Search

CN-121992096-A - Tuberculosis infection bimodal distinguishing microRNA combination, kit and detection system

CN121992096ACN 121992096 ACN121992096 ACN 121992096ACN-121992096-A

Abstract

The invention belongs to the technical field of tuberculosis detection, and provides a microRNA combination, a kit and a detection system for tuberculosis infection bimodal distinction. According to the invention, 9 specific microRNAs and 1 specific internal reference microRNAs are used as markers, the microRNAs in blood plasma are rapidly released by adopting a direct cleavage method, the relative expression quantity of the markers is synchronously detected by combining tail-added reverse transcription, four groups of primer grouping amplification and fluorescent PCR technology, and the comprehensive risk value is calculated by a built-in algorithm, so that latent tuberculosis infection, active infection and negative state are synchronously distinguished. The invention does not need sputum sample, has rapid and efficient detection, the detection sensitivity and specificity of active tuberculosis infection reach 92%, the detection sensitivity of latent tuberculosis infection reaches 93%, the specificity reaches 90%, the operation is convenient, and the application range is wide.

Inventors

  • LIU YITENG
  • GAO YIBO
  • XU NA
  • LIN LIHONG
  • CHEN XINCHUN
  • XU YUZHONG
  • HU YUNLONG

Assignees

  • 宁波市尚维高科有限公司

Dates

Publication Date
20260508
Application Date
20260108

Claims (10)

  1. 1. The microRNA combination for bimodal differentiation of tuberculosis infection is characterized by comprising 9 target microRNAs and 1 internal reference microRNA, wherein the 9 target microRNAs are hsa-let-7d-3p、hsa-miR-197-3p、hsa-miR-143-3p、hsa-miR-193a-3p、hsa-miR-16-2-3p、hsa-miR-92b-3p、hsa-miR-223-3p、hsa-miR-1290 and hsa-miR-320d, and the internal reference microRNAs are hsa-miR-151a-5p.
  2. 2. A primer probe combination, which is used for detecting the microRNA combination of claim 1, and comprises a specific reverse transcription primer group and a forward primer probe group; the reverse transcription primer group comprises nucleotide sequences shown as SEQ ID NO. 1-10; the forward primer probe group comprises nucleotide sequences shown as SEQ ID NO. 11-24.
  3. 3. The primer probe combination of claim 2, wherein the probe further carries a fluorescent group and/or a quenching group, wherein the fluorescent group comprises FAM, TET, JOE, HEX, VIC, ROX or Cy5, and wherein the quenching group comprises BHQ, MGB or TAMRA.
  4. 4. A kit for bimodal discrimination of tuberculosis infection, characterized in that it comprises a primer probe combination according to any one of claims 2-3.
  5. 5. The kit of claim 4, further comprising any one or more of a lysis buffer, a reverse transcription reaction solution, a reverse transcriptase, nuclease-free water, a PCR reaction solution, and a PCR enzyme.
  6. 6. The kit of claim 5, wherein the step of using the kit comprises: s1, directly cracking a sample to be detected and obtaining microRNA; s2, carrying out specific reverse transcription reaction on the microRNA by using a specific reverse transcription primer to obtain cDNA; S3, synchronously detecting the following biomarker combinations by using the cDNA as a template and adopting multi-target fluorescence PCR amplification, wherein microRNA is hsa-let-7d-3p、hsa-miR-197-3p、hsa-miR-143-3p、hsa-miR-193a-3p、hsa-miR-16-2-3p、hsa-miR-92b-3p、hsa-miR-223-3p、hsa-miR-1290、hsa-miR-320d and hsa-miR-151a-5p; S4, obtaining the Ct value of each biomarker, calculating the delta Ct value of each biomarker relative to an internal reference, and performing bimodal differential diagnosis of tuberculosis infection.
  7. 7. The kit of claim 6, wherein in S1, the lysate comprises protease K, naCl or KCl.
  8. 8. A dual-modality tuberculosis infection differentiation detection system, the detection system comprising: the sample processing unit is used for carrying out direct cleavage processing on a sample to be detected to obtain microRNA; A nucleic acid amplification unit, which pre-stores the primer probe combination according to any one of claims 2 to 3, and can automatically complete the specific reverse transcription reaction and the multi-target fluorescent PCR amplification; The data analysis unit is internally provided with a bimodal mathematical model, the model can receive the Ct values of the microRNAs output by the nucleic acid amplification unit, automatically calculate the delta Ct values of the 9 target microRNAs, calculate the active tuberculosis infection risk value and the latent tuberculosis infection risk value based on the delta Ct values, prestore double judgment thresholds and output distinguishing results through threshold comparison.
  9. 9. The test system of claim 8, wherein the sample to be tested comprises plasma, serum, whole blood, or peripheral blood free nucleic acid.
  10. 10. The detection system according to claim 8, wherein the determination is made that the active tuberculosis infection is determined when the active tuberculosis infection risk value is equal to or more than 0.5, the active tuberculosis infection risk value is <0.5, and the latent tuberculosis infection risk value is equal to or more than 0.5, and the determination is made that the determination is negative when both the active tuberculosis infection risk value and the active tuberculosis infection risk value are equal to or more than 0.5.

Description

Tuberculosis infection bimodal distinguishing microRNA combination, kit and detection system Technical Field The invention relates to the technical field of tuberculosis detection, in particular to a microRNA combination, a kit and a detection system for bimodal discrimination of tuberculosis infection. Background Tuberculosis is a chronic infectious disease caused by mycobacterium tuberculosis, has heavy global burden, and is important for prevention and control of latent tuberculosis infection and active tuberculosis in early stage identification. Although the conventional tuberculin test and gamma-interferon release test can detect infection, the conventional tubercle bacillus latent infection and active tuberculosis cannot be effectively distinguished, and the conventional sputum smear and culture method used as a gold standard have the limitations of low sensitivity, incapability of distinguishing latent tuberculosis patients, long time consumption, dependence on sputum samples and the like, so that a large number of patients are missed or delayed in treatment. Although the traditional molecular diagnosis technology improves the detection speed, the traditional molecular diagnosis technology is limited by equipment cost, electric power dependence, difficult sputum sampling and pretreatment, and difficult popularization in a basic layer. Therefore, development of a new rapid, accurate and convenient differential diagnosis method for basic use is needed. Micrornas (micrornas) are a class of short non-coding RNAs (about 21 to 23 nucleotides in length) produced by host cells with highly specific expression patterns during immune regulation and infection response. micrornas are stable in serum and easy to detect, and their concentration characteristics can reflect earlier changes in host immune status. In recent years, research shows that various microRNAs show specific expression changes in the tuberculosis infection process, and can be used as potential biomarkers for distinguishing latent tuberculosis infection and active tuberculosis. For example, chinese patent CN114231612A discloses a microRNA marker related to active tuberculosis and application thereof, the invention is based on 17 plasma microRNA markers for early diagnosis of active tuberculosis, the sensitivity of a validation set is 93 percent and the specificity is 71 percent, chinese patent CN107022643A provides application of miR-31-5p and miR-99a-5p in distinguishing active tuberculosis from tuberculosis latent infection, and the patent utilizes miR-31-5p and miR-99a-5p to distinguish active tuberculosis from latent infection, and the sensitivity is 74.5 percent and 70.6 percent respectively. The existing microRNA-based tuberculosis infection detection technology still has the defects of limited distinguishing capability and poor sensitivity and specificity, so that development of a tuberculosis infection bimodal distinguishing detection system with stronger sensitivity and specificity is needed at present. Disclosure of Invention The first aspect of the invention provides a microRNA combination for bimodal discrimination of tuberculosis infection, which consists of 9 target microRNAs and 1 internal reference microRNA, wherein the 9 target microRNAs are hsa-let-7d-3p、hsa-miR-197-3p、hsa-miR-143-3p、hsa-miR-193a-3p、hsa-miR-16-2-3p、hsa-miR-92b-3p、hsa-miR-223-3p、hsa-miR-1290 and hsa-miR-320d, and the internal reference microRNAs are hsa-miR-151a-5p. The second aspect of the invention provides a primer probe combination, which is used for detecting the microRNA combination and comprises a specific reverse transcription primer group and a forward primer probe group; the reverse transcription primer group comprises nucleotide sequences shown as SEQ ID NO. 1-10; the forward primer probe group comprises nucleotide sequences shown as SEQ ID NO. 11-24. Specifically, the probe also carries a fluorescent group and/or a quenching group, wherein the fluorescent group comprises any one or more of FAM, TET, JOE, HEX, VIC, ROX or Cy5, and the quenching group comprises any one or more of BHQ, MGB or TAMRA. In a third aspect, the invention provides a kit for bimodal discrimination of tuberculosis infection, the kit comprising the primer probe combination. Specifically, the kit further includes, but is not limited to, any one or more of a lysis buffer, a reverse transcription reaction solution, a reverse transcriptase, nuclease-free water, a PCR reaction solution, and a PCR enzyme. Further specifically, the kit comprises the following steps: s1, directly cracking a sample to be detected and obtaining microRNA; S2, carrying out specific reverse transcription reaction on the microRNA by using the specific reverse transcription primer to obtain cDNA; S3, synchronously detecting the following biomarker combinations by using the cDNA as a template and adopting multi-target fluorescence PCR amplification, wherein microRNA is hsa-let-7d-3p、hsa-miR-197-3p、hsa-miR-143-3p、