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CN-122017236-A - TB and NTM combined detection system and method based on multichannel immunodetection

CN122017236ACN 122017236 ACN122017236 ACN 122017236ACN-122017236-A

Abstract

The invention discloses a TB and NTM combined detection system and method based on multichannel immunodetection, and relates to the technical field of clinical detection. The method integrates the TB and NTM multi-target detection channels on the same immunochromatography platform, combines a specific antigen design, a multi-mode immunoreaction mechanism, an advanced signal processing algorithm and an expandable TDM (time division multiplexing) function, realizes the rapid, accurate and integrated identification of TB infection, NTM infection and co-infection states of the TB infection and the NTM infection, is suitable for various application scenes such as basic medical institutions, mobile screening vehicles, border inspection epidemic points, home self-tests and the like, and remarkably improves the early diagnosis efficiency and the accurate treatment level of mycobacteria infection.

Inventors

  • HE HUI
  • GU JUNQING
  • MI XING

Assignees

  • 恒燊中医科技(上海)有限公司

Dates

Publication Date
20260512
Application Date
20260304

Claims (10)

  1. 1. The TB and NTM combined detection system based on multichannel immunodetection is characterized by comprising a multichannel immunodetection card and a portable reading device; The multichannel immunity detection card is formed by sequentially laminating and fixing a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein 4-15 independent detection channels are arranged on the nitrocellulose membrane, signals are not interfered with each other through a physical isolation structure, the detection channels comprise at least one mycobacterium tuberculosis TB detection channel, at least one nontuberculous mycobacterium NTM detection channel, a quality control channel, a blank channel and a cross reaction monitoring channel, capture molecules aiming at TB specific antigens are coated in the TB detection channel, capture molecules aiming at NTM specific antigens are coated in the NTM detection channel, the width of each detection channel is 1.0-2.5 mm, and the length of each detection channel is 8-15 mm; The portable reading device comprises a signal acquisition module and an embedded processor, wherein the signal acquisition module is used for acquiring signals of all detection channels on the nitrocellulose membrane, the embedded processor is provided with a signal processing algorithm module and a interpretation stability index evaluation unit, and the signal processing algorithm module performs the following operations: S1, carrying out background subtraction and normalization processing on the collected original signals of all detection channels to obtain normalized signal intensity of all channels; S2, independently judging the normalized signal intensity of each channel based on a dynamic threshold mechanism, wherein the dynamic threshold is adjusted in real time based on a positive sample and negative sample distribution model continuously updated by a cloud database; S3, processing the comprehensive signal intensity of the TB detection channel and the comprehensive signal intensity of the NTM detection channel based on a cross channel deconvolution model to obtain a deconvolution signal value R s , wherein the cross channel deconvolution model is as follows: wherein, I TB is the normalized integrated signal intensity of the TB detection channel, I NTM is the normalized integrated signal intensity of the NTM detection channel, and alpha and beta are constant parameters obtained by training set calibration; S4, according to the comparison result of the deconvolution signal value R s , a first preset threshold value theta 1 and a second preset threshold value theta 2 , combining independent interpretation results of all channels, outputting a final diagnosis conclusion, wherein if the deconvolution signal value R s is larger than the first preset threshold value theta 1 , the determination is positive for TB, if the deconvolution signal value R s is smaller than the second preset threshold value theta 2 , the determination is positive for NTM, if the deconvolution signal value R s is larger than the second preset threshold value theta 2 and smaller than the first preset threshold value theta 1 , the comprehensive signal intensity I TB of the TB detection channel exceeds the independent interpretation threshold value, and meanwhile, the comprehensive signal intensity I NTM of the NTM detection channel exceeds the independent interpretation threshold value, the determination is double-infection for TB and NTM, and if the normalized signal intensity of all detection channels does not exceed the corresponding independent interpretation threshold value; The interpretation stability index evaluation unit generates by analyzing morphological characteristics of signal curves of all detection channels, wherein the morphological characteristics comprise signal peak width, rising edge slope and signal consistency among channels to obtain an interpretation stability index RSI, and when the interpretation stability index RSI is lower than a set threshold value, the system prompts resampling or retesting.
  2. 2. The combined TB and NTM detection system of claim 1, wherein said TB-specific antigen is selected from at least one of ESAT-6, CFP-10, ag85B and tuberculosis type lipoarabinomannan LAM, and said NTM-specific antigen is selected from at least one of NTM-LAM, mycobacterium avium complex MAC-specific antigen and Mycobacterium abscessum MAB-specific antigen.
  3. 3. The combined detection system for TB and NTM according to claim 1, wherein said TB detection channel adopts a sandwich immunoassay mode, a detection antibody for marking a signal molecule is preloaded in a binding pad, a capture molecule coated by the TB detection channel is an antibody for capturing a TB specific antigen, said NTM detection channel adopts a competition immunoassay mode, a NTM specific antigen-antibody complex marked with a signal molecule is preloaded in a binding pad, a capture molecule coated by the NTM detection channel is an antibody for capturing a NTM specific antigen, said signal molecule is colloidal gold particles or rare earth doped fluorescent microspheres, the particle size of said colloidal gold particles or rare earth doped fluorescent microspheres is controlled to be 20-100 nm, and the coating concentration of said capture molecule in the corresponding detection channel is 0.5-1.5 mg/mL.
  4. 4. The TB and NTM combined detection system according to claim 1 wherein the physical separation structure is a micron-sized groove formed in the surface of the nitrocellulose membrane, the groove having a depth of 10 to 30 μm and a width of 50 to 150 μm.
  5. 5. The combined TB and NTM detection system according to claim 1 wherein the TB detection channels are centrally disposed on one side and the NTM detection channels are centrally disposed on the other side, and wherein the TB detection channels and NTM detection channels are separated by at least a blank channel, a cross-reaction monitoring channel.
  6. 6. The combined TB and NTM detection system of claim 1, wherein said detection channel further comprises at least one drug monitoring channel, wherein said drug monitoring channel is internally coated with capture molecules for a specific therapeutic drug, wherein said conjugate pad is further preloaded with signal molecule complexes labeled with said specific therapeutic drug, and wherein said signal processing algorithm module of said portable reader device is further configured to quantitatively calculate the concentration of said specific therapeutic drug in a sample based on the competitive signal intensity of said drug monitoring channel and compare it to a predetermined therapeutic window range.
  7. 7. The TB and NTM combined detection system according to claim 6, wherein said specific therapeutic drug is selected from at least one of Bedaquinoline, delamani, pa Luo Mani, moxifloxacin and levofloxacin, and the capture molecule coated in said drug monitoring channel is a specific antibody or receptor protein for the corresponding drug, and the coating concentration is 0.8-1.2 mg/mL.
  8. 8. A detection method for detecting by using the TB and NTM combined detection system according to any one of claims 1 to 7, characterized in that the method comprises the steps of: (1) Providing a biological sample to be tested, and applying the biological sample to be tested to a sample pad of a multi-channel immunity detection card; (2) The biological sample to be detected sequentially flows through the binding pad, the nitrocellulose membrane and the water absorption pad under the capillary action, and specific immunoreactions occur in each detection channel of the nitrocellulose membrane; (3) Collecting signals of all detection channels on the nitrocellulose membrane by using a portable reading device; (4) Processing the acquired signals through a signal processing algorithm module arranged in the portable reading equipment, wherein the processing comprises background subtraction, normalization, independent channel interpretation based on a dynamic threshold value and comprehensive interpretation based on a cross channel deconvolution model; (5) Outputting a diagnosis conclusion of TB positive, NTM positive, TB and NTM double infection or negative and an interpretation stability index according to the signal processing result, and prompting resampling or retesting when the interpretation stability index is lower than a set threshold value.
  9. 9. The method according to claim 8, wherein the biological sample to be tested is whole blood, serum, plasma or urine, and when the biological sample to be tested is whole blood, a hemolysis buffer is added before or simultaneously with the application of the sample.
  10. 10. The method of claim 8, wherein when the detection system comprises a drug monitoring channel, the step (5) further quantitatively calculates and outputs a concentration evaluation result of the specific therapeutic drug in the sample based on the signal of the drug monitoring channel.

Description

TB and NTM combined detection system and method based on multichannel immunodetection Technical Field The invention relates to the technical field of clinical detection, in particular to a TB and NTM combined detection system and method based on multichannel immunodetection. Background Currently, tuberculosis (TB) remains a serious public health threat worldwide. NTM infection can cause pulmonary disease that is highly similar to tuberculosis, and patients often present with chronic cough, expectoration, hemoptysis, debilitation and low fever, with thoracic imaging characteristics (e.g., nodules, voids, fibroplasia, etc.) almost indistinguishable from tuberculosis. The overlapping of clinical and image results in clinical diagnosis with serious challenges, on one hand, NTM infection is easy to misdiagnose as tuberculosis, patients receive unnecessary long-term anti-tuberculosis treatment, adverse drug reaction is brought about, drug resistance is possibly induced, and on the other hand, active tuberculosis is misjudged as NTM disease, effective treatment is delayed, and the spreading risk and the disease deterioration possibility are increased. Therefore, accurate and rapid differentiation of TB and NTM infections has become an urgent need for clinical diagnosis and treatment. There are significant limitations to the prior art diagnostic techniques. For tuberculosis, the existing molecular detection tools such as GeneXpert are expensive in equipment, depend on laboratory environment, are difficult to popularize in areas with limited resources, have low sensitivity in traditional smear microscopy and are easy to miss diagnosis, and bacteria culture is a gold standard, but takes up to 2-6 weeks, so that treatment decisions are delayed. For NTM infection, the condition is more troublesome, namely, the rapid point of care testing (POCT) method special for NTM is lacking at present, the strain identification is carried out after the diagnosis is carried out in a multi-dependent culture mode, or the molecular techniques such as metagenome sequencing are relied on, and the methods have high cost and long period and require professional platforms and personnel. In the mixed infection or cross infection scenario, the problems are more complex, and the clinic often has difficulty in defining pathogen constitution in a short time, so that an accurate treatment scheme cannot be formulated. In addition, tuberculosis treatment drug monitoring (Therapeutic Drug Monitoring, TDM) is increasingly paid attention to optimizing curative effect and reducing drug resistance, but most detection systems at present fail to integrate pathogen identification and drug concentration monitoring functions, so that the whole-process management is disjointed. In summary, there is a urgent clinical need for a multi-channel fusion detection system capable of simultaneously achieving the following conditions, namely, firstly, achieving rapid detection within about 10 minutes, truly meeting POCT scene requirements, secondly, synchronously distinguishing mycobacterium tuberculosis from common NTM (such as M-intracellular mycobacteria complex (MAC), M-abscess Mycobacteria (MAB), M-Kansasii (KS) and the like), thirdly, identifying TB/NTM mixed infection, and fourthly, integrating therapeutic drug monitoring functions, and providing basis for individual medication. The system greatly improves the accuracy of early diagnosis, avoids mistreatment and misdiagnosis, controls drug resistance, optimizes patient management, and is particularly suitable for popularization and use in tuberculosis high-load areas, basic medical institutions and outpatient service. Disclosure of Invention The invention provides a TB and NTM combined detection system based on multichannel immunodetection, which aims to solve the technical problem that the composition of pathogens of lung diseases is difficult to be clarified in a short time in the prior clinical technology, so that an accurate treatment scheme cannot be formulated. The technical scheme adopted by the invention is as follows: a TB and NTM combined detection system based on multichannel immunodetection comprises a multichannel immunodetection card and a portable reading device; The multichannel immunity detection card is formed by sequentially laminating and fixing a sample pad, a combination pad, a nitrocellulose membrane, a water absorption pad and a bottom plate, wherein 4-15 independent detection channels are arranged on the nitrocellulose membrane, signals are not interfered with each other through a physical isolation structure, the detection channels comprise at least one mycobacterium tuberculosis TB detection channel, at least one nontuberculous mycobacterium NTM detection channel, a quality control channel, a blank channel and a cross reaction monitoring channel, capture molecules aiming at TB specific antigens are coated in the TB detection channel, capture molecules aiming at NTM specific antigens ar