Application of lncRNA-LOC100507195 in the diagnosis of sputum-negative pulmonary tuberculosis

Abstract

The present invention discloses the application of lncRNA-LOC100507195 in the diagnosis of septic tuberculosis. The present invention found that MTB infection with MΦ led to up-regulation of LOC100507195 expression; the test results of PBMC of 30 patients with septic tuberculosis showed that their expression level of LOC100507195 was significantly lower than that of healthy volunteers; this may be because LOC100507195, as a protective factor for macrophages against tuberculosis, was downregulated, resulting in susceptibility to tuberculosis. The results of the receiver operating characteristic curve analysis showed that LOC100507195 is expected to become a new target for the diagnosis of septic tuberculosis. By detecting the expression level of LOC100507195, it is possible to effectively distinguish between uninfected people and patients with septic tuberculosis.

Description

Technical Field

[0001] The present invention specifically relates to the application of lncRNA-LOC100507195 in the diagnosis of sputum-negative pulmonary tuberculosis. Background Art

[0002] Tuberculosis is still the infectious disease with the highest infection rate in the world, and the prevention and control situation is very severe. Rapid and sensitive diagnosis is the key to improving treatment effects and effectively controlling the spread of tuberculosis. Among the patients with pulmonary tuberculosis nationwide, patients with negative pulmonary tuberculosis account for 60% to 70% of active pulmonary tuberculosis patients, and the clinical symptoms and imaging features of many other lung diseases are difficult to distinguish from tuberculosis. The lack of effective diagnostic methods for negative pulmonary tuberculosis often causes delayed diagnosis or misdiagnosis of patients. Although my country has formulated several diagnostic standards, there are still many confusions in clinical application, and the problem of effective diagnosis of negative pulmonary tuberculosis has not been solved. Therefore, it has become a top priority to find new diagnostic markers for negative pulmonary tuberculosis and to develop new diagnostic methods!

[0003] Existing methods for diagnosing pulmonary tuberculosis mainly include sputum tuberculosis examination, tuberculin test, specific antibody determination, imaging examination and other methods. Sputum tuberculosis examination is time-consuming and has a high false negative rate; tuberculin test has a high false positive rate and is not easy to distinguish between people who have been vaccinated with BCG and people with tuberculosis; imaging examinations can hardly distinguish between pulmonary tuberculosis and other lung diseases. Finding nucleic acid as a diagnostic marker and using PCR technology as a diagnostic method have the advantages of being fast and sensitive, and are cheaper than specific antibodies, and are also convenient to prepare and test. Therefore, it is of great significance to find a nucleic acid marker that can be used as a diagnosis of negative pulmonary tuberculosis.

[0004] Mycobacterium tuberculosis (MTB) is an intracellular parasite that mainly parasitizes macrophages. Mycobacterium tuberculosis enters the lung tissue through the human respiratory tract in the form of aerosols and is phagocytosed by alveolar macrophages (AM), but MTB escapes the clearance of the body's immune system through corresponding mechanisms, and MTB will not be completely cleared by AM. Therefore, MTB can grow and multiply inside macrophages, and with the help of macrophage migration, continue to infect other uninfected macrophages. LncRNA (long noncoding RNA) is a type of RNA that is longer than 200nt and does not encode proteins. lncRNA can regulate gene expression at the epigenetic, transcriptional and post-transcriptional levels. It has a wide range of functions and participates in the regulation of various life activities of the body. It has been documented that lncRNA is involved in the regulation of the body's immunity.

[0005] There are already reports in the literature that lncRNA can be used as a diagnostic marker for cancer, but there are no reports on using lncRNA as a diagnostic marker for tuberculosis. Summary of the invention

[0006] The purpose of the present invention is to provide a new lncRNA diagnostic marker LOC100507195 for rapid diagnosis of sputum-negative pulmonary tuberculosis, and the price is lower than the current diagnostic methods.

[0007] The technical solution adopted by the present invention is:

[0008] Application of lncRNA nucleic acid marker in tuberculosis diagnosis, the nucleotide sequence of the lncRNA nucleic acid marker is shown in SEQ ID NO: 1, recorded as LOC100507195.

[0009] Preferably, the tuberculosis diagnosis is a pulmonary tuberculosis diagnosis.

[0010] Preferably, the tuberculosis diagnosis is a negative tuberculosis diagnosis.

[0011] A kit for diagnosing tuberculosis with negative bacteria, comprising a reagent capable of quantifying the expression amount of lncRNA-LOC100507195.

[0012] Use of a reagent capable of quantitatively detecting the expression level of lncRNA-LOC100507195 in the preparation of a tuberculosis diagnostic kit.

[0013] Preferably, the quantitative detection reagent contains a primer sequence capable of real-time fluorescence quantitative detection of lncRNA-LOC100507195.

[0014] Preferably, the pulmonary tuberculosis is bacterium-negative pulmonary tuberculosis.

[0015] The beneficial effects of the present invention are:

[0016] The present invention uses LOC100507195 obtained through early batch screening as a diagnostic marker for septic tuberculosis. By detecting the expression level of LOC100507195, uninfected people and septic tuberculosis patients can be effectively distinguished.

[0017] The present invention found that after MTB infects macrophages, it causes various reactions of macrophages, including gene transcription and protein synthesis, and the expression of lncRNA is also regulated. After MTB infects macrophages, it can cause the expression of LOC100507195 to be upregulated, indicating that LOC100507195 plays a corresponding role in the process of MTB infecting macrophages. Fluorescence quantitative PCR (qPCR) was used to detect PBMCs of patients with negative pulmonary tuberculosis and healthy normal people. Compared with PBMCs of healthy normal people, the expression of LOC100507195 in PBMCs of patients with negative pulmonary tuberculosis was downregulated, and there was a statistical difference between the two. This may be that LOC100507195 is a protective factor against tuberculosis in macrophages, and its expression is downregulated, causing the body to be susceptible to tuberculosis. Therefore, LOC100507195 can be used as a diagnostic marker for diagnosing patients with negative pulmonary tuberculosis and healthy normal people. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Real-time fluorescence quantitative PCR was used to detect the expression level of LOC100507195 in MΦ infected with MTB;

[0019] Figure 2 Real-time fluorescence quantitative PCR was used to detect the expression of lncRNA-LOC100507195 in PBMCs of patients with pulmonary tuberculosis with negative bacteria;

[0020] Figure 3 This is the ROC curve analysis. DETAILED DESCRIPTION

[0021] The present invention found that: ① MTB infection of MΦ leads to up-regulation of LOC100507195 expression; ② the detection results of PBMCs of 30 patients with negative pulmonary tuberculosis showed that the expression level of LOC100507195 was significantly lower than that of healthy volunteers; the receiver operating characteristic curve (ROC curve) analysis results showed that LOC100507195 is expected to become a new target for the diagnosis of negative pulmonary tuberculosis.

[0022] The present invention is further described below with reference to specific embodiments, but is not limited thereto.

[0023] Example 1 Upregulation of LOC100507195 expression in MΦ infected with MTB

[0024] Isolation of PBMC from healthy volunteers and immunomagnetic sorting of CD14 +Monocytes were induced to differentiate into MΦ using GM-CSF, and the resulting MΦ were infected with MTB. After 72 hours, the expression level of LOC100507195 in MΦ was detected by real-time fluorescence quantitative PCR.

[0025] Test results such as Figure 1 As shown, it can be seen that the expression level of LOC100507195 in MΦ after MTB infection was significantly upregulated, indicating that MTB infection promoted the expression of LOC100507195.

[0026] Example 2 Real-time fluorescence quantitative PCR detection of LOC100507195 expression in PBMC of patients with negative pulmonary tuberculosis

[0027] RNA was extracted from PBMCs of 30 sputum-negative pulmonary tuberculosis patients and 32 healthy volunteers, and the expression of lncRNA-LOC100507195 was detected by real-time fluorescence quantitative PCR.

[0028] Test results such as Figure 2 As shown, it can be seen that the expression of LOC100507195 in PBMCs of patients with negative pulmonary tuberculosis showed a significant down-regulation trend (P<0.001).

[0029] Example 3 LOC100507195 as a new target for diagnosis of tuberculosis with negative bacteria

[0030] This study found that compared with healthy people, the expression of LOC100507195 in PBMCs of tuberculosis patients showed a very significant down-regulation trend (P<0.001). In order to understand whether the expression changes of LOC100507195 in PBMCs can be used to distinguish healthy people from tuberculosis patients, we used ROC curves to analyze whether the expression changes of LOC100507195 in PBMCs can be used for the diagnosis of tuberculosis.

[0031] According to the relative expression of LOC100507195 in PBMC of healthy subjects and tuberculosis patients, a ROC curve with specificity as the abscissa and sensitivity as the ordinate was established using statistical analysis software. Figure 3 The results showed that the area under the ROC curve AUC (A) = 0.8594, indicating that LOC100507195 has a high accuracy in diagnosing tuberculosis patients ( Figure 3 ). Explanation of the meaning of the A value in the ROC curve: When A>0.5, the closer A is to 1, the better the diagnostic effect. A has a lower accuracy when it is 0.5-0.7, a certain accuracy when it is 0.7-0.9, and a higher accuracy when it is above 0.9.

[0032] The ROC curve (cumulative frequency distribution) was analyzed and the results are shown in Table 1.

[0033] Table 1 ROC curve analysis results

[0034]

[0035]

[0036] (The ellipsis above indicates that there is too much raw data, and only key data are listed in the table).

[0037] The results in Table 1 show that when the cut-off value (threshold value) of the transcription level of LOC100507195 is 7.368, the Youden's index (YI) is the largest (YI=62.5), and the detection sensitivity can reach 81.25%, while the specificity can reach 81.25%. Therefore, it is feasible to use LOC100507195 for the diagnosis of tuberculosis, which is of great significance for improving the diagnosis rate of tuberculosis.

[0038] In summary, lncRNA-LOC100507195 can be used as a new target for the diagnosis of sputum-negative pulmonary tuberculosis.

[0039] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention. <110> Southern Medical University <120> Application of lncRNA-LOC100507195 in the diagnosis of sputum-negative pulmonary tuberculosis <130> <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 574 <212> RNA <213> Homo sapiens <400> 1 gagaggctgt ggcaagggtc catgcctgaa tgtgtagaac aagaacagct ggaggctgtg 60 gctttctcca ggaagaggtt tctaaatctg cctcttagaa gatatcagaa agttaaagtt 120 tctcttgctg ccgccgtgta agaaggacct tttgcctcc accatgattc tagtcctcc 180 ccagccacgt ggaactgctt tcatttggtc tcttttcgaa tcagaagacc cagaaaactt ggatccactt ctggctctca tccctctggg cagtgtgatt tggattgagc agatgctccg 300 gctgccactc catcatatat tggaacagc attactttat agtaaatcat gaccacggtg 420. tatggcaatg aagttgcatc tgaagcagcc ggcaccctat gtgctagctg gcttttagc caggagactg tgaatctgtg gtcaggatct ctttagggct gctaaatata tactacccac 540. tatcactcgc tggagctgca gaccctagtt ttagtttc acttcttaaa gaccttcatc cttattaaaa tggtaatatc cttactgtca tcaa

Claims

1. Use of a reagent capable of quantitatively detecting the expression amount of lncRNA-LOC100507195 in the preparation of a diagnostic kit for tuberculosis with negative bacteria, wherein the nucleotide sequence of LOC100507195 is shown in SEQ ID NO: 1; The quantitative detection reagent contains a primer sequence capable of real-time fluorescence quantitative detection of lncRNA-LOC100507195, and the nucleotide sequence of LOC100507195 is shown in SEQ ID NO: 1.

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