Primer, probe and microarray chip for detecting mycobacterium tuberculosis and drug resistance of mycobacterium tuberculosis

Abstract

The invention discloses a primer, a probe and a microarray chip for detecting mycobacterium tuberculosis and drug resistance of the mycobacterium tuberculosis, the screened primer and the probe can be used for detecting the drug resistance of the mycobacterium tuberculosis and isoniazide and rifampicin through PCR (Polymerase Chain Reaction) amplification and fluorescence scanning by taking sputum as a sample; the screened probe can be specifically combined with a corresponding target gene. The microarray chip prepared by utilizing the screened probe can be specifically combined with a corresponding target gene, is relatively strong and stable in hybridization signal, can take a sputum sample as a detection object, and does not need invasion or anesthesia. The invention establishes a microarray chip detection method which is good in specificity, high in sensitivity, strong in stability, time-saving and labor-saving.

Description

(I) Technical field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to primers, probes and microarray chips for detecting isoniazid- and rifampicin-resistant Mycobacterium tuberculosis. (II) Background technology

[0002] Mycobacterium tuberculosis (TB) is the main pathogen of tuberculosis. Tuberculosis is a highly contagious disease with a high incidence rate. According to the World Health Organization (WHO), more than 10 million people are infected with Mycobacterium tuberculosis each year, and about 1.4 million people die from tuberculosis. In recent years, the number of new cases of drug-resistant tuberculosis has been on the rise. The emergence and spread of drug-resistant tuberculosis has brought huge challenges to the prevention and control of tuberculosis and has become a serious public health problem. Faced with such a situation, it is urgent to improve the level of tuberculosis detection and solve the thorny problems in tuberculosis prevention and control.

[0003] At present, the diagnosis methods of tuberculosis include clinical symptom assessment, laboratory tests and imaging examinations. Among them, laboratory tests mainly include sputum smear examination, tuberculosis culture and tuberculosis DNA detection. Although the sputum smear examination has a short detection time, it has low sensitivity, low accuracy and high false positive rate. It usually requires that the content of Mycobacterium tuberculosis in the sample to be tested is higher than 1000 / mL; the culture method has high sensitivity and specificity, but the culture time is long, usually requiring 14-21 days, which is not conducive to clinical application.

[0004] The diagnosis of tuberculosis DNA testing takes a long time, and the process of obtaining the sample - alveolar lavage fluid - requires anesthesia, which is cumbersome and has a strong sense of invasion from the nasal cavity into the lungs. Therefore, there is still a demand for faster, more accurate and convenient tuberculosis diagnosis methods and the determination of drug-resistant strains in the market, and the tuberculosis diagnosis market has huge potential and demand.

[0005] Currently, there is a molecular biological detection method using PCR amplification to detect Mycobacterium tuberculosis, which has the characteristics of high sensitivity, specificity, and rapidity. However, its sensitivity and specificity when using sputum are low, so it also requires alveolar lavage fluid for detection like the culture method. It is not suitable for people with weak constitutions such as the elderly and children who are not suitable for anesthesia. Therefore, it is crucial to design highly specific and sensitive primers and probes to improve the accuracy and specificity of sputum molecular biological detection results.

[0006] Multidrug-resistant tuberculosis (MDR-TB) refers to tuberculosis that is resistant to at least isoniazid and rifampicin. The widespread use of anti-tuberculosis drugs, especially the irregular use, leads to the occurrence and spread of drug-resistant tuberculosis. my country has 120,000 new cases of MDR-TB each year, accounting for 25% of the world's new cases of MDR-TB each year, making it the second largest country with a high burden of MDR-TB in the world. At present, there is no method for simultaneously detecting Mycobacterium tuberculosis and its isoniazid- and rifampicin-resistant forms in my country's tuberculosis diagnosis methods.

[0007] IS6110 is a multi-copy insertion element found in the Mycobacterium tuberculosis complex, which has become an important tool for distinguishing the Mycobacterium tuberculosis complex from other mycobacteria. IS6110 is widely used in tuberculosis nucleic acid diagnosis, but IS6110 is missing in a very small number of strains, which may lead to false negative results.

[0008] Oligonucleotide microarray chips are formed by spotting oligonucleotide probes in array form onto a treated glass substrate with active chemical groups (such as hydroxyl, aldehyde or amino) on the surface, reacting the probes with the active groups to form stable covalent bonds, and fixing the probes on the glass substrate. Under appropriate temperature and humidity conditions, specific probes are used to hybridize with the DNA of the sample to be tested that has been labeled in an appropriate manner through base complementary pairing, and then the location and strength of the hybridization signal are detected by corresponding detection equipment to determine the type of sample, complete disease detection and basic research work. This method has the advantages of high throughput, microquantification, parallelization, automation, and low cost. Therefore, it has been widely used in many fields such as diagnostic testing, differential expression analysis, and gene sequencing.

[0009] Currently, gene chip detection technology has been applied to the detection of many diseases, but there is a lack of technology for the parallel detection of Mycobacterium tuberculosis and its isoniazid- and rifampicin-resistant types. (III) Summary of the invention

[0010] The purpose of the present invention is to provide a primer, a probe and a microarray chip for detecting Mycobacterium tuberculosis and its drug-resistant types. The primers and probes provided by the present invention can be used to detect Mycobacterium tuberculosis and its isoniazid- and rifampicin-resistant types, and have the characteristics of high sensitivity and accuracy, high efficiency, rapidity and time-saving, and can be detected with sputum samples without invasion and anesthesia.

[0011] The technical solution adopted by the present invention is:

[0012] The invention provides primers and probes for detecting mycobacterium tuberculosis and its drug-resistant type. The primers include primers for the IS6110 gene of mycobacterium tuberculosis, primers for the mpt64 gene of mycobacterium tuberculosis, primers for the S315T mutation of the KatG gene of mycobacterium tuberculosis resistant to isoniazid, primers for the C15T mutation of the inhA gene of mycobacterium tuberculosis resistant to isoniazid, primers for the S531L and S531W mutations of the rpoB gene of mycobacterium tuberculosis resistant to rifampicin, and primers for the S6110 gene of mycobacterium tuberculosis resistant to isoniazid, ... Primers; the probes include a probe for the IS6110 gene of Mycobacterium tuberculosis, a probe for the mpt64 gene of Mycobacterium tuberculosis, a probe for the S315T mutation of the KatG gene of isoniazid-resistant Mycobacterium tuberculosis, a probe for the C15T mutation of the inhA gene of isoniazid-resistant Mycobacterium tuberculosis, a probe for the S531L mutation of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis, and a probe for the S531W mutation of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis;

[0013] The primers for the Mycobacterium tuberculosis IS6110 gene:

[0014] Upstream primer 5'-GAAGAATCCGCTGAGATAAAGC-3' (SEQ ID NO. 7),

[0015] Downstream primer 5′-GGTTGATGTGGTCGTAGTAGGT-3′ (SEQ ID NO. 8);

[0016] The primers for the Mycobacterium tuberculosis mpt64 gene:

[0017] Upstream primer 5'-GACTTCTGGTCGGGGTAGTAAC-3' (SEQ ID NO.9),

[0018] Downstream primer 5′-CTGTCGTTTTGCTCTGTTGTTC-3′ (SEQ ID NO. 10);

[0019] The primers for the S315T mutation of the KatG gene of isoniazid-resistant Mycobacterium tuberculosis are:

[0020] Upstream primer 5'-AAGAGCTCGTATGGCACCGG-3' (SEQ ID NO.11),

[0021] Downstream primer 5′-AGCGCCAGCAGGGCTCTTC-3′ (SEQ ID NO. 12);

[0022] The primer for the C15T mutation of the inhA gene of isoniazid-resistant Mycobacterium tuberculosis:

[0023] Upstream primer 5'-CGCTGCCCAGAAAGGGA-3' (SEQ ID NO. 13),

[0024] Downstream primer 5′-CCGGGTTTCCTCCGGT-3′ (SEQ ID NO. 14);

[0025] The primers for the rpoB gene S531L and S531W mutations of rifampicin-resistant Mycobacterium tuberculosis:

[0026] Upstream primer 5′-TGGTCCGCTTGCACGAGGGTCAGA-3′ (SEQ ID NO. 15),

[0027] Downstream primer 5′-CTCAGGGGTTTCGATCGGGCACAT-3′ (SEQ ID NO. 16);

[0028] Probe for the IS6110 gene of Mycobacterium tuberculosis 5′-TCACATAAGGCGTTGTCGAGGG-3′ (SEQ ID NO. 1);

[0029] Probe for the mpt64 gene of Mycobacterium tuberculosis 5′-TGTGGTGGCCGGCGTGTCCGCC-3′ (SEQ ID NO. 2);

[0030] Probe for the S315T mutation in the KatG gene of isoniazid-resistant Mycobacterium tuberculosis 5'-ATCACCACCGGCATC-3' (SEQ ID NO. 3);

[0031] Probe for the C15T mutation in the inhA gene of isoniazid-resistant Mycobacterium tuberculosis 5'-CGCGGCGAGATGATAGG-3' (SEQ ID NO. 4);

[0032] Probe for the S531L mutation in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis 5'-CTGTTGGCGCTGGGG-3' (SEQ ID NO. 5);

[0033] The probe for the S531W mutation in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis is 5'-CTGTGGGCGCTGGGG-3' (SEQ ID NO. 6).

[0034] Furthermore, the 3' end of the probes are all labeled with a fluorescent group 6-FAM, and the 5' end are all labeled with a quenching group BHQ1.

[0035] The invention also provides a microarray chip prepared based on the probe and primers for detecting Mycobacterium tuberculosis and its drug-resistant types.

[0036] Furthermore, the microarray chip is provided with a positive control area, a negative control area, a Mycobacterium tuberculosis IS6110 gene partition, a Mycobacterium tuberculosis mpt64 gene partition, an isoniazid-resistant Mycobacterium tuberculosis KatG gene S315T mutation partition, an isoniazid-resistant Mycobacterium tuberculosis inhA gene C15T mutation partition, a rifampicin-resistant Mycobacterium tuberculosis rpoB gene S531L mutation partition and a rifampicin-resistant Mycobacterium tuberculosis rpoB gene S531W mutation partition.

[0037] Furthermore, the negative control is 0.067 mol / L, pH 6.8 phosphate buffer; the positive control is a PCR reaction solution containing inactivated Mycobacterium tuberculosis genomic DNA, IS6110 gene probe, and mpt64 gene probe.

[0038] Further, 50 μL of PCR reaction solution consisted of: 5 μL of each 200 μmol / L deoxynucleoside triphosphate (dNTPs), 5 μL of 1.5 mmol / L MgCl2, 14 μL of 1× Taq DNA polymerase buffer, 2 units of Taq DNA polymerase, 400 μmol / L primers (primers in Table 3, 0.6 μL each), and 5 μL of genomic DNA sample extracted by the method of Example 3, supplemented to 50 μL with phosphate buffer (0.067 mol / L, pH 6.8).

[0039] Furthermore, the microarray chip is fixed with corresponding probes except for the negative control area and the positive control area.

[0040] The present invention also provides a method for detecting Mycobacterium tuberculosis and its drug-resistant types using the microarray chip, the method comprising the following steps: extracting genomic DNA from a sputum sample to be tested, performing PCR amplification using the primers, adding the DNA to a microarray chip with fixed probes, and performing constant temperature hybridization at 45°C for 3-4 hours; scanning using a laser confocal scanner, the negative control area has no fluorescence, the positive control area has fluorescence, and the fluorescence in each partition represents that the sample to be tested contains the corresponding gene.

[0041] Furthermore, the PCR amplified product was mixed with an equal volume of phosphate buffer, denatured at 95°C for 6 minutes, placed in an ice bath for 5 minutes, and then added to the microarray chip with fixed probes.

[0042] Furthermore, the genomic DNA is extracted as follows: a sputum sample is mixed with an equal volume of 1% NALC-NaOH, incubated at 20-25°C for 15 minutes, a phosphate buffer solution of 9 times the total volume of the mixture is added, centrifuged at a centrifugal force of 3000xg for 15 minutes, the precipitate is dissolved with distilled water, and then bathed in a water bath at 80°C for 40 minutes to kill bacteria, centrifuged at a centrifugal force of 8000xg for 1 minute, and the precipitate is collected to extract genomic DNA; the 1% NALC-NaOH is prepared by mixing a 4% NaOH aqueous solution with a 2.94% sodium citrate aqueous solution in a volume ratio of 7:3 to obtain a basic solution, adding N-acetyl-L-cysteine ​​to the basic solution, and adjusting the pH to between 2.0 and 2.8; the mass of the N-acetyl-L-cysteine ​​is 1 g / 100 mL based on the volume of the basic solution.

[0043] The present invention extracts genomic DNA from inactivated Mycobacterium tuberculosis and its isoniazid and rifampicin resistant types respectively; clones and sequences the specific conservative sequences of Mycobacterium tuberculosis and its isoniazid and rifampicin resistant types, and screens 6 oligonucleotide probes and 5 sets of primers. Using the fluorescent microarray method, the microarray chip is first prepared using the probe, and then the primers and the products amplified by the corresponding pathogens are specifically hybridized with the microarray chip with the fixed probes, and the hybridization results are judged according to the position and intensity of the fluorescent signal, so as to detect Mycobacterium tuberculosis and its isoniazid and rifampicin resistant types in the sample to be tested, and diagnose pulmonary tuberculosis in combination with clinical examination.

[0044] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in:

[0045] (1) The primers and probes screened by the present invention can be used to detect Mycobacterium tuberculosis and its isoniazid- and rifampicin-resistant types using sputum as a sample through PCR amplification and fluorescence scanning, and the screened probes can specifically bind to the corresponding target genes.

[0046] (2) The microarray chip prepared by the present invention using the screened probes can specifically bind to the corresponding target gene, the hybridization signal is strong and stable, and sputum samples can be used as the detection object without invasion and anesthesia.

[0047] (3) The present invention has established a microarray chip detection method with good specificity, high sensitivity, strong stability and time-saving and labor-saving, which can assist clinical detection and provide technical support for early detection and early treatment of tuberculosis. It is of great significance to meet the needs of tuberculosis prevention and control, control the spread of Mycobacterium tuberculosis and its isoniazid and rifampicin resistant types, and protect people's health. (IV) Description of the drawings

[0048] Figure 1Schematic diagram of probe distribution of microarray chip; P1-negative control partition; P2-positive control partition; 1-Mycobacterium tuberculosis IS6110 gene probe partition; 2-Mycobacterium tuberculosis mpt64 gene probe partition; 3-isoniazid-resistant Mycobacterium tuberculosis KatG gene S315T mutation probe partition; 4-isoniazid-resistant Mycobacterium tuberculosis inhA gene C-15-T mutation probe partition; 5-rifampicin-resistant Mycobacterium tuberculosis rpoB gene S531L mutation probe partition; 6-rifampicin-resistant Mycobacterium tuberculosis rpoB gene S531W mutation probe partition.

[0049] Figure 2 This is a laser confocal scanning image after hybridization of the microarray chip with the Mycobacterium tuberculosis IS6110 gene.

[0050] Figure 3 This is a laser confocal scanning image after hybridization of the microarray chip with the Mycobacterium tuberculosis mpt64 gene.

[0051] Figure 4 This is a laser confocal scanning image after hybridization of the microarray chip with the S315T mutation of the KatG gene of isoniazid-resistant Mycobacterium tuberculosis.

[0052] Figure 5 This is a laser confocal scanning image after hybridization of the microarray chip with the C-15-T mutation of the inhA gene of isoniazid-resistant Mycobacterium tuberculosis.

[0053] Figure 6 This is a laser confocal scanning image after hybridization of the microarray chip with the S531L mutation of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis.

[0054] Figure 7 This is a laser confocal scanning image after hybridization of the microarray chip with the S531W mutation of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis. (V) Specific implementation methods

[0055] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

[0056] Example 1. Design and screening of primers and probes for detecting isoniazid- and rifampicin-resistant Mycobacterium tuberculosis

[0057] 1. Initial screening

[0058] According to the complete gene sequence of Mycobacterium tuberculosis published on the Genbank website, PCR primers were designed using the Integrated DNA Technologies website. Following the principle that the probe should be as close to the upstream primer as possible, a better combination was selected from the alternative primers and probes provided on the website. After the better combination was selected, it was verified by Blast on the NCIBI website.

[0059] More than one hundred pairs of probe primer combinations were obtained through the above design steps, and then more than one hundred pairs of probe primer combinations obtained in the initial screening were synthesized through the solid phase phosphite amide triester method.

[0060] 2. Rescreening

[0061] The inactivated Mycobacterium tuberculosis nucleic acid extract (i.e., genomic DNA) was used as the detection object. The probes and their corresponding primers screened in step 1 were used to construct a real-time fluorescence PCR system according to Table 1, and added to the multi-well plate respectively. Positive samples (using the inactivated Mycobacterium tuberculosis nucleic acid extract as a template, the probes shown in SEQ ID NO.1 and SEQ ID NO.2 and the corresponding upstream and downstream primers were used to prepare the reaction system according to Table 1) and negative samples (phosphate (Tris-HCl) buffer 0.067 mol / L, pH 6.8) were set. The multi-well plate was placed in a PCR instrument, and the qPCR real-time fluorescence reaction was performed according to Table 2.

[0062] Table 1. Real-time fluorescence quantitative PCR reaction system for probe and primer screening

[0063]

[0064] Table 2. PCR reaction procedure

[0065]

[0066] The light signal was collected at 42°C and an excitation wavelength of 493 nm. The light signal values ​​obtained from the above experiment were sorted out, the data were compared with the positive control group, and the group with stronger signal values ​​was screened.

[0067] The final sequences of the primers and probes screened are shown in Table 3 below.

[0068] Table 3. Nucleotide sequences of primers and probes

[0069] name sequence IS6110 Probe 5'-TCACATAAGGCGTTGTCGAGGG-3'(SEQ ID NO.1) mpt64 probe 5'-TGTGGTGGCCGGCGTGTCCGCC-3'(SEQ ID NO.2) KatG gene S315T mutation probe 5'-ATCACCACCGGCATC-3'(SEQ ID NO.3) inhA gene C-15-T mutation probe 5'-CGCGGCGAGATGATAGG-3'(SEQ ID NO.4) rpoB gene S531L mutation probe 5'-CTGTTTGGCGCTGGGG-3'(SEQ ID NO.5) rpoB gene S531W mutation probe 5'-CTGTGGGCCGCTGGGG-3'(SEQ ID NO.6) IS6110-F 5'-GAAGAATCCGCTGAGATAAAGC-3' (SEQ ID NO.7) IS6110-R 5'-GGTTGATGTGGTCGTAGTAGGT-3'(SEQ ID NO.8) mpt64-F 5'-GACTTCTGGTCGGGGTAGTAAC-3'(SEQ ID NO.9) mpt64-R 5'-CTGTCGTTTTGCTCTGTTGTTC-3'(SEQ ID NO.10) KatG-F 5'-AAGAGCTCGTATGGCACCGG-3' (SEQ ID NO.11) KatG-R 5'-AGGCGCCAGCAGGGCTCTTC-3'(SEQ ID NO.12) inhA-F 5'-CGCTGCCCAGAAAGGGA-3'(SEQ ID NO.13) inhA-R 5'-CCGGGTTTCCTCCGGT-3'(SEQ ID NO.14) RpoB-F 5′-TGGTCCGCTTGCACGAGGGTCAGA-3′(SEQ ID NO.15) RpoB-R 5′-CTCAGGGGTTTCGATCGGGCACAT-3′(SEQ ID NO.16)

[0070] Example 2: Partitioning and preparation of a microarray chip for detecting isoniazid- and rifampicin-resistant Mycobacterium tuberculosis

[0071] 1. Partitioning of microarray chips

[0072] Referring to document 1 (Zhu Bin, Jing Fengxiang, Zhao Jianlong, Sun Yue, Chen Jifeng, Zhao Xintai, Xu Yuansen. Preparation of cDNA microarray using amino-modified glass slides [J]. Progress in Biochemistry and Biophysics, 2001, 28(1):121-124), a microarray chip was prepared using a glass slide modified with aminopropyltrimethoxysilane, which was 40 mm long, 10 mm wide, and 2 mm high, and an 8-row × 10-column microarray was set up, such as Figure 1 shown.

[0073] The probes on the microarray are distributed from top to bottom as follows: P1 partition (negative control), P2 partition (positive control), 1 partition (IS6110 gene probe of Mycobacterium tuberculosis), 2 partition (mpt64 gene probe of Mycobacterium tuberculosis), 3 partition (S315T mutation probe of KatG gene of Mycobacterium tuberculosis resistant to isoniazid), 4 partition (C15T mutation probe of inhA gene of Mycobacterium tuberculosis resistant to isoniazid), 5 partition (S531L mutation probe of rpoB gene of Mycobacterium tuberculosis resistant to rifampicin), 6 partition (S531W mutation probe of rpoB gene of Mycobacterium tuberculosis resistant to rifampicin), each partition has 1 row, and the corresponding points are 1 row×10 points. There is at least one microarray on each chip, and each point is loaded with samples according to Table 4, and each microarray can detect one sample.

[0074] 2. Preparation of microarray:

[0075] (1) Referring to Reference 1, a glass slide modified with aminopropyltrimethoxysilane was immersed in a 2 mol / L NaOH 70% ethanol solution for 2 h, washed several times with water, dried at 110°C and cooled to room temperature, immersed in a 1% 3-aminopropyltrimethoxysilane-95% acetone solution for 2 min, washed 10 times with acetone, dried at 110°C and cooled to room temperature for later use.

[0076] (2) According to the method in step 2.2 of reference 1, a microarray was prepared according to the partitions and the patterns in Table 4.

[0077] The composition of 50 μL of PCR reaction solution in Table 4 is as follows: 5 μL of each 200 μmol / L deoxynucleoside triphosphate (dNTPs), 5 μL of 1.5 mmol / L MgCl2, 14 μL of 1× Taq DNA polymerase buffer, 2 units of Taq DNA polymerase, 400 μmol / L primers (primers in Table 3, 0.6 μL of each), and 5 μL of genomic DNA sample extracted by the method of Example 3, supplemented to 50 μL with phosphate buffer (0.067 mol / L, pH 6.8).

[0078] Table 4 Probes, reagents and dosages used for partition spotting

[0079]

[0080] Example 3: Extraction of Mycobacterium tuberculosis and its isoniazid- and rifampicin-resistant genome

[0081] 1. Pretreatment of sputum samples:

[0082] (1) Preparation of 1% NALC-NaOH:

[0083] ① Preparation of basic solution: Mix 350 mL of 4% NaOH aqueous solution and 150 mL of 2.94% sodium citrate aqueous solution in a volume ratio of 7:3 to obtain 500 mL of basic solution.

[0084] ② Add N-acetyl-L-cysteine: Add 5 g of N-acetyl-L-cysteine ​​to 500 mL of the above base solution.

[0085] ③ Adjust the pH value: Use pH test paper or pH meter to monitor the pH value of the solution in step ②, and use NaOH or dilute acid (such as dilute hydrochloric acid) to adjust the pH to between 2.0 and 2.8 to obtain 1% NALC-NaOH.

[0086] (2) Genome extraction

[0087] Place 2.5 mL of sputum sample in a 50 mL centrifuge tube and add 2.5 mL of 1% NALC-NaOH. Then close the centrifuge tube and shake it, incubate it at 20-25°C for 15 minutes. After that, add phosphate buffer (0.067 mol / L, pH 6.8) to 45 mL of the centrifuge tube. Centrifuge at 3000xg for 15 minutes. After the precipitation process is completed, carefully discard the supernatant into a sterile bottle, and redissolve the remaining precipitate with 1 mL of distilled water and transfer it to a 1.5 mL microcentrifuge tube. Place the microcentrifuge tube in an 80°C water bath for 40 minutes to kill bacteria. Centrifuge at 8000xg for 1 minute. Carefully remove the supernatant. The precipitate uses the GF-1 Tissue DNA Extraction Kit to extract genomic DNA as a template. For specific operations, see the kit instructions.

[0088] Example 4: Detection of Mycobacterium tuberculosis and its isoniazid- and rifampicin-resistant genomic DNA using a fluorescent microarray chip

[0089] 1. 50 μL PCR reaction system: 5 μL of each 200 μmol / L deoxynucleoside triphosphate (dNTPs), 5 μL of 1.5 mmol / L MgCl2, 14 μL of 1× Taq DNA polymerase buffer, 2 units of Taq DNA polymerase, 400 μmol / L primers (primers in Table 3, 0.6 μL each), and 5 μL of genomic DNA sample extracted by the method of Example 3, supplemented to 50 μL with phosphate buffer (0.067 mol / L, pH 6.8).

[0090] 2. Amplification: Place the prepared reaction solution in a PCR amplifier and perform the PCR reaction according to the PCR reaction program in Table 2. The PCR product should be kept away from light. For short-term storage, place it in a 4°C refrigerator.

[0091] 3. Hybridization

[0092] (1) 20 μL hybridization solution: 10 μL phosphate buffer (0.067 mol / L, pH 6.8), 10 μL PCR amplification product from step 2. First, thaw the phosphate buffer at 42°C before preparing the hybridization solution. Denature the hybridization solution at 95°C for 6 minutes and place it on ice for 5 minutes to obtain the denatured hybridization solution for later use.

[0093] (2) Hybridization

[0094] Use a pipette to slowly inject 18μL of denatured hybridization solution through the sample well of the cover slip to each partition of the microarray chip. The hybridization solution will form a liquid film between the convex platform under the cover slip and the chip surface by relying on the surface tension of the liquid. Be careful not to shake the cover slip or chip to avoid damaging the liquid film. Cover the cover slip tightly. Put it in a 45℃ constant temperature water bath, let it stand, and hybridize for 3-4 hours.

[0095] After hybridization, take out the chip and place it in the washing solution I preheated at 42℃, shake and wash at 42℃ for 5 minutes, then use the washing solution II preheated at 42℃, shake and wash at 42℃ for 5 minutes, then use the washing solution III preheated at 42℃, shake and wash at 42℃ for 5 minutes, rinse with ultrapure water preheated at 42℃ for 5 times, soak in ethanol for 1min, and finally put it in a 37℃ incubator to dry. This chip can be stored away from light and is valid for scanning within 4 hours.

[0096] Washing solution I: The final concentration of SSC is 1×, and the final concentration of SDS is 0.03%.

[0097] Wash solution II: The final concentration of SSC is 0.2×.

[0098] Wash solution III: The final concentration of SSC is 0.05×.

[0099] Table 5. SSC lotion composition and content

[0100] Lotion Element SSC 1× NaCl 0.15mol / L, sodium citrate 0.015mol / L SSC 0.2× NaCl 0.03mol / L, sodium citrate 0.003mol / L SSC 0.05× NaCl 0.0075mol / L, sodium citrate 0.00075mol / L

[0101] 4. Scanning and result interpretation

[0102] The washed chip was scanned and analyzed under a laser confocal scanner to remove the negative control background and determine the results.

[0103] like Figure 2 As shown, partition P1 has no fluorescence, while partition P2 and partition 1 have fluorescence, indicating that partition 1 is the PCR amplification product of Mycobacterium tuberculosis IS6110 genomic DNA;

[0104] like Figure 3 As shown, partition P1 has no fluorescence, while partition P2 and partition 2 have fluorescence, indicating that partition 2 is the PCR amplification product of Mycobacterium tuberculosis mpt64 genomic DNA;

[0105] like Figure 4 As shown, partition P1 has no fluorescence, while partitions P2 and 3 have fluorescence, indicating that partition 3 is the PCR amplification product of the DNA of the KatG gene S315T mutation group of isoniazid-resistant Mycobacterium tuberculosis;

[0106] like Figure 5 As shown, partition P1 has no fluorescence, while partition P2 and partition 4 have fluorescence, indicating that partition 4 is the PCR amplification product of the C-15-T mutation group DNA of the inhA gene of isoniazid-resistant Mycobacterium tuberculosis;

[0107] like Figure 6 As shown, partition P1 has no fluorescence, while partition P2 and partition 5 have fluorescence, indicating that partition 5 is the PCR amplification product of the S531L mutation group DNA of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis;

[0108] like Figure 7 As shown, partition P1 has no fluorescence, while partition P2 and partition 6 have fluorescence, indicating that partition 6 is the PCR amplification product of the S531W mutant group DNA of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis.

[0109] 5. Waste disposal

[0110] Dispose of used and unused reagents and contaminated waste in accordance with local or national regulations for the disposal of infectious and potentially infectious waste.

[0111] Example 5: Application of fluorescence microarray chip for detection of Mycobacterium tuberculosis and its drug-resistant forms

[0112] Clinical samples were collected from a hospital in Zhejiang Province and were sputum samples from patients with pulmonary tuberculosis, lung cancer, nontuberculous mycobacterial infection, fungal infection and other easily confused diseases. A total of 1,223 clinical samples were obtained, of which 76 cases were diagnosed by bronchoalveolar lavage fluid culture combined with molecular biology, including 76 cases of non-resistant Mycobacterium tuberculosis infection, 39 cases of isoniazid-resistant Mycobacterium tuberculosis infection caused by KatG gene mutation, 62 cases of isoniazid-resistant Mycobacterium tuberculosis infection caused by inhA gene mutation, and 49 cases of rpoB gene mutation (including S531L and S531W mutations) caused rifampicin-resistant Mycobacterium tuberculosis infection. Sample collection was in accordance with the patient's informed consent, protection of patient privacy and medical ethics requirements.

[0113] The method of Example 4 was used for detection, and the results are shown in Table 6.

[0114] Table 6 Comparison of fluorescence microarray method, bronchoalveolar lavage fluid culture combined with molecular biology detection method for detection of Mycobacterium tuberculosis and its drug-resistant forms

[0115]

[0116] According to the clinical experimental results in the above table, among 1214 suspected cases of pulmonary tuberculosis, the detection compliance rate of the microarray chip of the present invention exceeded 95%.

[0117] At present, the diagnosis methods of tuberculosis include clinical symptom assessment, laboratory tests and imaging examinations. The clinical symptoms of pulmonary tuberculosis are cough, hemoptysis, fever, weight loss and loss of appetite, which are easily confused with lung cancer, pulmonary nodules, pneumonia caused by non-tuberculous mycobacterium infection, etc., and are difficult to distinguish. Laboratory tests mainly include sputum smear examination, tuberculosis culture and tuberculosis DNA detection. Although the sputum smear examination has a short detection time, it has low sensitivity, low accuracy and high false positive rate. It usually requires the content of Mycobacterium tuberculosis in the sample to be tested to be higher than 1000 / mL; the culture method has high sensitivity and specificity, but the culture time is long, usually requiring 14-21 days, which is not conducive to clinical application. Imaging examinations are difficult to detect patients with occult pulmonary tuberculosis (small lung cavities and unclear symptoms) or in the early stages of the disease.

[0118] In summary, the microarray chip of the present invention has good consistency with the existing methods, and has greatly improved sensitivity and specificity compared with other detection methods, and is easy to operate, which can meet clinical needs.

Claims

1. A primer and a probe for detecting Mycobacterium tuberculosis and its drug-resistant forms, characterized in that: The primers and probes include primers and probes for the IS6110 gene of Mycobacterium tuberculosis, primers and probes for the mpt64 gene of Mycobacterium tuberculosis, primers and probes for the S315T mutation of the KatG gene of isoniazid-resistant Mycobacterium tuberculosis, primers and probes for the C15T mutation of the inhA gene of isoniazid-resistant Mycobacterium tuberculosis, primers and probes for the S531L mutation of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis, and primers and probes for the S531W mutation of the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis; Primers and probes for the IS6110 gene of Mycobacterium tuberculosis: Upstream primer 5′-GAAGAATCCGCTGAGATAAAGC-3′, Downstream primer 5'-GGTTGATGTGGTCGTAGTAGGT-3', Probe 5′-TCACATAAGGCGTTGTCGAGGG-3′; Primers and probes for the mpt64 gene of Mycobacterium tuberculosis: Upstream primer 5′-GACTTCTGGTCGGGGTAGTAAC-3′, Downstream primer 5'-CTGTCGTTTTGCTCTGTTGTTC-3', Probe 5′-TGTGGTGGCCGGCGTGTCCGCC-3′; Primers and probes for the S315T mutation in the KatG gene of isoniazid-resistant Mycobacterium tuberculosis: Upstream primer 5'-AAGAGCTCGTATGGCACCGG-3', Downstream primer 5'-AGCGCCAGCAGGGCTCTTC-3', Probe 5′-ATCACCACCGGCATC-3′; Primers and probes for the C15T mutation in the inhA gene of isoniazid-resistant Mycobacterium tuberculosis: Upstream primer 5′-CGCTGCCCAGAAAGGGA-3′, Downstream primer 5'-CCGGGTTTCCTCCGGT-3', Probe 5′-CGCGGCGAGATGATAGG-3′; Primers for rpoB gene S531L and S531W mutations in rifampicin-resistant Mycobacterium tuberculosis: Upstream primer 5′-TGGTCCGCTTGCACGAGGGTCAGA-3′, Downstream primer 5′-CTCAGGGGTTTCGATCGGGCACAT-3′, The probe for the S531L mutation in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis is 5'-CTGTTGGCGCTGGGG-3'. The probe targeting the S531W mutation in the rpoB gene of rifampicin-resistant Mycobacterium tuberculosis is 5'-CTGTGGGCGCTGGGG-3'.

2. The primer and probe according to claim 1, characterized in that: The 3' end of the probes are all labeled with a fluorescent group 6-FAM, and the 5' end is all labeled with a quenching group BHQ1.

3. A microarray chip for detecting Mycobacterium tuberculosis and its drug-resistant forms prepared based on the probes and primers according to claim 1.

4. The microarray chip as claimed in claim 3, characterized in that, The microarray chip is provided with a positive control area, a negative control area, a Mycobacterium tuberculosis IS6110 gene partition, a Mycobacterium tuberculosis mpt64 gene partition, an isoniazid-resistant Mycobacterium tuberculosis KatG gene S315T mutation partition, an isoniazid-resistant Mycobacterium tuberculosis inhA gene C15T mutation partition, a rifampicin-resistant Mycobacterium tuberculosis rpoB gene S531L mutation partition and a rifampicin-resistant Mycobacterium tuberculosis rpoB gene S531W mutation partition.

5. The microarray chip as claimed in claim 4, characterized in that, The negative control is 0.067 mol / L, pH 6.8 phosphate buffer; the positive control is a PCR reaction solution containing inactivated Mycobacterium tuberculosis genomic DNA, IS6110 gene probe, and mpt64 gene probe.

6. The microarray chip as claimed in claim 3, characterized in that, The microarray chip is fixed with corresponding probes except for the negative control area and the positive control area.

7. A method for detecting Mycobacterium tuberculosis and its drug-resistant types using the microarray chip of claim 3, the method comprising the following steps: extracting genomic DNA from a sputum sample to be tested, amplifying the genomic DNA using the primers, adding the DNA to a microarray chip with fixed probes, and hybridizing at 45°C for 3-4 hours; scanning the sample using a laser confocal scanner, wherein the negative control area has no fluorescence, the positive control area has fluorescence, and the fluorescence in each partition indicates that the sample to be tested contains the corresponding gene.

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