CN106367511A - Application of lncRNA-LOC100507195 in mycobacterium tuberculosis negative pulmonary tuberculosis diagnosis - Google Patents

Abstract

The invention discloses application of lncRNA-LOC100507195 in mycobacterium tuberculosis negative pulmonary tuberculosis diagnosis. It is found that after MTB is infected with macrophage, consequently LOC100507195 expression is up-regulated. The detection results on the PBMC of 30 mycobacterium tuberculosis negative pulmonary tuberculosis patients show that the expression level of LOC100507195 is remarkably lower than that of a healthy volunteer; LOC100507195 is possibly adopted as an antituberculous protective factor of the macrophage, the expression of LOC100507195 is down-regulated, and consequently the body is likely to be infected with tuberculosis. The receiver operating characteristic curve analysis result shows that LOC100507195 is expected to become a new target spot of mycobacterium tuberculosis negative pulmonary tuberculosis diagnosis. Uninfected people and the mycobacterium tuberculosis negative pulmonary tuberculosis patients can be effectively distinguished by detecting the expression level of LOC100507195.

Description

Application in bacterium the moon diagnosis of pulmonary tuberculosis for the lncrna-loc100507195

Technical field

Present invention relates particularly to application in bacterium the moon diagnosis of pulmonary tuberculosis for the lncrna-loc100507195.

Background technology

Tuberculosis are still infection rate highest infectious disease in global range so far, prevention and control situation very severe.Quickly, sensitive Diagnosis is to improve therapeutic effect, the key of effective control transmission of tuberculosis.In national lunger, bacterium the moon lunger Account for the 60%～70% of active tuberculosiss patient, and the clinical symptoms of other pulmonary disease multiple and Features be difficult to Tuberculosis distinguish.The method of bacterium the moon pulmonary tuberculosis efficient diagnosis is lacked, often results in patient diagnosis delay or mistaken diagnosis.Though China Formulate some standards of diagnosis, but still suffer from many puzzlements in clinical practice, and unresolved efficient diagnosis bacterium the moon pulmonary tuberculosis This difficult problem.Therefore, find bacterium the moon diagnosis of pulmonary tuberculosis new logo, start new diagnostic method and become the task of top priority！

Existing diagnosis of pulmonary tuberculosis method is mainly sputum examination for tubercle bacillus, tuberculin test, its specific antibody determination, image Learn the methods such as inspection.Time-consuming for sputum examination for tubercle bacillus, and false negative is high；Tuberculin test false positive is high to be inoculated it is not easy to distinguish Bacillus calmette-guerin vaccine crowd and tuberculosis crowd；Imaging examination is difficult to differentiate between pulmonary tuberculosis and other pulmonary disease.And find nucleic acid and make For diagnosis marker, pcr technology is used as diagnostic method, has the advantages that quick, sensitive, and price compares specific antibody Cheaply, prepare and detection operation is also convenient for.Therefore, finding can be non-as the nucleic acids marker of bacterium the moon diagnosis of pulmonary tuberculosis, meaning All.

Mycobacterium tuberculosis (mycobacterium tuberculosis, mtb) belong to intracellular bacterial parasite, main parasitic in Macrophage (macrophage).Mycobacterium tuberculosis in the form of an aerosol, enter lung tissue through human respiratory tract, by alveolar Macrophage phagocytic (am), but mtb escapes the removing of body immune system by corresponding mechanism, and mtb will not be completely clear by am Remove.Therefore mtb can be in macrophage intracellular growth and breeding, and migrating by macrophage, and other are not infected to continue infection Macrophage.Lncrna (long noncoding rna) is the rna of the not encoding proteins that a class length is more than 200nt, Lncrna can be on epigenetic, regulate and control gene expression on transcription and post-transcriptional level, and its function extensively, participates in regulation and control body Various vital movements.Document has been had to prove, lncrna participates in the regulation and control of immunity of organism.

There is document report at present, lncrna can be used as cancer diagnosis mark.But not yet have using lncrna as The phthisical report of diagnostic markers analyte detection.

Content of the invention

It is an object of the invention to provide a kind of brand-new lncrna diagnosis marker loc100507195, for quickly examining Disconnected bacterium the moon pulmonary tuberculosis, and price is cheaper than current diagnostic method.

The technical solution used in the present invention is:

Application in Diagnosis of Tuberculosis for the lncrna nucleic acids marker, the nucleotide sequence of this lncrna nucleic acids marker is such as Shown in seq id no:1, it is designated as loc100507195.

Preferably, described Diagnosis of Tuberculosis is diagnosis of pulmonary tuberculosis.

Preferably, described diagnosis of pulmonary tuberculosis is bacterium the moon diagnosis of pulmonary tuberculosis.

A kind of bacterium the moon diagnosis of pulmonary tuberculosis test kit, this test kit contains can be to the expression of lncrna-loc100507195 Amount carries out quantitative reagent.

Diagnosis of pulmonary tuberculosis examination prepared by the reagent that the expression of lncrna-loc100507195 can be carried out with detection by quantitative Application in agent box.

Preferably, the reagent of described detection by quantitative contains and can detect lncrna-loc100507195 by real time fluorescent quantitative Primer sequence.

Preferably, described pulmonary tuberculosis are bacterium the moon pulmonary tuberculosis.

The invention has the beneficial effects as follows:

The batch screening by early stage for the present invention, the loc100507195 therefrom screening acquisition examines as bacterium the moon is phthisical Disconnected mark, by detecting the expression of loc100507195, can effectively be distinguished the crowd of being uninfected by and be suffered from bacterium the moon pulmonary tuberculosis Person.

Present invention discover that: after mtb infection macrophage, cause the various reactions of macrophage, turning including gene Record and the synthesis of albumen, the expression of lncrna is also regulated and controled.After mtb infection macrophage, can cause loc100507195's Up-regulated, illustrates that loc100507195 plays corresponding effect during mtb infection macrophage.Use fluorescent quantitation Pcr (qpcr) detects bacterium the moon pulmonary tuberculosis patient and the pbmc of healthy normal person, with respect to the pbmc of healthy normal person, Loc100507195 is but to lower in bacterium the moon pulmonary tuberculosis patient pbmc expression, and both have significant difference, and this is probably Loc100507195, as the antiphthisic protective factors of macrophage, its down-regulated expression, leads to body susceptible to tuberculosis.Therefore Loc100507195 can be used as the diagnosis marker of diagnosis bacterium the moon pulmonary tuberculosis patient and healthy normal person.

Brief description

Fig. 1 detects the expression of loc100507195 in the m φ of mtb infection for real time fluorescent quantitative pcr；

Fig. 2 detects the table of lncrna-loc100507195 in bacterium the moon lunger pbmc for real time fluorescent quantitative pcr Reach；

Fig. 3 is roc tracing analysiss.

Specific embodiment

Present invention discover that: 1. lead to loc100507195 up-regulated after mtb infection m φ；2. to 30 bacterium the moon pulmonary tuberculosis The testing result of patient pbmc then shows, the expression of its loc100507195 is substantially less than healthy volunteer；Experimenter's work Make characteristic curve (receiver operating characteristic curve, abbreviation roc curve) analysis result to show, Loc100507195 is expected to become bacterium the moon diagnosis of pulmonary tuberculosis novel targets.

With reference to specific embodiment, the present invention is described further, but is not limited thereto.

The up-regulated of loc100507195 in the m φ of embodiment 1 infection mtb

The pbmc of separating health volunteer, immunological magnetic bead sorting cd14⁺Mononuclear cell, with gm-csf induced monocyte to M φ breaks up, and the m φ of gained is infected mtb.After 72h, real time fluorescent quantitative pcr detects the expression water of loc100507195 in m φ Flat.

Testing result is as shown in figure 1, there it can be seen that the expression of the loc100507195 of mtb metainfective m φ Substantially raise, illustrate that mtb infection promotes the expression of loc100507195.

Embodiment 2 real time fluorescent quantitative pcr detects the expression of loc100507195 in bacterium the moon lunger pbmc

Extract the rna of 30 bacterium the moon lungers and 32 healthy volunteer pbmc, real time fluorescent quantitative pcr detects The expression of lncrna-loc100507195.

Testing result as shown in Fig. 2 there it can be seen that bacterium the moon lunger pbmc in loc100507195 expression be in Now notable down regulation trend (p < 0.001).

Embodiment 3loc100507195 is as the novel targets of bacterium the moon diagnosis of pulmonary tuberculosis

The study find that, compared with Healthy People, loc100507195 express in tubercular pbmc present extremely notable under Tune trend (p < 0.001).In order to understand loc100507195 pbmc expression change whether can be used for distinguish Healthy People with Tubercular, using roc tracing analysiss loc100507195, whether the expression change in pbmc examines for lungy for we Disconnected.

According to relative expression quantity in Healthy People and tubercular pbmc for the loc100507195, soft using statistical analysiss Part, set up abscissa be specificity, vertical coordinate be sensitivity roc curve, see Fig. 3.Result shows, roc area under curve auc A ()=0.8594, illustrates that loc100507195 has higher accuracy (Fig. 3) for tuberculosis patient diagnosis.A in roc curve Value meaning explanation: in a > in the case of 0.5, a is closer to 1, illustrates that diagnosis effect is better.A has relatively low standard when 0.5～0.7 Really property, has certain accuracy when 0.7～0.9, has high accuracy when more than 0.9.

Roc curve (cumulative frequency distribution) is analyzed, the results are shown in Table 1.

Table 1roc tracing analysiss result

(above ellipsis represents that initial data is too many, only lists critical data in table).

Table 1 interpretation of result is found: when transcriptional level cut-off value (thresholding) of loc100507195 takes 7.368, about Mounting index (youden ' s indx, yi) maximum (yi=62.5), now detection sensitivity can reach 81.25%, and specificity Reach 81.25%.So, it is feasible that loc100507195 is used for diagnosis lungy, for raising diagnosis lungy Rate is significant.

In sum: lncrna-loc100507195 can be used as bacterium the moon diagnosis of pulmonary tuberculosis novel targets.

Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not subject to above-described embodiment Limit, other any spirit without departing from the present invention and the change made under principle, modification, replacement, combine, simplify, All should be equivalent substitute mode, be included within protection scope of the present invention.

<110>Nanfang Medical Univ

<120>application in bacterium the moon diagnosis of pulmonary tuberculosis for the lncrna-loc100507195

<130>

<160> 1

<170> patentin version 3.5

<210> 1

<211> 574

<212> rna

<213>people (homo sapiens)

<400> 1

gagaggctgt ggcaagggtc catgcctgaa tgtgtagaac aagaacagct ggaggctgtg 60

gctttctcca ggaagaggtt tctaaatctg cctcttagaa gatatcagaa agttaaagtt 120

tctcttgctg ccgccgtgta agaaggacct tttgcctccc accatgattc taagtcctcc 180

ccagccacgt ggaactgctt tcatttggtc tcttttcgaa tcagaagacc cagaaaactt 240

ggatccactt ctggctctca tccctctggg cagtgtgatt tggattgagc agatgctccg 300

gctgccactc catcatatat tggaaacagc attactttat agtaaatcat gaccacggtg 360

tatggcaatg aagttgcatc tgaagcagcc ggcaccctat gtgctagctg gcttttagac 420

caggagactg tgaatctgtg gtcaggatct ctttagggct gctaaatata tactacccac 480

tatcactcgc tggagctgca gaccctagtt ttagagtttc acttcttaaa gaccttcatc 540

cttattaaaa tggtaatatc cttactgtca tcaa 574

Claims (7)

1. Application in Diagnosis of Tuberculosis for the 1.lncrna nucleic acids marker it is characterised in that: the nucleoside of this lncrna nucleic acids marker Acid sequence, as shown in seq id no:1, is designated as loc100507195.
2. 2. according to claim 1 application it is characterised in that: described Diagnosis of Tuberculosis be diagnosis of pulmonary tuberculosis.
3. 3. according to claim 2 application it is characterised in that: described diagnosis of pulmonary tuberculosis be bacterium the moon diagnosis of pulmonary tuberculosis.
4. 4. a kind of bacterium the moon diagnosis of pulmonary tuberculosis test kit it is characterised in that: this test kit contains can be to lncrna- The expression of loc100507195 carries out quantitative reagent.
5. 5. diagnosis of pulmonary tuberculosis reagent prepared by the reagent that the expression of lncrna-loc100507195 can be carried out with detection by quantitative Application in box.
6. 6. application according to claim 5 it is characterised in that: the reagent of described detection by quantitative contain can real-time fluorescence fixed The primer sequence of amount detection lncrna-loc100507195.
7. 7. according to claim 5 application it is characterised in that: described pulmonary tuberculosis be bacterium the moon pulmonary tuberculosis.