, 59.4℃, 60.5℃, 61.6℃, 62.7℃, 63.8℃). Digital PCR detection was performed according to Table 9 and Example 1 to examine the signal separation degree of the S531L mutant plasmid in the FAM and VIC channels at different annealing temperatures.

[0131] Experimental results are as follows Figure 6As shown in the figure, within the annealing temperature range of 56.1℃ to 60.5℃, the signals of positive and negative droplets in the FAM channel were well separated; when the annealing temperature was higher than 60.5℃, the signal intensity of positive droplets in the FAM channel significantly weakened, and the signal separation decreased. Within the annealing temperature range of 59.4℃ to 62.7℃, all droplets in the VIC channel were negative, indicating that the probe corresponding to the mutation site could not be effectively annealed, which is consistent with the expected results of the drop-off method; when the annealing temperature was lower than 59.4℃ or higher than 62.7℃, weak positive signals appeared, indicating that the single-base mismatch probe and the template bound nonspecifically, and the probe specificity was greatly reduced. Therefore, considering the above two factors, 60℃ is selected as the optimal annealing temperature.

[0132] Example 4: Performance Verification of Heterogeneous Drug Resistance Detection Each mutant plasmid (L511P, D516V, S522L, H526D, S531L) was mixed with the wild-type plasmid at a certain ratio to prepare a series of samples with mutation ratios of 50%, 10%, 5%, 1%, and 0%, respectively. The total copy number of each sample ratio was fixed at 10,000 copies / reaction. Digital PCR detection was performed according to the steps shown in Table 9 and Example 1, with each ratio repeated three times. The specific copy numbers of the wild-type plasmid and the mutant plasmid were calculated to obtain the linear relationship between the measured mutation ratio and the theoretical value.

[0133] The experimental results are attached. Figure 7 As shown, for those located in rpoB For different mutation sites within the RRDR region of the gene (corresponding to the five mutation sites of the five probes), this method achieved a linear regression determination coefficient (R²) > 0.99 between the measured and theoretical values ​​within a mutation ratio range of 1%-50%, with a quantitative deviation ≤ 15%. Accurate quantification was still possible even when the mutation ratio decreased to 1%. These results demonstrate that this method exhibits good quantitative accuracy against heterogeneous drug resistance in different mutant strains.

[0134] To verify the quantitative accuracy under different copy numbers, simulated samples were prepared using wild-type and S531L mutant plasmids for the most common drug-resistant mutation site, S531L. The total copy number for each reaction was controlled at 50,000, 10,000, 5,000, 1,000, 500, and 100 copies / reaction. Simultaneously, under different copy number conditions, the ratio of wild-type to mutant plasmids was varied to obtain a series of simulated samples with mutation ratios of 50%, 10%, 5%, 2.5%, 1%, and 0.1%, to evaluate the quantitative accuracy under different template input amounts. Detection was performed according to the steps shown in Table 9 and Example 1, with each concentration repeated three times. The specific copy numbers of the wild-type and mutant plasmids were calculated, thus obtaining the relationship between the measured mutation ratio and the theoretical value.

[0135] The experimental results are attached. Figure 8 As shown, within the template concentration range of 100–50,000 copies / reaction, the linear regression coefficient of determination (R²) between the measured and theoretical copy numbers is >0.98, indicating a good linear relationship. Under different template input conditions, this method exhibits good linearity (R²>0.90) within the mutation ratio range of 1%–50%; the linear correlation coefficient is even higher when the 0.1% mutation ratio is not considered. These results demonstrate that this method can stably detect drug resistance mutation ratios of over 1% within a relatively wide dynamic range (100–50,000 copies / reaction).

[0136] Example 5: Validation of detection performance for low-bacterial samples This embodiment evaluates the detection performance of the method of the present invention on formalin-fixed paraffin-embedded tissue (FFPE) samples by preparing FFPE simulants. Clinically confirmed Mycobacterium tuberculosis-negative FFPE tissue samples (such as healthy lung tissue or lymph node tissue) were obtained, and DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Germany) according to the manufacturer's instructions to obtain a negative FFPE sample extract. In subsequent preparation of simulants, a known concentration of mutant or wild-type plasmid can be added to the negative FFPE sample extract and thoroughly mixed to ensure that the matrix composition of the final simulant sample is consistent with that of the real FFPE sample extract, thereby simulating the detection environment of nucleic acid degradation and the presence of inhibitors in real FFPE samples.

[0137] Linearity determination: For the most common drug-resistant mutation site S531L, FFPE simulated samples were prepared using the S531L mutant plasmid and negative FFPE extract, and then serially diluted to obtain simulated samples with copy number concentrations of 5, 50, 500, 5000, and 50000 copies / reaction, for a total of five gradients. Each gradient was repeated three times. Digital PCR detection was performed according to the steps shown in Table 9 and Example 1, and the linear relationship between the measured total copy number and the theoretical total copy number was calculated.

[0138] Determination of the limit of blank (LOB): Using a template-free control (NTC) as a blank sample, the experiment was repeated 20 times. Digital PCR detection was performed according to the steps shown in Example 1, and the copy number corresponding to the 5 probes was recorded. The LOB for each probe was calculated independently, and the 95th percentile, which is the 95th percentile value of the positive signal copy number distribution in the blank sample, was taken as the final LOB.

[0139] Determination of the limit of detection (LOD): For the S531L locus, six low-concentration series samples were prepared by serial dilution using FFPE simulated samples, with copy number concentrations of 50, 25, 12.5, 7.5, 5, and 2.5 copies / reaction. Each concentration gradient was repeated 20 times. Digital PCR detection was performed according to the steps shown in Example 1, and the detection rate of mutants at each concentration was calculated. Probit regression analysis was used to calculate the lowest mutant copy number corresponding to a 95% detection rate as the limit of detection (LOD).

[0140] Precision determination: Two concentrations of wild-type FFPE simulants were selected: high concentration (5000 copies / reaction) and low concentration (500 copies / reaction). Each concentration sample was tested 10 times within a day, and the mean, standard deviation, and coefficient of variation (CV%) of the measured copy numbers were calculated. Simultaneously, the samples were tested repeatedly over 5 consecutive independent experimental days (once daily, 3 replicates each time), and the inter-day coefficient of variation was calculated.

[0141] The experimental results for determining the linear range are attached. Figure 9 As shown, within the range of 5 to 50,000 copies / reaction, the linear regression equation between the measured copy number and the theoretical copy number is Y = 1.012X - 0.14, with a coefficient of determination R² = 0.9967, indicating that the method of this invention has a good linear relationship over a wide dynamic range.

[0142] The results of the blank limit determination show that the LOB of this method is 2.5 copies / reaction, meaning that when the mutant copy number is less than 2.5 copies / reaction, it cannot be reliably distinguished from the background signal. This result indicates that the method of the present invention has good specificity.

[0143] The results of the limit of detection (LOD) determination are shown in Table 12. Probit regression analysis was performed on the data in Table 12: since the detection rate was 100% at both 50 copies / reaction and 25 copies / reaction, the data point at 50 copies / reaction was discarded. The logarithms of the sample concentration values ​​for the remaining five concentration points were taken, and regression analysis was performed with the Probit values ​​corresponding to the detection rates at each concentration. The lowest mutant copy number corresponding to a 95% detection rate was taken as the LOD. The results are attached. Figure 10 As shown in the figure. The results indicate that the limit of detection (LOD) of this method is 11.9 copies / reaction, with a 95% confidence interval of (8.2, 17.1). This detection limit meets the clinical needs for detecting sparsely bacterial samples.

[0144] Table 12 Experimental results of FFPE sample detection limit determination

[0145] Precision measurement results showed that the intraday CV% for high-concentration samples was 4.8%, and the interday CV% was 7.2%; while the intraday CV% for low-concentration samples was 9.5%, and the interday CV% was 13.6%. All met the precision requirement of CV% < 25%, indicating that the method of this invention has good repeatability and reproducibility.

[0146] Example 6: Detection of Clinical Samples 6.1 Samples and Detection FFPE tissue samples were collected from 20 tuberculosis patients. All samples were confirmed as Mycobacterium tuberculosis infection by GeneXpert MTB / RIF, but rifampicin resistance was unclear. DNA was extracted using the QIAamp DNA FFPETissue Kit (QIAGEN, Germany) according to the manufacturer's instructions. After DNA extraction, digital PCR was performed using the probe composition described in Example 1 and the reaction system in Table 9, with an annealing temperature of 60°C. Exogenous and internal controls were included in the results analysis after passing quality control. Drug resistance determination and heterogeneity calculation were the same as in Example 1. Sputum samples were also collected from the same patients, and rifampicin resistance was confirmed using GeneXpert MTB / RIF.

[0147] 6.2 Results Analysis All exogenous internal control samples of 20 FFPE samples passed the quality control test, with a detection efficiency of 100%. The dPCR results are as follows: (1) Sensitive: 11 cases; (2) Drug resistant: 9 cases.

[0148] A retrospective analysis was conducted on the clinical outcomes of 20 patients. All 20 patients initially received a standard HRZE regimen containing rifampin. Eleven sensitive patients had a good prognosis; the nine resistant patients experienced disease progression within 1–3 months, and their regimens were subsequently adjusted. Furthermore, GeneXpert MTB / RIF testing of sputum samples from the nine resistant patients confirmed rifampin resistance.

[0149] This embodiment demonstrates that the method accurately determined rifampicin resistance status in 20 FFPE samples, showcasing the unique advantages of this method in retrospective detection of drug resistance and early warning of treatment prognosis using FFPE samples.

[0150] Verification of the effect of proportional probe locking nucleic acid modification This embodiment evaluates the impact of locked nucleic acid modification on the probe's ability to distinguish single-base mismatches, specifically targeting Mycobacterium tuberculosis. rpoBFor the three common mutation sites in the RRDR region of the gene (corresponding to codons 512-517, 523-528, and 529-533, respectively), two sets of probes with identical sequences but different modifications were designed: one set consisted of conventional DNA probes without LNA modification (SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14), and the other set consisted of probes with LNA modification introduced at the corresponding sites (SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5). LNA modification sites were marked with "+". The sequences of the three LNA-modified probes are shown in Table 1, and the sequences of the three unmodified probes are shown below: Unmodified probe 1 (corresponding codons 512-517, SEQ ID NO:12): Q705-CCAATTCATGGACC-BHQ3 Unmodified probe 2 (corresponding to codons 523-528, SEQ ID NO:13): ROX-TGTTGACCCACAAGCGC-BHQ2 Unmodified probe 3 (corresponding codons 529-533, SEQ ID NO:14): VIC-CGACTGTCGGCGCTG-BHQ1 Two reaction systems were prepared: one group containing the probes modified with locked nucleic acids as the experimental group, and the other group containing the corresponding probes without LNA modification as the control group (the remaining components were the same as the experimental group). Both the control and experimental groups were further divided into three subgroups, each containing a 1:1 mixture of wild-type and D516V, H526D, and S531L mutant plasmids (as described in Example 1) with a total copy number of 5000 copies / reaction as the mixed plasmid template. Digital PCR detection was performed according to the steps described in Example 1. The fluorescence intensity distribution of positive droplets in the corresponding channels (VIC, ROX, Q705) for each probe was recorded, and the signal separation between wild-type and mutant positive droplets was calculated using the following formula:

[0151] In the formula: μ1 and μ2 are the average fluorescence intensities of the wild-type droplet population and the mutant droplet population, respectively, and σ1 and σ2 are the standard deviations of the fluorescence intensities of the wild-type droplet population and the mutant droplet population, respectively.

[0152] The experimental results are shown in Table 13 below. Taking the probe group corresponding to the VIC channel as an example, in the control group, the fluorescence signals of wild-type and mutant positive droplets partially overlapped, with a low separation of 1.4; in the experimental group, the fluorescence signals of wild-type and mutant positive droplets were clearly separated, with a separation of 3.3, which was significantly improved compared to the control group. The results for the probe groups corresponding to the remaining two channels were similar, with the separation of the experimental group being significantly higher than that of the control group.

[0153] Table 13 Signal separation of probes with and without locked nucleic acid modification in corresponding channels

[0154] The above results indicate that locked nucleic acid modification significantly enhances the probe's ability to distinguish single-base mismatches and improves the signal separation between wild-type and mutant positive droplets. Therefore, the present invention preferably uses locked nucleic acid modified probes shown in SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:5 to ensure the accuracy and stability of heterogeneous drug resistance detection.

[0155] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0156] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A probe assembly, characterized in that, It includes 5 drop-off probes, the target regions of which continuously cover Mycobacterium tuberculosis. rpoB Rifampicin resistance-determining regions in genes; The tuberculosis mycobacterium rpoB The rifampicin resistance-determining region of the gene includes codons 507-533.

2. The probe assembly according to claim 1, characterized in that, The probe set has a nucleic acid sequence as shown in any of SEQ ID NO:1~5; Optionally, the reference probe for any one drop-off probe in the probe group is the other four drop-off probes; Optionally, at least one drop-off probe in the probe set targets the Mycobacterium tuberculosis. rpoB The antisense strand of the rifampicin resistance-determining region of the gene.

3. The probe assembly according to claim 1, characterized in that, The probe set is modified with locked nucleic acid; Optionally, the probe set has fluorescent groups or quenching groups labeled at both ends; Optionally, the fluorescent label includes at least one of the following: FAM, Q705, CY5, ROX, or VIC; Optionally, the quenching group includes at least one of the following: BHQ1, BHQ2, or BHQ3.

4. A probe composition, characterized in that, Includes the probe set as described in any one of claims 1 to 3.

5. The probe composition according to claim 4, characterized in that, The probe composition further includes: a first primer, the first primer being used to amplify the Mycobacterium tuberculosis. rpoB Rifampicin resistance-determining regions in genes; Optionally, the probe composition further includes: an exogenous internal reference plasmid template, a second primer, and an exogenous internal reference probe, wherein the second primer is used to amplify the exogenous internal reference plasmid template, and the exogenous internal reference probe has fluorescent groups or quenching groups labeled at both ends; Optionally, the first primer has a nucleic acid sequence as shown in any of SEQ ID NO:6~7; Optionally, the second primer has a nucleic acid sequence as shown in any of SEQ ID NO:8-9; Optionally, the exogenous internal reference plasmid template has a nucleic acid sequence as shown in SEQ ID NO:10; Optionally, the exogenous internal control probe has a nucleic acid sequence as shown in SEQ ID NO:11; Optionally, the exogenous internal reference probe is labeled with the fluorescent group Q705, and the exogenous internal reference probe is used to determine the detection effectiveness and calibrate spectral crosstalk.

6. Use of the probe set according to any one of claims 1 to 3 or the probe composition according to any one of claims 4 to 5 in the preparation of a kit for detecting rifampicin resistance in Mycobacterium tuberculosis.

7. A reagent kit, characterized in that, It includes the probe set according to any one of claims 1 to 3 or the probe composition according to any one of claims 4 to 5.

8. A method for detecting rifampicin resistance in Mycobacterium tuberculosis, characterized in that, include: The nucleic acid to be tested is subjected to droplet generation, wherein the droplets contain the probe composition according to claim 5; The droplets are subjected to PCR amplification to obtain fluorescence signals, which include: a first fluorescence channel signal, which is the channel signal corresponding to the 5 drop-off probes in the probe set; and a second fluorescence channel signal, which is the channel signal corresponding to the exogenous internal reference probe. Based on the fluorescence signal, the rifampicin resistance of Mycobacterium tuberculosis was evaluated.

9. The method according to claim 8, characterized in that, The nucleic acid to be tested is not less than 8 copies / reaction; Optionally, the nucleic acid to be tested is derived from at least one of the following: sputum, culture, tissue, cerebrospinal fluid, or bronchoalveolar lavage fluid; Optionally, the tissue is formalin-fixed paraffin-embedded tissue; Optionally, the annealing temperature of the method is 56°C to 63°C; Optionally, the drug resistance evaluation includes at least one of the following: rifampicin resistance heterogeneity rate in Mycobacterium tuberculosis, rifampicin resistance in Mycobacterium tuberculosis, or the effectiveness of the detection.

10. The method according to any one of claims 8 or 9, characterized in that, The rules for determining drug resistance include: If the signal in the second fluorescence channel is abnormal, the detection is deemed invalid. If the detection is valid, and the copy number corresponding to all the first fluorescent channel signals in the five first fluorescent channel signals is <12 copies / reaction, then Mycobacterium tuberculosis is determined to be undetectable. If the detection is valid, and in the five first fluorescence channel signals, the copy number corresponding to all the first fluorescence channel signals is ≥12 copies / reaction, and the copy number corresponding to the five first fluorescence channel signals is equal after correction, then the Mycobacterium tuberculosis in the sample to be tested is determined to be sensitive to rifampicin. If the detection is valid, and among the five signals of the first fluorescent channel, at least one corresponds to a copy number ≥ 12 copies / reaction, and at least one corresponds to a copy number < 12 copies / reaction, then the Mycobacterium tuberculosis in the sample to be tested is determined to be resistant to rifampicin. If the detection is valid, and in all five signals of the first fluorescence channel, the corresponding copy number is ≥12 copies / reaction, Furthermore, if the copy numbers corresponding to the five first fluorescence channel signals are not completely consistent after correction, it is determined that the Mycobacterium tuberculosis in the test sample is resistant to rifampicin and exhibits heterogeneous resistance. Optionally, when the test sample exhibits heterogeneous drug resistance, and the highest copy number among the corrected copy numbers of the five first fluorescence channel signals is ≥100 copies / reaction, the corrected copy numbers of the five first fluorescence channel signals are sorted from high to low. Channels with copy numbers lower than the maximum copy number are designated as low copy number channels, and their corresponding copy numbers are denoted as A1, A2, ..., A1. N where N is 1 to 4; Let the highest copy number be denoted as B; The heterogeneity corresponding to each low copy number channel is calculated using the following formula: Heterogeneity rate = (BA) N ) / B 100%.

11. The method according to claim 10, characterized in that, The correction includes: Sort the original copy numbers of the five signals from the first fluorescence channel from high to low; For fluorescence channels where the relative deviation between the original copy number and the highest original copy number is not higher than a preset threshold, the arithmetic mean is taken to obtain the corrected highest copy number. Optionally, the preset threshold is 1%.

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