CN102925541B - A kind of method and test kit detecting tuberculosis latent infection state - Google Patents

Abstract

The invention provides a method and a kit for detecting and discriminating the infection state of tuberculosis. The content of the characteristic gene is quantitatively detected by the real-time quantitative fluorescence amplification method. The multiple of difference in gene expression was used to determine the status of tuberculosis latent infection.

Description

A method and kit for detecting tuberculosis latent infection status

technical field

The invention relates to a method and kit for detecting tuberculosis latent infection, and also relates to a detection reagent used in the method.

Background technique

Tuberculosis, also known as tuberculosis and white plague, is a chronic infectious disease caused by tubercle bacillus infection. It is an ancient disease with a history of thousands of years and was once the main killer of human beings. Although effective anti-tuberculosis drugs have been discovered since the 1950s, and the epidemic has been controlled to a certain extent, in recent years, due to the neglect of tuberculosis in many countries, financial investment has been reduced, coupled with population growth and floating population. The increase and other factors have slowed the decline of tuberculosis prevalence, and even some countries and regions have rebounded. Therefore, the World Health Organization (WHO) declared a "global tuberculosis emergency" in 1993, and determined that March 24 every year is the "World Tuberculosis Prevention and Control Day". day". In my country, tuberculosis is also a major public health problem today. According to the fourth national epidemiological survey, there are about 1 million new cases in my country every year, more than 400 million people are infected, and 130,000 people die of tuberculosis every year.

An important problem in tuberculosis control is the latent infection of tuberculosis. About 20-30% of the people in contact with patients are latent tuberculosis infected people. They are healthy and have no clinical symptoms, nor are they in a disease state, and the bacteria are in a latent state. Although the vast majority of these people will never get sick, about 10% of those infected will develop active tuberculosis in the next few years. Therefore, the detection and identification of tuberculosis infection status plays a very important role in the prevention of tuberculosis.

At present, there are many laboratory methods for detecting tuberculosis infection, including traditional sputum smears of acid-fast bacteria, rapid culture of mycobacteria, amplification of tuberculosis-specific nucleic acid fragments (quantitative and qualitative), and tuberculin skin test (PPD). And T cell immune release method of gamma-interferon (IGRA) and so on. The PPD method is based on the strength of the body’s type IV delayed immune response to tuberculosis antigens to diagnose the status of tuberculosis infection; IGRA uses tuberculosis-specific antigens to stimulate human peripheral blood lymphocytes, and the T lymphocytes of tuberculosis-infected patients have been activated. , will secrete a large amount of γ-interferon, and then detect the γ-interferon by different methods to diagnose tuberculosis infection; the diagnostic kits using this method include QuantiFERON-TBGoldIn-tube (Cellestis, Australia) and T-SPOT.TB (Oxford Immunotech , UK).

Both PPD and IGRA methods are based on cell-based immunodiagnostic methods. Although this type of method has been widely used, there is an obvious defect that has not been resolved. What they diagnose is not tuberculosis but tuberculosis infection. This means that they cannot differentiate between patients with active TB and those with latent TB infection, both groups who are positive and indistinguishable using the above methods. Although patients with active tuberculosis have clinical symptoms, when patients with atypical clinical symptoms and suspected tuberculosis need to be diagnosed or are used in areas with a high rate of latent infection, accurate diagnosis is often not possible, which affects the application value of this method.

Latency after tuberculosis infection is caused by the protective immune response of the host, and there are also great differences in the host immune response compared to active tuberculosis. Therefore, the identification of latent infection and active disease status can be achieved by using new immune molecules Markup was improved.

Therefore, considering the large base of TB-infected people in my country and the high rate of latent infection, it is of great value to find a method or kit that can accurately identify the state of latent infection and active TB.

Contents of the invention

Aiming at the deficiencies of the existing cellular immune technology in identifying tuberculosis latent infection and active tuberculosis, the present invention provides a non-invasive method, by detecting the specific gene expression level of mononuclear cells in biological samples after contact with tuberculin In order to determine whether there is tuberculosis latent infection in the biological sample, in order to realize the method of the present invention, a detection kit used in the method is also provided.

The first object of the present invention is to provide a method for detecting latent infection of tuberculosis in biological samples; make tuberculin contact with mononuclear cells isolated from biological samples, then extract the total ribonucleic acid (RNA) of the cells, and perform Methods Synthesize the RNA into cDNA, then quantitatively detect the content of characteristic genes by real-time quantitative fluorescence amplification method, and finally determine the tuberculosis latency by comparing and analyzing the difference in gene expression levels between tuberculin and non-tuberculin infection status.

Wherein, the detected characteristic gene can be:

i) Any nucleotide sequence in SEQIDNo.1~4 or any combination of said nucleotide sequences;

ii) The complementary sequence or any combination of complementary sequences of any nucleotide sequence in SEQIDNo.1~4;

iii) Any polynucleotide that hybridizes to the above-mentioned nucleotide sequence or the above-mentioned complementary sequence under stringent conditions, preferably, the hybridized polynucleotide is at least 15 consecutive nucleotides.

Among them, nucleic acid fluorescent quantitative PCR detection of each gene includes oligonucleotide primers that can bind to the target gene. The design of the primers is based on SEQ ID No. 1~4 as a template, and the primers are designed by methods known in the industry.

Wherein, the biological sample detected in the present invention can be a sample derived from a human, or a sample derived from other mammals, which is characterized by containing living mononuclear cells. Some of the more common biological samples that can be detected include peripheral blood, lymph fluid, pleural effusion, pleural effusion, nodules, liver, spleen, lung and other organs. A preferred embodiment detects mononuclear cells obtained by separating and purifying human peripheral blood.

According to a preferred embodiment of the present invention, the oligonucleotide primers used are the oligonucleotide sequences described in SEQ ID No.5-12 respectively.

According to a preferred embodiment of the gene chip of the present invention, wherein said quantitatively detected characteristic genes include SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4. More preferred is the combination of SEQ ID No.1 and SEQ ID No.4.

Latency after tuberculosis infection is caused by the protective immune response of the host, and there are also great differences in the host immune response compared to active tuberculosis. Therefore, the identification of latent infection and active disease status can be achieved by using new immune molecules Markup was improved. The implementation effect of the present invention is that it can detect the unique immune response of tuberculosis latent infection, and then distinguish the latent infection state from the active disease state through the immune response. The invention includes two very important technical links, one of which is the use of tuberculin Mononuclear cells in biological samples are contacted and incubated. As a result of latent infection, the expression of some specific immune molecules is altered after tuberculin stimulation. The second is the quantitative detection and comparison of specific molecules. As mentioned above, the genes represented by SEQIDNo.1~4 are the specific molecules discovered. By detecting the expression changes of these molecules, information on the status of tuberculosis infection can be obtained indirectly , has an important reference role in the control and diagnosis of tuberculosis. The above-mentioned incubation of mononuclear cells with tuberculin is generally 2 to 12 hours, and a typical incubation time is 4 hours as described in the implementation.

The judging standard of the difference multiple of gene expression in the present invention is to carry out receiver operating characteristic curve analysis (ROC curve) according to the gene in two groups of populations of active tuberculosis and latent infection, and select the optimal value as the gene to distinguish active tuberculosis and the threshold of latent tuberculosis infection. As in the examples, the preferred criteria for four genes are the expression difference multiple of SEQIDNo.1<1.77, the expression difference multiple of SEQIDNo.2>0.896, the expression difference multiple of SEQIDNo.3<1.088, the expression difference multiple of SEQIDNo.4> 2.222, can be judged as latent infection.

Another object of the present invention is to provide a test kit for detecting latent infection of tuberculosis, specifically, the test kit includes but is not limited to: (1) tuberculin, cell culture plate, RNA extract, reverse transcriptase Reaction system, real-PCR amplification reaction system and primers, negative control samples, positive control samples, RNA positive control substances, and (2) packaging boxes for separate and centralized packaging of reagent bottles or tubes.

The kit for detecting tuberculosis infection status of the present invention, the primers are designed with the following gene sequence as the target gene:

Any nucleotide sequence in SEQIDNo.1~4, or the complementary sequence of the nucleotide sequence, or one or more of polynucleotides that hybridize to the nucleotide sequence or its complementary sequence under stringent conditions .

In the kit for detecting tuberculosis infection status of the present invention, the target gene is a combination of SEQ ID No.1 and SEQ ID No.4.

In the kit for detecting tuberculosis infection status of the present invention, the primers preferably comprise one or more nucleotides in SEQ ID No.5-12.

The above-mentioned SEQIDNo.1～12 sequence of the present invention is as follows, wherein, NCBI is the National Center for Biotechnology Information of the United States:

SEQ ID No.1 (NCBI registration number: NM_001565.2):

gtacaaaaaagcaggctccaccatgaatcaaactgccattctgatttgctgccttatctttctgactctaagtggcattcaaggagtacctctctctagaactgtacgctgtacctgcatcagcattagtaatcaacctgttaatccaaggtctttagaaaaacttgaaattattcctgcaagccaattttgtccacgtgttgagatcattgctacaatgaaaaagaagggtgagaagagatgtctgaatccagaatcgaaggccatcaagaatttactgaaagcagttagcaaggaaaggtctaaaagatctcctttggacccagctttcttgtac

SEQ ID No.2 (NCBI registration number: NM_024490.1):

ccgcaggcggggagctcgcaccgccgcgcccgggccgcgagtgatgataacctaagaggccggcgcgggcgggcgtgagcggcggaggagccgggcgcggcgacacgcggccatggagcgggagccggcggggaccgaggagcccgggcctccgggacggcggaggcgccgagagggcaggacgcgcacggtgcgctccaacctgctgccgcccccgggcgccgaggaccctgcggctggcgcggccaagggcgagcggcgacggcggcgcgggtgtgcccagcacctggccgacaaccggctcaagactaccaagtacacgctgctgtccttcctgcccaagaacctgttcgagcagttccaccgcccggccaacgtgtactttgtcttcatcgcgctgctcaacttcgtgccggcggtgaacgccttccagcccggcctggcactggcgccggtgctcttcatcctggccatcacggccttcagggacctgtgggaggactacagccgccaccgctccgaccacaagatcaaccacctgggctgcctggtcttcagcagggaagaaaagaaatacgtgaaccgattctggaaagaaatccacgtgggagactttgtgcgtcttcgctgcaacgaaatcttccctgcggacattctgctgctctcctccagtgaccccgacgggctatgccacatcgagaccgccaacctggatggagagaccaacctgaagcggcggcaggtggtccgcggcttctcggagcttgtctccgaattcaatcctttgacgttcaccagcgtgatcgaatgcgagaagccaaacaacgacctgagtaggtttcgcggctgcatcatacatgacaacgggaaaaaggccgggctgtataaagaaaacctgctgctgaggggctgcacccttaggaacacggacgcagtcgtcggcattgtcatctacgcaggacatgaaaccaaggctctgctgaacaacagtgggccccgc tacaagcgcagcaagctggagaggcagatgaactgcgacgtgctctggtgtgtcctgctccttgtttgcatgtctctgttttcagcagtcggacatggactgtggatatggcggtatcaagagaagaagtcattattttatgtccccaagtctgatggaagctccttatccccagtcacagctgcagtttactcatttttaacaatgataatagttctgcaggttttgatcccaatttccttatacgtttccattgaaattgttaaagcatgccaagtgtacttcattaaccaggacatgcagttgtatgacgaagaaacagactcgcagctgcagtgccgagctctgaacatcacggaagacttaggacagatacagtacattttctcagataaaactggcactttgacagagaataagatggttttccgaagatgcactgtgtctggtgtagaatattctcatgatgcaaatgcgcagcgtctggccaggtaccaagaggcagactcggaggaggaggaggtggtgcccagagggggctcggtgtcccagcgcggcagcatcggcagccaccagagtgtccgggtggtgcacagaacccagagcaccaagtcccaccggcgcacgggcagccgggccgaggccaagagggccagcatgctgtccaagcacacggccttcagcagccccatggagaaggatatcacgcccgacccaaagctgctggagaaggtgagtgagtgtgacaagagcctagccgtggcgaggcatcaggagcacctgctggcccacctctcgcccgagctgtctgacgtctttgatttcttcatcgcactcaccatctgcaacacagtcgtcgtcacgtccccggatcagccacgaacaaaggtgagggtgaggtttgagctgaagtccccggtgaagacgatagaagacttcctgcggaggttcacacccagctgcctgacctcaggctgcagcagcatcgggagcctggccg ccaacaagtccagccacaagttgggctccagcttcccgtccaccccgtccagcgacggcatgcttctcaggctggaggagaggctgggccagcccacctcggccatcgccagcaacggctacagcagccaggcggacaactgggcctcggagcttgctcaggagcaggagtcagagcgcgagctgcggtacgaggcggagagcccggatgaggccgcactggtgtatgcggccagagcctacaactgcgtgcttgtggagcggctgcacgaccaagtgtcagtggagctgccccacctgggcaggctcaccttcgagctcctgcacacactgggtttcgattccgtccgcaagaggatgtcagtggtgatccggcacccgcttaccgatgagatcaacgtctacaccaagggggccgactcagtggtcatggatctcctgcagccctgctcttcagttgacgccagagggaggcatcaaaaaaagattcggagcaaaactcagaattacctcaacgtgtatgcggcggaaggcctgcgcaccttgtgcatcgccaagagagttctgagtaaagaagagtatgcctgctggttgcaaagccacctagaagccgaatcctccctggaaaacagcgaggagctcctcttccagtctgccattcgcctggagaccaacctgcacttgttaggtgccactgggattgaagaccgcctgcaggacggagtccctgaaactatttctaaattgcgtcaagcgggcctgcagatttgggttctcactggtgacaaacaagaaacagctgtcaacattgcatatgcctgcaaactgctggaccacgacgaggaggtcatcaccctgaatgccacctcccaggaggcgtgtgcagccctgctagaccagtgcctatgctacgtgcagtccagaggcctccagagagcccctgagaagaccaagggcaaagtgagcatgaggttctcctctctctgcccaccctccacgtc cactgcctctggccgcagacccagcctcgtgatcgatgggagaagcctggcctacgctctcgagaaaaacctggaggacaaattcctcttccttgccaagcagtgccgctccgtcctctgctgtcggtcgacgcctctgcagaagagcatggtggtgaagctggtgcggagcaagctcaaggccatgaccctggccataggtgatggagccaatgatgtcagcatgatccaggtggcagatgtgggtgtgggaatctccggccaggagggtatgcaggcagtgatggccagcgactttgcagtgccgaaattccgatacctggagaggctcttgattcttcacgggcattggtgctactcccgacttgccaacatggtgctgtacttcttctacaaaaacacaatgttcgtgggcctcctgttttggttccagtttttctgtggcttctctgcatctaccatgattgaccagtggtatctaatcttctttaatctgctcttctcgtcacttcccccgctcgtgactggggtgctggacagggatgtgccagccaatgtgctgctgaccaacccgcagctctacaagagtggccagaacatggaggaataccggccacgaacgttctggtttaacatggccgacgccgccttccagagcctggtttgcttttccattccttacctggcctactatgactcgaacgtggacctgtttacctgggggacccctattgtgacaatcgcgctgctcactttcctgctccacctgggcattgaaaccaaaacctggacctggctcaactggataacgtgtggcttcagtgtccttttgtttttcaccgtggctttgatttacaatgcgtcttgtgccacgtgctatcctccgtccaacccttactggactatgcaagccttactgggtgacccagtgttttacttgacttgcctgatgacgcctgtcgctgcactgctgcccagattgtttttcagatccctccag gggagggttttccccacacaacttcagctggcacgtcagttgaccaggaagtcccccaggagatgcagtgctcccaaagagacctttgctcagggacgcctcccgaaggactcgggaaccgagcactcatcagggaggacagtcaagacctctgtgcccctgtcccagccttcttggcacacacagcagccggtctgctccctggaggccagcggggagcccagcacagtggacatgagcatgccagtgagggagcacaccctgctggaggggctgagcgcaccggcccccatgtcctctgcgccaggggaggctgtcctgaggagtccaggagggtgtcctgaggagtccaaggtgagagctgccagcaccggcagggtgacccccctgtcttccctcttcagcctgcctaccttcagcttactcaactggatttcctcctggtcgctggtcagcaggctggggagtgtcttacagttctcccggacggagcagcttgcagatggacaagcgggacgtggacttcctgtccagccccactcaggccgatcaggacttcaagggccagaccacagactacttataggagcatcttcaaggcggtcacagtgaaaaccttgaaatggccttttttaatatatataaataaatgttaatattatttatgtttattatttgcacagaagagttctagggagatgtatttctaaatgtttcccaggctaatacaggaaacaagaggtaccaaaaaagaaagtttattttttaaaattctaagtagagtatattgaaaagaaaaagaagagccttaacatatataaaagtttaaagaagagtaacacttgaaaagtgtgtttagatttattttttcatctcatttttaagaacaagcagtacgatttgttttcttcaacatgtgtgactgcgcactgagtacaaatgtgtgactgctcatggttaatgcaggcaggtgtgaacatgggggaacaatgagcagaga tggcagagggcagagcacatggcccccagaggcttccagtctcactgacacaggagggctgggctccacttcatccagatgaaggaaaggaagacctcaagaaaaattcacagttgagtgcatcccagcattctgttccgggcaggcatttcaggaagaccgccttgtaggtattacatccctggtgtcgtattttgcctgttaaatcgtaacaagcaataaacaactttcactttgcaaaaaaaaaaaaaaaaaaa

SEQ ID No.3 (NCBI registration number: NM_006068.2):

ccctttaggatagccactgcaacatcatgaccaaagacaaagaacctattgttaaaagcttccattttgtttgccttatgatcataatagttggaaccagaatccagttctccgacggaaatgaatttgcagtagacaagtcaaaaagaggtcttattcatgttccaaaagacctaccgctgaaaaccaaagtcttagatatgtctcagaactacatcgctgagcttcaggtctctgacatgagctttctatcagagttgacagttttgagactttcccataacagaatccagctacttgatttaagtgttttcaagttcaaccaggatttagaatatttggatttatctcataatcagttgcaaaagatatcctgccatcctattgtgagtttcaggcatttagatctctcattcaatgatttcaaggccctgcccatctgtaaggaatttggcaacttatcacaactgaatttcttgggattgagtgctatgaagctgcaaaaattagatttgctgccaattgctcacttgcatctaagttatatccttctggatttaagaaattattatataaaagaaaatgagacagaaagtctacaaattctgaatgcaaaaacccttcaccttgtttttcacccaactagtttattcgctatccaagtgaacatatcagttaatactttagggtgcttacaactgactaatattaaattgaatgatgacaactgtcaagttttcattaaatttttatcagaactcaccagaggtccaaccttactgaattttaccctcaaccacatagaaacgacttggaaatgcctggtcagagtctttcaatttctttggcccaaacctgtggaatatctcaatatttacaatttaacaataattgaaagcattcgtgaagaagattttacttattctaaaacgacattgaaagcattgacaatagaacatatcacgaaccaagtttttctgttttcacagacagctttgta caccgtgttttctgagatgaacattatgatgttaaccatttcagatacaccttttatacacatgctgtgtcctcatgcaccaagcacattcaagtttttgaactttacgcagaacgttttcacagatagtatttttgaaaaatgttccacgttagttaaattggagacacttatctttgttttgtctcccaactttgtccagagtgagtggtgccattacgaactctattttgcccatcacaatctctttcatgaaggatctaataacttaatcctcatcttactggaacccattccacagaacagcattcccaacaagtaccacaagctgaaggctctcatgacgcagcggacttatttgcagtggcccaaggagaaaagcaaacgtgggctcttttgggctaacattagagccgcttttaatatgaaattaacactagtcactgaaaacaatgatgtgaaatcttaaaaaaatttaggaaattcaacttaagaaaccattatttacttggatgatggtgaatagtacagtcgtaagtaactgtctggaggtgcctccattatcctcatgccttcaggaaagacttaacaaaaacaatgtttcatctggggaactgagctaggcggt

SEQ ID No.4 (NCBI registration number: NM_000417.1):

Gtacaaaaaagcagaagggccgtcaaggcccaccatggattcatacctgctgatgtggggactgctcacgttcatcatggtgcctggctgccaggcagagctctgtgacgatgacccgccagagatcccacacgccacattcaaagccatggcctacaaggaaggaaccatgttgaactgtgaatgcaagagaggtttccgcagaataaaaagcgggtcactctatatgctctgtacaggaaactctagccactcgtcctgggacaaccaatgtcaatgcacaagctctgccactcggaacacaacgaaacaagtgacacctcaacctgaagaacagaaagaaaggaaaaccacagaaatgcaaagtccaatgcagccagtggaccaagcgagccttccaggtcactgcagggaacctccaccatgggaaaatgaagccacagagagaatttatcatttcgtggtggggcagatggtttattatcagtgcgtccagggatacagggctctacacagaggtcctgctgagagcgtctgcaaaatgacccacgggaagacaaggtggacccagccccagctcatatgcacaggtgaaatggagaccagtcagtttccaggtgaagagaagcctcaggcaagccccgaaggccgtcctgagagtgagacttcctgcctcgtcacaacaacagattttcaaatacagacagaaatggctgcaaccatggagacgtccatatttacaacagagtaccaggtagcagtggccggctgtgttttcctgctgatcagcgtcctcctcctgagtgggctcacctggcagcggagacagaggaagagtagaagaacaatctaaggcctcatgggcccagctttcttgtac

SEQ ID No. 5: TGCCATTCTGATTTGCTGCCTT

SEQ ID No. 6: GGCTTGCAGGAATAATTTCAAGTTTTTT

SEQ ID No. 7: ACCGCTCCGACCACAAGATCA

SEQ ID No. 8: TGTCCGCAGGGAAGATTTCGT

SEQ ID No. 9: TCCCATAACAGAATCCAGCTACTTG

SEQ ID No. 10: CCTTGAAATCATTGAATGAGAGATCT

SEQ ID No. 11: CACAGAGGTCCTGCTGAGAGCG

SEQ ID No. 12: CTCTTCACCTGGAAACTGACTGGT

Target gene and primer relationship:

amplified target gene upstream primer downstream primer SEQ ID No.1 SEQ ID No.5 SEQ ID No.6 SEQ ID No.2 SEQ ID No.7 SEQ ID No.8 SEQ ID No.3 SEQ ID No.9 SEQ ID No.10 SEQ ID No.4 SEQ ID No.11 SEQ ID No.12

Description of drawings

Figure 1 shows the expression difference of SEQIDNo.1~4 genes detected by real-time quantitative fluorescent PCR between active tuberculosis (n=25) and tuberculosis latent infection population (n=36); ** in the figure indicates that the difference is very significant.

detailed description

In the following, the gene chip of the present invention will be introduced and illustrated in detail through specific examples, so as to better understand the scope of the present invention, but the following examples do not limit the scope of the present invention.

In the present embodiment, the detection of tuberculosis infection status is carried out using the kit of the present invention, the method is as follows:

Step 1: Isolation, stimulation and RNA extraction of mononuclear cells:

Take venous blood into a blood collection tube, centrifuge at 3000rpm for 10min, absorb the white cells in the middle layer, and resuspend them in 8ml of cell culture medium RPMI1640.

Add 4ml of Ficoll lymphocyte separation medium, and add the above cell suspension to the upper layer of the lymphocyte separation medium. Centrifuge at 3000rpm room temperature for 20min.

Absorb the cells in the middle layer after centrifugation into a new centrifuge tube, add RPMI1640 to resuspend, mix well, and centrifuge at 2000rpm for 5min.

Discard the supernatant, add 5ml red blood cell lysate, and incubate at room temperature for 10min.

Add RPMI1640 to resuspend, mix well, centrifuge at 2000rpm for 5min, and discard the supernatant.

The cells were resuspended in the culture medium, and the cells were divided into two groups, one group was added with 10 mg/ml tuberculin, and the other group was added with the culture medium as a control.

Total RNA was extracted from mononuclear cells using TRIzol reagent.

Step 2: Real-time quantitative fluorescent PCR detection of gene expression differences:

RNA was reverse-transcribed into cDNA using the PeverseTranscriptionKit kit. The reaction system was: 5×PrimeScriptBuffer2ul, RTEnzymeMixI0.5ul, OligodTPrimer25pmol final concentration, Random6mers50pmol final concentration, total RNA less than 500ng, and RNasefree water to make up the system to 10ul. Using real-timePCR primers designed based on SEQIDNo.1~4 genes, use the SYBRGreenPCRMasterMix kit to amplify SEQIDNo.1~4 and GAPDH genes (control housekeeping gene) with a standard real-time quantitative fluorescence program. The reaction system is: 2 ×SYBRGreenPCRMix10ul, upstream and downstream primers 8pmol each, ROXReferenceDye0.4ul, cDNA template 1ul, make up the system to 20ul with RNasefree water.

The primers used in this example are listed in Table 1.

The fluorescent quantitative PCR reaction program is a 2-step method, carried out on ABI7500, the reaction conditions are: 95°C for 2min, 40 cycles, each cycle includes 95°C for 10 seconds, 60°C for 30 seconds.

Since the fluorescent quantitative PCR adopts the double-stranded chimeric fuel method (SYBRGreenI), the calculation method of the gene expression level change value is a relative quantitative method, and the expression of the housekeeping gene GAPDH is used as a quantitative reference, and the △△Ct statistical method (refer to LivakK . & Schmittgen, Methods 25, 402-408 (2001)) to calculate the relative expression levels of the above genes.

Table 1, primers used in this embodiment

amplified target gene upstream primer downstream primer SEQ ID No.1 TGCCATTCTGATTTGCTGCCTT GGCTTGCAGGAATAATTTCAAGTTTTTT SEQ ID No.2 ACCGCTCCGACCACAAGATCA TGTCCGCAGGGAAGATTTCGT SEQ ID No.3 TCCCATAACAGAATCCAGCTACTTG CCTTGAAATCATTGAATGAGAGATCT SEQ ID No.4 CACAGAGGTCCTGCTGAGAGCG CTCTTCACCTGGAAACTGACTGGT

The relative gene expression level after PPD stimulation was divided by the relative gene expression level of the control stimulation group to obtain the gene expression change factor after PPD stimulation.

Receiver operating characteristic curve analysis (ROC curve) was performed on the above four genes in the two groups of people, and the optimal value was selected as the threshold for distinguishing active tuberculosis from latent tuberculosis infection. In the present embodiment, the four genes preferably determine the tuberculosis latent infection standard as the expression difference multiple of SEQIDNo.1<1.77, the expression difference multiple of SEQIDNo.2>0.896, the expression difference multiple of SEQIDNo.3<1.088, and the expression difference of SEQIDNo.4 Multiple >2.222.

The results of real-time quantitative fluorescent PCR were analyzed and counted (see Figure 1), the expression of mononuclear cells after PPD stimulation in active tuberculosis patients (n=25) and tuberculosis latent infection patients (n=36) .1~4 gene comparison found that active tuberculosis patients had higher gene expressions of SEQIDNo.1 and 4 than those with latent tuberculosis infection, while gene expression changes of SEQIDNo.2 and 3 were lower than those of latent infection group. Expression changes were significantly different between the two groups (Table 2).

According to the analysis of the ROC curve analysis, the optimal value is selected as the detection threshold. The verification results of active tuberculosis (n=25) and tuberculosis latent infection (n=36) are shown in Table 3. The four genes have the ability to distinguish active tuberculosis and tuberculosis. The role of latent tuberculosis infection. Among them, the combined detection of SEQID No.1 and 4 genes has the best effect, with a sensitivity of 92%, a specificity of 97%, and an accuracy of 95%. It can distinguish the immune status very well and fully meets the requirements of clinical testing.

Table 2 Differences in gene expression between active tuberculosis and latent tuberculosis infection populations compared by real-time quantitative fluorescent PCR

Gene gene description Differences in gene expression in the TB group Differences in gene expression in LTBI groups TB/LTBI ratio P valuea SEQ ID No.4 interleukin 2 receptor A 5.6457 4.0139 1.4065 0.007* SEQ ID No.2 ATPase, type 10A 0.8258 0.9051 0.9124 0.002* SEQ ID No.1 Interferon gamma-induced 10kDa protein 6.9644 1.0565 6.5922 0.004* SEQ ID No.3 Toll-like receptor 6 0.8122 0.9912 0.8194 0.010*

a Non-parametric U test, p<0.01 means the difference is very significant.

Table 3 Combined detection of gene expression differences of SEQIDNo.1~4 to distinguish between active tuberculosis and latent tuberculosis infection

Combinations of genes detected sensitivity specificity Correct rate(%) SEQ ID Nos. 1, 2 and 3 88 94 92 SEQ ID No.1 and 2 92 97 95 SEQ ID No. 1 and 4 88 92 90 SEQ ID No.1 and 3 88 97 93 SEQ ID No.2 and 4 80 97 90 SEQ ID No.2 and 3 92 94 93 SEQ ID No.3 and 4 88 92 90

It can be seen from the above detection that the kit provided by the present invention has good effect and correct rate in detecting tuberculosis infection status.

Through the description of the above embodiments, those skilled in the art can understand that the polynucleotides that use the complementary sequences of the above-mentioned SEQ ID No. 1 to 4 sequences, or hybridize with the above-mentioned sequences or the complementary sequences under stringent conditions can also play the same role. The detection effect and the detection method are the same as described in the above-mentioned embodiment 1.

The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, equivalent changes and modifications made without departing from the spirit and scope of the present invention shall fall within the scope of the present invention.

SEQUENCELISTING

<110> Huashan Hospital Affiliated to Fudan University

<120> A method and kit for detecting tuberculosis latent infection status

<160>12

<170>PatentInversion3.3

<210>1

<211>295

<212>DNA

<213>Homosapiens

<400>1

atgaatcaaactgccattctgatttgctgccttatctttctgactctaagtggcattcaa60

ggagtacctctctctagaactgtacgctgtacctgcatcagcattagtaatcaacctgtt120

aatccaaggtctttagaaaaacttgaaattattcctgcaagccaattttgtccacgtgtt180

gagatcattgctacaatgaaaaagaagggtgagaagagatgtctgaatccagaatcgaag240

gccatcaagaatttactgaaagcagttagcaaggaaaggtctaaaagatctcctt295

<210>2

<211>5257

<212>DNA

<213>Homosapiens

<400>2

ccgcaggcggggagctcgcaccgccgcgcccgggccgcgagtgatgataacctaagaggc60

cggcgcgggcgggcgtgagcggcggaggagccgggcgcggcgacacgcggccatggagcg120

ggagccggcggggaccgaggagcccggggcctccgggacggcgggaggcgccgagaggggcag180

gacgcgcacggtgcgctccaacctgctgccgcccccgggcgccgaggaccctgcggctgg240

cgcggccaagggcgagcggcgacggcggcgcgggtgtgcccagcacctggccgacaaccg300

gctcaagactaccaagtacacgctgctgtccttcctgcccaagaacctgttcgagcagtt360

ccaccgcccggccaacgtgtactttgtcttcatcgcgctgctcaacttcgtgccggcggt420

gaacgccttccagcccggcctggcactggcgccggtgctcttcatcctggccatcacggc480

cttcagggacctgtgggaggactacagccgccaccgctccgaccacaagatcaaccacct540

gggctgcctggtcttcagcagggaagaaaagaaatacgtgaaccgattctggaaagaaat600

ccacgtgggagactttgtgcgtcttcgctgcaacgaaatcttccctgcggacattctgct660

gctctcctccagtgaccccgacgggctatgccacatcgagaccgccaacctggatggaga720

gaccaacctgaagcggcggcaggtggtccgcggcttctcggagcttgtctccgaattcaa780

tcctttgacgttcaccagcgtgatcgaatgcgagaagccaaacaacgacctgagtaggtt840

tcgcggctgcatcatacatgacaacgggaaaaaggccgggctgtataaagaaaacctgct900

gctgaggggctgcacccttaggaacacggacgcagtcgtcggcattgtcatctacgcagg960

acatgaaaccaaggctctgctgaacaacagtgggccccgctacaagcgcagcaagctgga1020

gaggcagatgaactgcgacgtgctctggtgtgtcctgctccttgtttgcatgtctctgtt1080

ttcagcagtcggacatggactgtggatatggcggtatcaagagaagaagtcattatttta1140

tgtccccaagtctgatggaagctccttatccccagtcacagctgcagtttactcattttt1200

aacaatgataatagttctgcaggttttgatcccaatttccttatacgtttccattgaaat1260

tgttaaagcatgccaagtgtacttcattaaccaggacatgcagttgtatgacgaagaaac1320

agactcgcagctgcagtgccgagctctgaacatcacggaagacttaggacagatacagta1380

cattttctcagataaaactggcactttgacagagaataagatggttttccgaagatgcac1440

tgtgtctggtgtgtagaatattctcatgatgcaaatgcgcagcgtctggccaggtaccaaga1500

ggcagactcggaggaggaggaggtggtgcccagaggggggctcggtgtcccagcgcggcag1560

catcggcagccaccagagtgtccgggtggtgcacagaacccagagcaccaagtcccaccg1620

gcgcacgggcagccgggccgaggccaagagggccagcatgctgtccaagcacacggcctt1680

cagcagccccatggagaaggatatcacgcccgacccaaagctgctggagaaggtgagtga1740

gtgtgacaagagcctagccgtggcgaggcatcaggagcacctgctggcccacctctcgcc1800

cgagctgtctgacgtctttgatttcttcatcgcactcaccatctgcaacacagtcgtcgt1860

cacgtccccggatcagccacgaacaaaggtgagggtgaggtttgagctgaagtccccggt1920

gaagacgatagaagacttcctgcggaggttcacacccagctgcctgacctcaggctgcag1980

cagcatcgggagcctggccgccaacaagtccagccacaagttgggctccagcttcccgtc2040

caccccgtccagcgacggcatgcttctcaggctggaggagaggctgggccagcccacctc2100

ggccatcgccagcaacggctacagcagccaggcggacaactgggcctcggagcttgctca2160

ggagcaggagtcagagcgcgagctgcggtacgaggcggagagcccggatgaggccgcact2220

ggtgtatgcggccagagcctacaactgcgtgcttgtggagcggctgcacgaccaagtgtc2280

agtggagctgccccacctgggcaggctcaccttcgagctcctgcacacactgggtttcga2340

ttccgtccgcaagaggatgtcagtggtgatccggcacccgcttaccgatgagatcaacgt2400

cctacaccaagggggccgactcagtggtcatggatctcctgcagccctgctcttcagttga2460

cgccagagggaggcatcaaaaaaagattcggagcaaaactcagaattacctcaacgtgta2520

tgcggcggaaggcctgcgcaccttgtgcatcgccaagagagttctgagtaaagaagagta2580

tgcctgctggttgcaaagccacctagaagccgaatcctccctggaaaacagcgaggagct2640

cctcttccagtctgccattcgcctggagaccaacctgcacttgttaggtgccactgggat2700

tgaagaccgcctgcaggacggagtccctgaaactatttctaaattgcgtcaagcggggcct2760

gcagatttgggttctcactggtgacaaacaagaaacagctgtcaacattgcatatgcctg2820

caaactgctggaccacgacgaggaggtcatcaccctgaatgccacctcccaggaggcgtg2880

tgcagccctgctagaccagtgcctatgctacgtgcagtccagaggcctccagagagcccc2940

tgagaagaccaagggcaaagtgagcatgaggttctcctctctctgcccaccctccacgtc3000

cactgcctctggccgcagacccagcctcgtgatcgatgggagaagcctggcctacgctct3060

cgagaaaaacctggaggacaaattcctcttccttgccaagcagtgccgctccgtcctctg3120

ctgtcggtcgacgcctctgcagaagagcatggtggtgaagctggtgcggagcaagctcaa3180

ggccatgaccctggccataggtgatggagccaatgatgtcagcatgatccaggtggcaga3240

tgtgggtgtgggaatctccggccaggagggtatgcaggcagtgatggccagcgactttgc3300

agtgccgaaattccgatacctggagaggctcttgattcttcacgggcattggtgctactc3360

ccgacttgccaacatggtgctgtacttcttctacaaaaacacaatgttcgtgggcctcct3420

gttttggttccagtttttctgtggcttctctgcatctaccatgattgaccagtggtatct3480

aatcttctttaatctgctcttctcgtcacttcccccgctcgtgactggggtgctggacag3540

ggatgtgccagccaatgtgctgctgaccaacccgcagctctacaagagtggccagaacat3600

ggaggaataccggccacgaacgttctggtttaacatggccgacgccgccttccagagcct3660

ggtttgcttttccattccttacctggcctactatgactcgaacgtggacctgtttacctg3720

ggggacccctattgtgacaatcgcgctgctcactttcctgctccacctgggcattgaaac3780

caaaacctggacctggctcaactggataacgtgtggcttcagtgtccttttgtttttcac3840

cgtggctttgattacaatgcgtcttgtgccacgtgctatcctccgtccaacccttactg3900

gactatgcaagccttactgggtgacccagtgttttacttgacttgcctgatgacgcctgt3960

cgctgcactgctgcccagattgtttttcagatccctccaggggggagggttttccccacaca4020

acttcagctggcacgtcagttgaccaggaagtcccccaggagatgcagtgctcccaaaga4080

gacctttgctcagggacgcctcccgaaggactcgggaaccgagcactcatcagggaggac4140

agtcaagacctctgtgcccctgtcccagccttcttggcacacacagcagccggtctgctc4200

cctggaggccagcggggagcccagcacagtggacatgagcatgccagtgaggggagcacac4260

cctgctggaggggctgagcgcaccggcccccatgtcctctgcgccagggggaggctgtcct4320

gaggagtccaggagggtgtcctgaggagtccaaggtgagagctgccagcaccggcagggt4380

gacccccctgtcttccctcttcagcctgcctaccttcagcttactcaactggatttcctc4440

ctggtcgctggtcagcaggctggggagtgtcttacagttctcccggacggagcagcttgc4500

agatggacaagcgggacgtggacttcctgtccagccccactcaggccgatcaggacttca4560

agggccagaccacagactacttataggagcatcttcaaggcggtcacagtgaaaaccttg4620

aaatggccttttttaatatatataaataaatgttaatattattttgtttattatttgca4680

cagaagagttctagggagatgtatttctaaatgtttcccaggctaatacaggaaacaaga4740

ggtaccaaaaaagaaagtttattttttaaaattctaagtagaggtatattgaaaagaaaaa4800

gaagagccttaacatatataaaagtttaaagaagagtaacacttgaaaagtgtgtttaga4860

tttattttttcatctcatttttaagaacaagcagtacgatttgttttcttcaacatgtgt4920

gactgcgcactgagtacaaatgtgtgactgctcatggttaatgcaggcaggtgtgaacat4980

gggggaacaatgagcagagatggcagaggggcagagcacatggcccccagaggcttccagt5040

ctcactgacacaggagggctgggctccacttcatccagatgaaggaaaggaagacctcaa5100

gaaaaattcacagttgagtgcatcccagcattctgttccgggcaggcatttcaggaagac5160

cgccttgtagttattacatccctggtgtcgtattttgcctgttaaatcgtaacaagcaat5220

aaacaactttcactttgcaaaaaaaaaaaaaaaaaaaaa5257

<210>3

<211>1628

<212>DNA

<213>Homosapiens

<400>3

ccctttaggatagccactgcaacatcatgaccaaagacaaagaacctattgttaaaagct60

tccattttgtttgccttatgatcataatagttggaaccagaatccagttctccgacggaa120

atgaatttgcagtagacaagtcaaaaagaggtcttattcatgttccaaaagacctaccgc180

tgaaaaccaaagtcttagatatgtctcagaactacatcgctgagcttcaggtctctgaca240

tgagctttctatcagagttgacagttttgagactttcccataacagaatccagctacttg300

atttaagtgttttcaagttcaaccaggatttagaatatttggattttctcataatcagt360

tgcaaaagatatcctgccatcctattgtgagtttcaggcatttagatctctcattcaatg420

atttcaaggccctgcccatctgtaaggaatttggcaacttatcacaactgaatttcttgg480

gattgagtgctatgaagctgcaaaaattagatttgctgccaattgctcacttgcatctaa540

gttatatccttctggatttaagaaattattatataaaagaaaatgagacagaaagtctac600

aaattctgaatgcaaaaacccttcaccttgtttttcacccaactagtttatcgctatcc660

aagtgaacatatcagttaatactttagggtgcttacaactgactaatattaaattgaatg720

atgacaactgtcaagttttcattaaatttttatcagaactcaccagaggtccaaccttac780

tgaattttaccctcaaccacatagaaacgacttggaaatgcctggtcagagtctttcaat840

ttctttggcccaaacctgtggaatatctcaatatttacaatttaacaataattgaaagca900

ttcgtgaagaagattttacttattctaaaacgacattgaaagcattgacaatagaacata960

tcacgaaccaagtttttctgttttcacagacagctttgtacaccgtgttttctgagatga1020

acattatgatgttaaccatttcagatacaccttttatacacatgctgtgtcctcatgcac1080

caagcacattcaagtttttgaactttacgcagaacgttttcacagatagtatttttgaaa1140

aatgttccacgttagttaaattggagaacacttatctttgttttgtctcccaactttgtcc1200

agagtgagtggtgccattacgaactctattttgcccatcacaatctctttcatgaaggat1260

ctaataacttaatcctcatcttactggaacccattccacagaacagcattcccaacaagt1320

accacaagctgaaggctctcatgacgcagcggacttatttgcagtggcccaaggagaaaa1380

gcaaacgtgggctcttttgggctaacattagagccgcttttaatatgaaattaacactag1440

tcactgaaaacaatgatgtgaaatcttaaaaaaatttaggaaattcaacttaagaaacca1500

ttatttacttggatgatggtgaatagtacagtcgtaagtaactgtctggaggtgcctcca1560

ttatcctcatgccttcaggaaagacttaacaaaaacaatgtttcatctggggaactgagc1620

taggcggt1628

<210>4

<211>880

<212>DNA

<213>Homosapiens

<400>4

gtacaaaaaagcagaagggccgtcaaggcccaccatggattcatacctgctgatgtgggg60

actgctcacgttcatcatggtgcctggctgccaggcagagctctgtgacgatgacccgcc120

agagatcccacacgccacattcaaagccatggcctacaaggaaggaaccatgttgaactg180

tgaatgcaagagaggtttccgcagaataaaaagcgggtcactctatatgctctgtacagg240

aaactctagccactcgtcctgggacaaccaatgtcaatgcacaagctctgccactcggaa300

cacaacgaaacaagtgacacctcaacctgaagaacagaaagaaaggaaaaccacagaaat360

gcaaagtccaatgcagccagtggaccaagcgagccttccaggtcactgcagggaacctcc420

accatgggaaaatgaagccacagagagaatttatcattcgtggtggggcagatggttta480

ttatcagtgcgtccagggatacagggctctacacagaggtcctgctgagagcgtctgcaa540

aatgacccacgggaagacaaggtggacccagccccagctcatatgcacaggtgaaatgga600

gaccagtcagtttccaggtgaagagaagcctcaggcaagccccgaaggccgtcctgagag660

tgagacttcctgcctcgtcacaacaacagattttcaaatacagacagaaatggctgcaac720

catggagacgtccatatttacaacagagtaccaggtagcagtggccggctgtgttttcct780

gctgatcagcgtcctcctcctgagtgggctcacctggcagcggagacagaggaagagtag840

aagaacaatctaaggcctcatgggcccagctttcttgtac880

<210>5

<211>22

<212>DNA

<213> Synthetic

<220>

<223> Primer

<400>5

tgccattctgatttgctgcctt22

<210>6

<211>27

<212>DNA

<213> Synthetic

<220>

<223> Primer

<400>6

ggcttgcaggaataatttcaagttttt27

<210>7

<211>21

<212>DNA

<213> Synthetic

<220>

<223> Primer

<400>7

accgctccgaccacaagatca21

<210>8

<211>21

<212>DNA

<213> Synthetic

<220>

<223> Primer

<400>8

tgtccgcagggaagatttcgt21

<210>9

<211>25

<212>DNA

<213> Synthetic

<220>

<223> Primer

<400>9

tcccataacagaatccagctacttg25

<210>10

<211>26

<212>DNA

<213> Synthetic

<220>

<223> primer

<400>10

ccttgaaatcattgaatgagagatct26

<210>11

<211>22

<212>DNA

<213> Synthetic

<220>

<223> primer

<400>11

cacagaggtcctgctgagagcg22

<210>12

<211>24

<212>DNA

<213> Synthetic

<220>

<223> Primer

<400>12

ctcttcacctggaaactgactggt24

Claims (7)

1. detect a test kit for tuberculosis latent infection state, it is characterized in that, described test kit comprises pcr amplification primer, and described primer designs for target gene, and described target-gene sequence comprises the combination of SEQIDNo.1 and 2.

2. test kit according to claim 1, is characterized in that, described target gene also comprises:

With one or more in the polynucleotide of described nucleotide sequence or its complementary sequence hybridization under nucleotide sequence shown in SEQIDNo.1 or described nucleotide sequence complementary sequence or stringent condition.

3. test kit according to claim 1, is characterized in that, described target gene also comprises:

With one or more in the polynucleotide of described nucleotide sequence or its complementary sequence hybridization under nucleotide sequence shown in SEQIDNo.2 or described nucleotide sequence complementary sequence or stringent condition.

4. test kit according to claim 1, is characterized in that, described target gene also comprises:

With one or more in the polynucleotide of described nucleotide sequence or its complementary sequence hybridization under nucleotide sequence shown in SEQIDNo.3 or described nucleotide sequence complementary sequence or stringent condition.

5. test kit according to claim 1, is characterized in that, described target gene also comprises:

With one or more in the polynucleotide of described nucleotide sequence or its complementary sequence hybridization under nucleotide sequence shown in SEQIDNo.4 or described nucleotide sequence complementary sequence or stringent condition.

6. test kit according to claim 1, is characterized in that, described primer comprises following one or more pairs of primer:

Upstream primer: TGCCATTCTGATTTGCTGCCTT,

Downstream primer: GGCTTGCAGGAATAATTTCAAGTTTTT;

Upstream primer: ACCGCTCCGACCACAAGATCA,

Downstream primer: TGTCCGCAGGGAAGATTTCGT;

Upstream primer: TCCCATAACAGAATCCAGCTACTTG,

Downstream primer: CCTTGAAATCATTGAATGAGAGATCT;

Upstream primer: CACAGAGGTCCTGCTGAGAGCG,

Downstream primer: CTCTTCACCTGGAAACTGACTGGT.

7. according to the test kit in claim 1 ~ 6 described in any one claim, it is characterized in that, also comprise:

Tuberculin, Tissue Culture Plate, RNA extract, reverse transcriptase reaction system, real-PCR amplification reaction system, negative control sample, positive control sample, RNA positive reference substance, and separate and concentrate the packing box packing reagent bottle or pipe.