Application of lncRNA-MIR3945HG V1 in the diagnosis of sputum-negative pulmonary tuberculosis

Abstract

The present invention discloses the application of lncRNA-MIR3945HG V1 in the diagnosis of septic tuberculosis. The present invention found that MTB infection with MΦ led to upregulation of MIR3945HG V1 expression; the test results of PBMC of 30 patients with septic tuberculosis also showed that the expression level of MIR3945HG V1 was significantly higher than that of healthy volunteers; the results of the receiver operating characteristic curve analysis showed that MIR3945HG V1 is expected to become a new target for the diagnosis of septic tuberculosis. By detecting the expression level of MIR3945HG V1, it is possible to effectively distinguish between uninfected people and septic tuberculosis patients.

Description

Technical Field

[0001] The present invention specifically relates to the application of lncRNA-MIR3945HGV1 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 lncRNA diagnostic marker MIR3945HGV1 for rapid diagnosis of sputum-negative pulmonary tuberculosis, and the price is lower than the current diagnostic method.

[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 MIR3945HGV1.

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

[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-MIR3945HGV1.

[0012] Application of a reagent capable of quantitatively detecting the expression level of lncRNA-MIR3945HGV1 in the preparation of a tuberculosis diagnosis kit.

[0013] Preferably, the quantitative detection reagent contains a primer sequence capable of real-time fluorescence quantitative detection of lncRNA-MIR3945HG V1.

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

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

[0016] The present invention uses batch screening in the early stage to screen and obtain lncRNA-MIR3945HGV1 as a diagnostic marker for septic tuberculosis. By detecting the expression level of MIR3945HG V1, uninfected people and septic tuberculosis patients can be effectively distinguished.

[0017] The present invention found that after MTB infects macrophages (MΦ), it causes various reactions of MΦ, including gene transcription and protein synthesis, and the expression of lncRNA is also regulated. After MTB infects MΦ, it can cause the expression of MIR3945HGV1 to be upregulated, indicating that MIR3945HG V1 plays a corresponding role in the process of MTB infecting MΦ. Fluorescence quantitative PCR (qPCR) was used to detect PBMCs of patients with negative pulmonary tuberculosis and healthy normal people. Compared with healthy normal people, MIR3945HG V1 was upregulated in PBMCs of patients with negative pulmonary tuberculosis, and there was a statistical difference between the two. Therefore, MIR3945HGV1 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 MIR3945HGV1 in MΦ infected with MTB;

[0019] Figure 2 Real-time fluorescence quantitative PCR was used to detect the expression of lncRNA-MIR3945HGV1 in PBMCs of patients with pulmonary tuberculosis. Figure 3 This is the ROC curve analysis. DETAILED DESCRIPTION

[0020] The present invention found that: ①MTB infection of macrophages MΦ leads to up-regulation of MIR3945HG V1 expression; ②the detection results of PBMCs of 30 patients with negative pulmonary tuberculosis also showed that the expression level of MIR3945HG V1 was significantly higher than that of healthy volunteers; the receiver operating characteristic curve (ROC curve) analysis results showed that MIR3945HGV1 is expected to become a new target for the diagnosis of negative pulmonary tuberculosis.

[0021] The present invention is further described below with reference to specific embodiments, but is not limited thereto.

[0022] Example 1 Upregulation of MIR3945HG V1 expression in MΦ infected with MTB

[0023] Isolation of PBMC from healthy volunteers and immunomagnetic sorting of CD14 + Monocytes were induced to differentiate into MΦ with GM-CSF, and the obtained MΦ were infected with MTB. After 72 hours, the expression level of MIR3945HGV1 in MΦ was detected by real-time fluorescence quantitative PCR.

[0024] Test results such as Figure 1As shown, it can be seen that the expression level of MIR3945HGV1 in MΦ after MTB infection was significantly upregulated, indicating that MTB infection promoted the expression of MIR3945HGV1.

[0025] Example 2 Real-time fluorescence quantitative PCR detection of MIR3945HG V1 expression in PBMC of patients with negative pulmonary tuberculosis

[0026] RNA was extracted from PBMCs of 30 sputum-negative pulmonary tuberculosis patients and 32 healthy volunteers, and the expression level of lncRNA-MIR3945HGV1 was detected by real-time fluorescence quantitative PCR.

[0027] Test results such as Figure 2 As shown, it can be seen that the expression of MIR3945HG V1 in PBMCs of patients with negative pulmonary tuberculosis showed a significant upregulation trend (P<0.0001).

[0028] Example 3 MIR3945HGV1 as a new target for diagnosis of tuberculosis

[0029] This study found that compared with healthy people, the expression of MIR3945HG V1 in PBMCs of tuberculosis patients showed a very significant up-regulation trend (P<0.0001). In order to understand whether the expression changes of MIR3945HGV1 in PBMCs can be used to distinguish healthy people from tuberculosis patients, we used ROC curves to analyze whether the expression changes of MIR3945HGV1 in PBMCs can be used for the diagnosis of tuberculosis.

[0030] According to the relative expression of MIR3945HG V1 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) = 0.9258, indicating that MIR3945HGV1 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.

[0031] The ROC curve (cumulative frequency distribution) was analyzed and the results are shown in Table 1.

[0032] Table 1 ROC curve analysis results

[0033]

[0034]

[0035] (The ellipsis above indicates that there is too much raw data, and only key data are listed in the table).

[0036] Analysis of the results in Table 1 shows that when the cut-off value (threshold value) of the transcription level of MIR3945HGV1 is 11.25, the Youden's index (YI) is the largest (YI=75), at which time the detection sensitivity can reach 100% and the specificity can reach 75%. Therefore, it is feasible to use MIR3945HG V1 for the diagnosis of tuberculosis, which is of great significance for improving the diagnosis rate of tuberculosis.

[0037] In summary, lncRNA-MIR3945HGV1 can be used as a new target for the diagnosis of sputum-negative pulmonary tuberculosis.

[0038] 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-MIR3945HG V1 in the diagnosis of sputum-negative pulmonary tuberculosis <130> <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 3390 <212> RNA <213> Homo sapiens <400> 1 atgtgggtag tctgaggccc acggtgtcag aagccctggt tattcaggct cctgaatggt 60 gactggtttg tcccctcctg ctggagtcat gtgtcctctg aactggccat ggagcagaag 120 aggttgcatg gccagaagga aaggaaaact gctcctgtct ccccatcccc tagaatcgtt 180 glycaaatga gtgacactgc cctgggatct gggtctctgg ttgaggcctt ggagctactg 240 cctctgcctg tgaactcacc catcatcgct aacaggttgc tccacagcct acagatctca 300 atatttcgct agcacaaac ccaagcggcc agcacggtg gctcacgcct gtaatcccag 360 cactttggga ggccgaggcg ggtggatcac ttgaggtcag gagttcgaga ccagcctgac 420 siacatggag aaaccccgtc tctaaaa atacaaaatt agccggcgt ggtgacacgt 480 gcccgtaatc ccagctactt gggaggctga ggcaggagaa ttgctgaac accggggtg 540 gaggttgtgg tgagccgaga ttgcaccact gcactccagt ctgggagaca gagagagacc 600 ccatctcaaa agaaaaaaaaaaaaagac cagggactt ccggaacta aacatctca 660 gccctttcat cgccagtgtc ttcatcttac aagcagaaaa ggaaaaaag cagtgggaag 720 cagtaagaag ctaccgctga ctcactatga attactgctg actttgaatt atttagcagc 780 tgtggaaaag ccatctttg tttaaaaaa aaataggtt ttctgaatg ctttttattt 840 ttttattgt ataataccct gcccagaaca gtcaagtcaa ctgacatctg ctaatgact 900 gagacactgt ggaatttgta tactggcatg gtgtgcagtt tcgtgcaagt ataccacag 960 agacagacaa atctctatca ttgccacaga ctgcttatag caaattcca gagctcctgt 1020 cattggtctt taatagttgt gctttgttgc tttgttacc acgtgtgcat ttgtgtcaac 1080 attatacaaa gcactctttc tcacagagc tagtttcact ctacaaccat actgtaagac 1140 aggtattgtt ttagatca gaaaccaagg ctctgagagg tttagtgact cacctagat 1200 catgaactaa taataagtga cggatcagggg acttgacctg ggacattgat gccaattcca 1260 gggcaccttc caccgtgtca tatggcactt gctctacata tgtgccaggc agcacccgca 1320 tggctttcc tcatgaaca acgtggacc agcctac agcagttgtg agaaagtgcc 1380 tccagctggg agcgcatctg gcccagggca ccagctgcca ctccatgctc tgaatccatc 1440 ctggcctgtg ctgacccc ccctttccat gggctgctcc ccaccgtga tggggtgg 1500 caggggcact aggcagcccc cttcctgaga gataggggc tcctctgcct agaatgttgt 1560 ctcagcactt ctctctctgcc ttgctgaact ttcctagag ccaggaggc ctccatacaa 1620 cctttcctct ccttcatttg gagtcagact tgcactgcag tctaatggtc atcccacttt 1680 tttccagctc cctgataaaa tctatggctt ctgcttcttg gaggacccag attatcacag 1740 cttatctcat tcctttgcag cagatgcccc tgcaggccat ctctccaggc tcaggaacct 1800 aaagaagaat ctacccagat gtggtggctc acacctgtga tcccaccact ttgggaggct 1860 gaggcaagag gatcacttga gcccaggagt tggagaccaa cgtaggcaat acagcaagac 1920 tcccatctct acaaaaaaaa aaaagaaaga aagaaagaaa gaaaagaaat tagctggctg 1980 tggtgctgca catttgtaat accagctact ctagaggctg aggctggagg atcacttgag 2040 ccgaggagtg agctatgacc acaccattgc atttcagcct cagtgacaga gcaaggccct 2100 gcctcaaaaa aaaaaaaaaa aaaagaaaag aataagataa aataaataat acaggagaac 2160 ctagtgctca ggttctctag tgaagaggaa ggcaaaagat agaaaggtca gacaaactta 2220 aacatgcatt ccaagatcgg cctgggtccg cccgtagaca tgaaggaatc ttctggtgga 2280 aagttaggca gggaaacgga cactgctatg agcatgtctt cctgggcagc ttgcgatggt 2340 caggaggagg tggttgttcc tcagcagcgg tgcttccgat gaagccccat gttgtagtgg 2400 aagacaagcc attcctgtct gaccttctgt gccgcgccta ggcttccaaa agctgctatc 2460 gccaagatcc atttccgttc acctttaaac tgcttgttat ggggaggagt cagccagcca 2520 tgatgcatac ttgttacaga aagaaacgcc cacgttgagc tctcctgata agaagagaga 2580 gcattcgtca ttgtctgtat ctgtcatctg aagaaggaac caatgtcaaa aatcccacac 2640 attctcctcc cgcaagtcac agagtaagcc tcccattctt gagctcgttc attcaccaaa 2700 cacattctga tctctcctgt gcccaggcgg tgggattcag agaagaagtg ctgcctagta 2760 gtcagcaccc agtgccagga aaacaggaca aggggagttg tcgggggaga caccaggatt 2820 gaagttgctt ttactaaaaa acattttttt tatcttgtta acaaatatat ttaccatggt 2880 acatggtggc aatgctctca gttttaaaac acagcaatta tcttccagtg gtcactaatt 2940 aatccaggtg tggctaggaa cagctggata ctgatctgtg tgatgatggc acctctatca 3000 tcaggctgcc attgctggca ctttgggcaa ggtggactgg ggttctgctc tggggctgtg 3060 tgcccaaggt ttacacctca ctattttaca gcacactgga gctcccaaca gtccccgaag 3120 gtctggagat caccaatggg caggagagaa gggaggggtt tagacatcag ggagccaagt 3180 tctacagcat cttctagatc caggctatcg agcacttgaa atgcggctaa gtctgaattg 3240 agatgtgctg tgtgtaagag acacaacaga ttttgaagac agtatgagaa aaagaacata 3300 aaatatctca gtaattttat attgattaca tgttgaaatg atactaattt ttgatatact 3360 gggttaaata aaatagatta ttaaaattaa 3390

Claims

1. Use of a reagent capable of quantitatively detecting the expression amount of lncRNA-MIR3945HG V1 in preparing a diagnostic kit for sputum-negative pulmonary tuberculosis, wherein the nucleotide sequence of the nucleic acid marker of lncRNA-MIR3945HG V1 is shown in SEQ ID NO: 1; The quantitative detection reagent contains a primer sequence capable of real-time fluorescence quantitative detection of lncRNA-MIR3945HG V1.

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