Method capable of realizing rapid detection of mycobacterium tuberculosis

Abstract

The invention relates to the technical field of biological detection and medical diagnosis, and discloses a method capable of rapidly detecting mycobacterium tuberculosis, which comprises the following steps: S1, providing a detection test strip which sequentially comprises a sample pad, a combination pad, a chromatography membrane and a water absorption pad; wherein the combination pad is coated with a labeled mycobacterium tuberculosis specific nucleic acid probe, the chromatography membrane is provided with a detection line and a quality control line, capture molecules are fixed on the detection line and the quality control line, the method integrates a nucleic acid probe chromatography technology and simple sample treatment, expensive instruments are not needed, and a result can be obtained by visual inspection within 15 minutes. The kit remarkably reduces the detection threshold and cost, is simple and convenient to operate and high in sensitivity, can synchronously identify drug resistance, perfectly meets the urgent demand of primary medical scenes on rapid and accurate diagnosis of tuberculosis, and assists in early intervention and epidemic prevention and control.

Description

Technical Field

[0001] This invention relates to the fields of biological detection technology and medical diagnostic technology, specifically to a method for rapid detection of Mycobacterium tuberculosis. Background Technology

[0002] Tuberculosis (TB) is a serious chronic infectious disease caused by Mycobacterium tuberculosis, and it is the leading cause of death from a single infectious disease worldwide. The World Health Organization reports that nearly one-third of the global population has been infected with TB, with over ten million new cases and millions of deaths annually. Therefore, developing rapid, accurate, and convenient TB detection technologies is of paramount importance for the early diagnosis, epidemic control, and effective treatment of TB.

[0003] While smear microscopy is currently simple and inexpensive, its sensitivity is low, typically requiring at least 5,000-10,000 bacteria per milliliter of sputum for detection, making it prone to false negatives. Meanwhile, solid or liquid culture is considered the "gold standard" for diagnosis and allows for drug sensitivity testing. However, this method is time-consuming, with long culture cycles, failing to provide timely evidence for early diagnosis and treatment, severely delaying the condition, especially for drug-resistant tuberculosis patients. Furthermore, by detecting the specific nucleic acid sequence of Mycobacterium tuberculosis, high sensitivity and specificity are achieved, and rifampicin resistance can be identified simultaneously. Despite this, this method has extremely high requirements for equipment, laboratory environment, and operator expertise, and is expensive, making it difficult to implement in resource-scarce rural and remote areas. Therefore, we propose a rapid detection method for Mycobacterium tuberculosis to address these problems. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a rapid detection method for Mycobacterium tuberculosis, solving the problems of high false negative rates caused by low sensitivity of smear microscopy; treatment delays caused by excessively long solid or liquid culture methods; and the stringent requirements of molecular nucleic acid detection for expensive equipment and professional environments, making it difficult to popularize in resource-scarce areas.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for rapid detection of Mycobacterium tuberculosis, comprising the following steps:

[0006] S1. A test strip is provided, wherein the test strip comprises, in sequence, a sample pad, a conjugate pad, a chromatography membrane, and an absorbent pad; wherein the conjugate pad is coated with a labeled Mycobacterium tuberculosis-specific nucleic acid probe, and the chromatography membrane is provided with a detection line and a control line immobilized with capture molecules;

[0007] S2. Process the sample to be tested to release the target nucleic acid of Mycobacterium tuberculosis in the sample;

[0008] S3. Add the processed sample to the sample pad of the test strip;

[0009] S4. By observing whether a visible color signal appears on the detection line within a predetermined time, it can be determined whether the sample contains Mycobacterium tuberculosis.

[0010] Preferably, in step S1, the marker is selected from colloidal gold, colored latex microspheres, quantum dots, upconversion luminescent materials, or fluorescent microspheres; the visible color signal is a color band corresponding to the marker.

[0011] Preferably, in S1, the sequence targeted by the Mycobacterium tuberculosis-specific nucleic acid probe is at least one of the following: a Mycobacterium tuberculosis complex-specific sequence, an IS6110 insertion sequence, and a rifampicin resistance-related rpoB gene mutation hotspot region sequence.

[0012] Preferably, in step S2, the processing includes extracting or isothermally amplifying nucleic acids from the sample.

[0013] Preferably, the isothermal amplification is loop-mediated isothermal amplification, recombinase polymerase amplification, or strand displacement amplification.

[0014] Preferably, the nucleic acid extraction or isothermal amplification step is performed at a single temperature not exceeding 65°C.

[0015] Preferably, when the nucleic acid probe targets the rpoB gene mutation hotspot region associated with rifampicin resistance, the presence of Mycobacterium tuberculosis and its resistance to rifampicin can be determined by observing whether the detection line changes color.

[0016] Beneficial effects

[0017] This invention provides a method for rapid detection of Mycobacterium tuberculosis. Compared with existing technologies, it has the following advantages:

[0018] This rapid detection method for Mycobacterium tuberculosis combines specific nucleic acid probe labeling with chromatography technology. Detection can be completed within 15 minutes simply by visually observing the chromogenic bands, eliminating the need for complex instruments and significantly reducing equipment dependence and operational barriers, making it particularly suitable for grassroots applications. Furthermore, by integrating simplified sample lysis with optional single-temperature isothermal amplification steps, the pretreatment process is further simplified while ensuring high sensitivity and specificity. This enables rapid, accurate, and low-cost detection of Mycobacterium tuberculosis and its drug resistance, providing efficient technical support for early diagnosis and epidemic control. Attached Figure Description

[0019] Figure 1 This is a flowchart of a method for rapid detection of Mycobacterium tuberculosis according to the present invention. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] like Figure 1 As shown:

[0022] A method for rapid detection of Mycobacterium tuberculosis includes the following steps:

[0023] S1. A disposable test strip is provided. Its structure, along the sample flow direction, includes a sample pad, a conjugate pad, a chromatography membrane, and an absorbent pad. The components are tightly connected. The conjugate pad is made of glass fiber and is pre-coated with a Mycobacterium tuberculosis-specific nucleic acid probe labeled with a marker. After drying, it is sealed and stored. The chromatography membrane is made of nitrocellulose and has two parallel functional lines: a detection line and a control line. The detection line has a capture molecule, such as a complementary nucleic acid sequence or antibody, that can specifically bind to the target nucleic acid complex of the labeled probe. The control line has a control molecule that can specifically bind to the free labeled probe to verify the effectiveness of the test strip.

[0024] The nucleic acid probe markers on the pad are selected from colloidal gold, which develops as red bands, or colored latex microspheres, which can be selected as blue or purple bands. No additional instruments are required, and the color signal can be observed with the naked eye. It is suitable for grassroots scenarios without equipment.

[0025] S2. Preprocess the sample to be tested. The core purpose is to release the target nucleic acid of Mycobacterium tuberculosis in the sample. The specific process is as follows:

[0026] Take 0.5-2 mL of sample, add an equal volume of lysis buffer, and let stand at room temperature for 5-15 min to destroy the cell wall and cell membrane of Mycobacterium tuberculosis.

[0027] If the bacterial count in the sample is small, isothermal amplification reagent can be added, and loop-mediated isothermal amplification, recombinase polymerase amplification, or chain displacement amplification can be selected. No temperature cycling is required; only a constant temperature water bath is needed.

[0028] Centrifuge for 5 minutes, and use the supernatant as the test solution. No purification is required; and nucleic acid extraction and isothermal amplification can be completed at a single temperature of ≤65℃, reducing equipment dependence.

[0029] S3. Use a pipette to draw 100-150 μL of the test solution and drop it into the center of the sample pad of the test strip, avoiding liquid overflow; place the test strip horizontally in a light-proof environment at 20-25℃. Relying on the capillary action of the absorbent pad, the test solution will migrate along the sample pad, conjugate pad, chromatography membrane and absorbent pad. The reaction takes 10-15 minutes and no additional operation is required during this period.

[0030] S4. Within 15 minutes after sample addition, observe the test line (T) and control line (C), and the judgment rules are as follows:

[0031] C-line and T-line color development indicate that the sample contains Mycobacterium tuberculosis;

[0032] The presence of a C-line and no T-line indicates that the sample does not contain Mycobacterium tuberculosis.

[0033] If line C does not show color, it indicates that the test strip is invalid and needs to be tested again.

[0034] If the probe targets the hotspot region of rpoB gene mutation, add wild-type rpoBT1 line and mutant rpoBT2 line. If the C line is colored, T1 is colored, and T2 is not colored, it indicates sensitivity. If the C line is colored, T1 is not colored, and T2 is colored, it indicates resistance. If the C line is colored, T1 and T2 are both colored, it indicates a mixed strain of wild-type and mutant.

[0035] It should be noted that the nucleic acid probe labels on the binding pad are selected from quantum dots, upconversion luminescent materials or fluorescent microspheres. Quantum dots, upconversion luminescent materials and fluorescent microspheres can be used for quantitative detection, and a simple reader is required to effectively improve the detection sensitivity.

[0036] In an optional embodiment, the sequence targeted by the Mycobacterium tuberculosis-specific nucleic acid probe is at least one of the following: a Mycobacterium tuberculosis complex-specific sequence, an IS6110 insertion sequence, and a rifampicin resistance-related rpoB gene mutation hotspot region sequence.

[0037] In this embodiment, the Mycobacterium tuberculosis-specific nucleic acid probe targets at least one of the following sequences to ensure detection specificity and avoid cross-reaction with non-tuberculous mycobacteria: For Mycobacterium tuberculosis complex-specific sequences, such as the conserved region of the 16S rRNA gene, it can cover pathogenic strains such as Mycobacterium tuberculosis and Mycobacterium bovis; For the IS6110 insertion sequence, a Mycobacterium tuberculosis-specific insertion sequence, each bacterial cell contains 10-15 copies, which can improve detection sensitivity; For the rifampicin resistance-related rpoB gene mutation hotspot region sequence: the rpoB gene is the gene encoding the β subunit of Mycobacterium tuberculosis RNA polymerase, and mutations in its 81bp core region are directly related to rifampicin resistance. Probes targeting this region can simultaneously achieve bacterial detection and drug resistance identification.

[0038] Example 1:

[0039] The IS6110 sequence probe labeled with colloidal gold was coated with a pad, and the complementary IS6110 sequence was fixed on the T line of the chromatographic membrane, while the anti-colloidal gold antibody was fixed on the C line.

[0040] Take 1 mL of sputum sample, add 1 mL of lysis buffer, let stand at room temperature for 8 min, centrifuge at 8000 rpm for 5 min, and take 100 μL of supernatant;

[0041] The supernatant was added to the sample pad and left at room temperature for 15 minutes. Both the C line and the T line turned red, indicating a positive result for Mycobacterium tuberculosis. A sputum sample from a healthy person was also tested, and only the C line turned red, indicating a negative result.

[0042] Example 2:

[0043] Using colored latex microspheres coated with a pad labeled with an rpoB gene probe, the T1 line of the chromatography membrane was immobilized with the wild-type rpoB complementary sequence, the T2 line with the mutant rpoB complementary sequence, and the C line with anti-latex microsphere antibody. 0.5 mL of bronchial lavage fluid was taken, lysis buffer was added, and then RPA amplification reagent was added. The mixture was incubated at 37°C for 20 min to obtain the amplified sample. 150 μL of the amplified sample was added, and after 15 min, color development was observed at the C line and T2 line, but no color development was observed at the T1 line, indicating a positive result for Mycobacterium tuberculosis and resistance to rifampin.

[0044] This solution combines specific nucleic acid probe labeling with chromatography technology, allowing for detection within 15 minutes with only visual observation of the colored bands. It eliminates the need for complex instruments, significantly reducing equipment dependence and operational barriers, making it particularly suitable for grassroots applications. Furthermore, by integrating simplified sample lysis with optional single-temperature isothermal amplification steps, it further simplifies the pretreatment process while maintaining high sensitivity and specificity. This enables rapid, accurate, and low-cost detection of Mycobacterium tuberculosis and its drug resistance, providing efficient technical support for early diagnosis and epidemic control.

[0045] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for rapid detection of Mycobacterium tuberculosis, characterized in that: The method comprises the following steps: S1, providing a test strip, the test strip comprising a sample pad, a conjugate pad, a chromatographic membrane and a water absorption pad in sequence; wherein the conjugate pad is coated with a labeled Mycobacterium tuberculosis specific nucleic acid probe, and the chromatographic membrane is provided with a detection line and a quality control line on which capture molecules are immobilized; S2, processing a sample to be tested to release Mycobacterium tuberculosis target nucleic acid in the sample; S3, adding the processed sample to the sample pad of the test strip; S4, observing whether a visible color signal appears on the detection line within a predetermined time to determine whether Mycobacterium tuberculosis is contained in the sample.

2. The method as claimed in claim 1, wherein the said method is capable of detecting Mycobacterium tuberculosis rapidly. In S1, the label is selected from colloidal gold, colored latex microspheres, quantum dots, up-conversion luminescent materials or fluorescent microspheres; and the visible color signal is a color band corresponding to the label.

3. The method as claimed in claim 2, wherein the said method is capable of detecting Mycobacterium tuberculosis in a time period of 2-3 hours. In S1, the Mycobacterium tuberculosis specific nucleic acid probe targets at least one of a Mycobacterium tuberculosis complex specific sequence, an IS6110 insertion sequence, and a rifampicin resistance related rpoB gene mutation hotspot region sequence.

4. The method as claimed in claim 2, wherein the said method is capable of detecting Mycobacterium tuberculosis in a time period of 2-3 hours. In S2, the processing comprises extracting or isothermal amplifying nucleic acid in the sample.

5. The method as claimed in claim 4, wherein the said method is capable of detecting Mycobacterium tuberculosis in a time period of 2-3 hours. The isothermal amplification is loop-mediated isothermal amplification, recombinase polymerase amplification or strand displacement amplification.

6. The method as claimed in claim 4, wherein the said method is capable of detecting Mycobacterium tuberculosis in a time period of 2-3 hours. The nucleic acid extraction or isothermal amplification step is completed at a single temperature of no more than 65℃.

7. The method as claimed in claim 1, wherein the said method is capable of detecting Mycobacterium tuberculosis in a time period of 2 hours. When the nucleic acid probe targets the rifampicin resistance related rpoB gene mutation hotspot region, observing whether the detection line changes color determines the presence of Mycobacterium tuberculosis and its resistance to rifampicin at the same time.

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