Rapid detection primer combination based on second-line drug-resistant mutation site of mycobacterium tuberculosis and application of rapid detection primer combination

Abstract

The invention discloses a rapid detection primer combination based on mycobacterium tuberculosis second-line drug-resistant mutation sites and application thereof, and belongs to the technical field of molecular diagnosis. On one hand, the invention provides a rapid detection primer combination based on the mycobacterium tuberculosis second-line drug-resistant mutation site, and on the other hand, the invention provides rapid detection of the mycobacterium tuberculosis second-line drug-resistant mutation site by using the primer combination. The optimized system is high in stability, high in sensitivity and high in specificity; an integrated detection platform and a detection reagent are matched, detection of 384 samples is completed within 6-7 h in the whole process, operation is easy, convenient and rapid, result analysis is visual, cost is low, accuracy is high, detection flux is high, large-scale application and popularization are easy, performance is more excellent compared with existing related products based on qPCR or NGS methodology, and practicability is outstanding.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular diagnosis, and in particular relates to a rapid detection primer combination based on second-line drug-resistant mutation sites of Mycobacterium tuberculosis and its application. Background Art

[0002] The prevalence of multidrug-resistant tuberculosis (MDR-TB) has become a global public health challenge, particularly in parts of my country, India, and Russia. MDR-TB patients may require treatment with second-line anti-TB drugs, commonly used such as fluoroquinolones (FQ), aminoglycosides, cyclic peptides, thioamides, and para-aminosalicylic acid. However, with the widespread use of second-line drugs, MTB resistant to these drugs has been detected worldwide, and the current situation of second-line drug resistance in my country is also not optimistic. It has been reported that 72.6% of multidrug-resistant MTB in Shandong Province from 2004 to 2007 were resistant to second-line anti-tuberculosis drugs. During the same period, the resistance rate of MDR-TB patients in Henan Province to the main second-line anti-tuberculosis drugs (ofloxacin and amikacin) reached 48.1%, indicating that the resistance to second-line anti-tuberculosis drugs in my country has become very serious, and the detection of drug-resistant gene mutation sites has become increasingly important.

[0003] MDR-TB spreads rapidly, progresses rapidly, and has a high mortality rate. In general clinical laboratories, drug susceptibility testing for Mycobacterium tuberculosis still relies on traditional methods such as absolute concentration, ratio, and resistance ratio. These methods, which rely on traditional bacterial culture and drug susceptibility testing, are clearly no longer effective. Research has shown that rapid nucleic acid detection of the tuberculosis pathogen is more sensitive than traditional sputum smear and culture tests, and is more conducive to early clinical diagnosis.

[0004] Current options for mycobacterial nucleic acid testing include semi-nested real-time quantitative PCR, probe melting curve analysis, gene chip analysis, reverse hybridization, and nucleic acid mass spectrometry. Semi-nested real-time quantitative PCR offers high sensitivity, but it only detects rifampicin resistance and does not report specific resistance mutations, leading to high testing costs. The probe melting curve analysis avoids cross-contamination and laboratory contamination, yet similarly does not report specific resistance mutations. Six kits are required for strain identification and detection of different resistance gene mutations. Gene chip analysis can reveal the site and nature of mutations, but the hybridization and detection process is cumbersome and can only detect rifampicin and isoniazid resistance mutations. Reverse hybridization can reveal the site and nature of mutations, but it is an open-label assay, potentially contaminating the amplified product and leading to false reports of resistance, and the hybridization development process is cumbersome. Compared to these detection methods, time-of-flight nucleic acid mass spectrometry, as a medium-throughput technology in the field of genetic testing, offers a wide detection range, can identify the site and nature of mutations, and can detect multiple drug-resistance mutations simultaneously. This makes it a highly efficient and cost-effective medium-throughput genotyping platform that balances sensitivity and specificity.

[0005] At present, there have been many detailed studies on the detection of resistance gene sites and resistance mechanisms of first-line tuberculosis drugs, but there are few multiplex detection methods for second-line drug resistance gene sites. The present invention fills the gap in this field and leverages the advantages of the time-of-flight nucleic acid mass spectrometry platform to develop a primer set and method for detecting resistance gene sites for second-line anti-tuberculosis drugs, providing an effective solution for better guiding the use of drugs for tuberculosis patients in the future. Summary of the Invention

[0006] To address the challenges of the existing technology, the present invention aims to provide a rapid detection primer combination based on second-line drug-resistant mutation sites in Mycobacterium tuberculosis and its application. This primer combination allows for simultaneous detection of drug-resistant mutations in multiple second-line anti-tuberculosis drugs in a single test.

[0007] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0008] In response to the variable mutations in the extended primer region sequence, the present invention creatively divides each detection site into two groups to avoid mutual interference between adjacent mutation sites. The two-tube reaction can complete the detection of all sites simultaneously, and obtain all bacterial species information and drug-resistant mutation site information at a one-time low cost, avoiding the economic burden caused by repeated testing.

[0009] The first aspect of the present invention provides a rapid detection primer combination based on the second-line drug-resistant mutation site of Mycobacterium tuberculosis, which includes PCR amplification primer sets 1-2 and single-base extension primer sets 1-2;

[0010] The PCR amplification primer set 1 includes five first-group 1st-PCR primers and five first-group 2st-PCR primers, the nucleotide sequences of the first-group 1st-PCR primers are shown in SEQ ID NOs. 1 to 5, and the nucleotide sequences of the first-group 2st-PCR primers are shown in SEQ ID NOs. 15 to 19;

[0011] The PCR amplification primer set 2 includes 9 second group 1st-PCR primers and 9 second group 2st-PCR primers, the nucleotide sequences of the second group 1st-PCR primers are shown in SEQ ID NOs. 6 to 14, and the nucleotide sequences of the second group 2st-PCR primers are shown in SEQ ID NOs. 20 to 28;

[0012] The single-base extension primer set 1 includes 10 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 29 to 38;

[0013] The single-base extension primer set 2 includes 13 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 39 to 51.

[0014] Furthermore, the second-line drug-resistant sites and drug-resistant gene sites of Mycobacterium tuberculosis detected by PCR amplification primer set 1 and single-base extension primer set 1 include: ext_rd9, gyrB500_2, gyrB500_1, IS6110, gyrA94_1EV1, gyrA94_1EV2, gyrA94_2EV1, gyrA94_2EV5, rplC460 and rplC546.

[0015] Furthermore, the second-line drug-resistant sites and drug-resistant gene sites of Mycobacterium tuberculosis detected by PCR amplification primer set 2 and single-base extension primer set 2 include: gyrA_A90VEV1, gyrA_A90VEV2, gyrA_S91P, alr261, alr337, cycA188, cycA318, cycA406, cycA508, cycA521, ddlA92, Rv0678_193 and Rv0678_466.

[0016] The second aspect of the present invention provides the use of the above primer combination in preparing a rapid detection product for second-line drug-resistant mutation sites in Mycobacterium tuberculosis, wherein the second-line drug resistance is fluoroquinolone, linezolid, cycloserine and clofazimine.

[0017] The third aspect of the present invention provides a kit containing the above primer combination.

[0018] Furthermore, the kit is a kit for detecting second-line drug-resistant mutation sites of Mycobacterium tuberculosis, and the second-line drug resistance is fluoroquinolone, linezolid, cycloserine and clofazimine.

[0019] Furthermore, the kit also contains reagents required for PCR amplification reaction, SAP reaction and / or single base extension reaction.

[0020] Furthermore, the kit also includes reagents required for time-of-flight mass spectrometry detection.

[0021] Furthermore, the kit also includes reagents required for extracting sample DNA.

[0022] Furthermore, the reagents required for the PCR amplification reaction in the kit include a PCR reaction mixture and a PCR enzyme mixture.

[0023] Furthermore, the reagents required for the SAP reaction in the kit include a SAP reaction mixture and a SAP enzyme mixture.

[0024] Furthermore, the reagents required for the single-base extension reaction in the kit include an extension reaction mixture and an extension enzyme mixture.

[0025] A fourth aspect of the present invention provides a method for using the above-mentioned kit, or a method for detecting second-line drug-resistant mutation sites in Mycobacterium tuberculosis for non-disease diagnosis purposes, comprising the following steps:

[0026] 1) using the genomic DNA of the sample to be tested as a template, and using the PCR amplification primer sets 1 to 2 to perform PCR amplification reactions respectively to obtain PCR reaction products;

[0027] 2) performing SAP reaction on the PCR reaction products respectively to obtain SAP reaction products;

[0028] 3) using the obtained SAP reaction product as a template, mixing it with the single-base extension primer sets 1 to 2 in sequence according to the numbering to perform a single-base extension reaction to obtain a single-base extension reaction product;

[0029] 4) performing time-of-flight mass spectrometry analysis and result judgment on the obtained single base extension reaction product.

[0030] The present invention has the following beneficial effects:

[0031] (1) The present invention provides a method for rapid detection of second-line drug-resistant mutation sites in Mycobacterium tuberculosis using a primer combination. The optimized system has strong stability, high sensitivity, and strong specificity. It is equipped with an integrated detection platform and detection reagents, and the entire process can complete the detection of 384 samples within 6-7 hours. The operation is simple and fast, the result analysis is intuitive, the cost is low, the accuracy is high, the detection throughput is high, and it is easy to promote and apply on a large scale. It has better performance than existing related products based on qPCR or NGS methodologies and has outstanding practicality.

[0032] (2) The present invention refers to the globally shared database NCBI (https: / / www.ncbi.nlm.nih.gov / ), the pathogenic microorganism database (https: / / mycobrowser.epfl.ch / ), the WHO guidelines, and relevant domestic guidelines and expert consensus in China to select and determine the gene site information of second-line drug-resistant mutations of Mycobacterium tuberculosis, and determine the rplC and rrl genes related to linezolid resistance, the alr, cyc, and ddlA genes related to cycloserine resistance, the gyrA and gyrB genes related to fluoroquinolones, and the Rv0678 gene related to clofazimine resistance.

[0033] (3) Based on the detection principle of the time-of-flight nucleic acid mass spectrometry (DP-TOF) platform, multiple PCR amplification primers and extension primers are designed, and the sites can realize multi-well synchronous detection; a nucleic acid mass spectrometry multiple detection reaction system is established and optimized, and the result determination method is determined.

[0034] (4) The premixing of multiple components simplifies the difficulty of system preparation and subsequent testing for operators in clinical applications, significantly improves the stability and repeatability of micro-system preparation and testing, is easy to operate, effectively reduces the threshold for use and the difficulty of getting started, and correctly interprets the test results, thereby improving the diagnosis and treatment level of drug-resistant tuberculosis and accelerating the control of drug-resistant tuberculosis; for the examinees, repeated testing can be avoided, the diagnosis rate can be improved, and medical expenses can be reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the result of primer set 1 detection for sample 1. The figure shows the peaks detected at all 10 sites using primer set 1. The DP-TOF nucleic acid mass spectrometer detection software automatically determines the base type at each site based on the peaks of the extension products in each well.

[0036] Figure 2 This is the result of primer set 2 testing for sample 1. The figure shows the peaks detected by primer set 2 at all 13 sites. The DP-TOF nucleic acid mass spectrometer detection software automatically determines the base type at each site based on the peaks of the extension products in each well.

[0037] Figure 3Figure 4 shows the accuracy test for second-line tuberculosis drug resistance testing.

[0038] Figure 4 These are the test results of gyrA94_2EV1 100% wild type, 20% mutant type, 5% mutant type, and 100% mutant type in implementation case 6. DETAILED DESCRIPTION

[0039] The present invention will be further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] Example 1:

[0041] The PCR amplification primers and extension primers for the detection of second-line drug resistance genes of Mycobacterium tuberculosis were synthesized by Shanghai Bio-Tech Co., Ltd. and are as follows:

[0042] (1) Amplification primers

[0043]

[0044]

[0045] (2) Extending primer sequence

[0046]

[0047] Example 2:

[0048] Preparation of a kit for detecting second-line drug-resistant mutation sites in Mycobacterium tuberculosis

[0049] (1) Nucleic acid sample pretreatment reagents for time-of-flight nucleic acid mass spectrometry detection system include the following main components:

[0050]

[0051] (2) Amplification reaction primer premix: a mixture of the two sets of nucleotide sequences in claim 1, with each primer concentration being 0.3 to 3 μM;

[0052] (3) Single base extension reaction primer premix: a mixture of the two sets of nucleotide sequences as described in claim 2, wherein the concentration of each primer is between 5 and 30 μM;

[0053] (4) Desalination resin: including cation exchange resin powder for removing salt ions from the elongation reaction solution;

[0054] (5) Detection chip: includes a silicon-based chip containing 384 pre-dotted matrices.

[0055] Example 3:

[0056] Method for detecting drug-resistant mutation sites in Mycobacterium tuberculosis.

[0057] 1. Nucleic Acid Extraction from Clinical Samples

[0058] a) Sample type: sputum, alveolar lavage fluid, etc.

[0059] b) Sample pretreatment: If there are sticky solids, they must be liquefied using a liquefaction solution first.

[0060] Take 1 ml of sample and follow the instructions of the following two extraction kits for subsequent operations:

[0061] c) Nucleic acid extraction: Extract nucleic acid according to the instructions of the Novagen VAMNE Magnetic Pathogen DNA / RNA Kit (Cat. No. RM603-C5) or the Kangwei Century Magbead Pathogenic Microbiome DNA / RNA Kit (Cat. No.: CW3061S).

[0062] 2. Detection of drug-resistant gene loci based on time-of-flight mass spectrometry platform

[0063] 1) PCR reagent preparation (reagent preparation area)

[0064] Remove 2x Multiplex PCR mix, UNG enzyme, dUTP solution, and amplification primers (including 1st-PCR and 2nd-PCR primers) from the kit. Thaw and vortex at room temperature to mix thoroughly. Centrifuge at 2000 rpm for 10 seconds. Calculate the number of reaction reagents needed and prepare two aliquots for wells 1 and 2.

[0065] Each test reaction system was prepared as follows:

[0066] Reagents Dosage Reagents Dosage 2*Multiplex PCR mix 15 μL dUTP solution 0.1μL UNG enzyme 0.5μL Well 1 / well 2 PCR primers 6μL

[0067] Calculate the amount of each reagent to be used, mix thoroughly, and dispense 21.6 μL into PCR reaction tubes, which are then transferred to the sample processing area.

[0068] 2) Sample loading (sample processing area)

[0069] 8.4 μL of sample DNA solution and blank control NTC (8.4 μL of ultrapure water) were added respectively, the reaction tubes were tightly capped, and transferred to the nucleic acid amplification area.

[0070] 3) PCR amplification (nucleic acid amplification region)

[0071] Place the reaction tubes in a certain order on the PCR instrument and perform PCR amplification according to the following procedure:

[0072]

[0073] *Note: The PCR product will not be used for the next experimental step and can be stored at 4°C overnight.

[0074] 4) SAP reagent preparation (reagent preparation area)

[0075] Remove SAP Buffer and SAP enzyme from the kit, thaw each at room temperature, shake and mix thoroughly, and centrifuge at 2000 rpm for 10 seconds. Calculate the number of reaction reagents needed.

[0076] Each test reaction system was prepared as follows:

[0077] Reagents SAP Buffer SAP enzyme Ultrapure water Dosage 0.17μL 0.30μL 1.53μL

[0078] 5) Add SAP reagent (nucleic acid amplification area)

[0079] Take out 5 μL of the PCR product from step 3, add 2 μL of the above-mentioned SAP reaction solution to each of the 5 μL products taken out, and cover the reaction tube tightly.

[0080] Place the reaction tubes in a certain order on the PCR instrument and perform SAP digestion according to the following procedure:

[0081]

[0082] *Note: The SAP product should be immediately processed into the next step. It is not recommended to store it at 4°C overnight.

[0083] 6) Extension reagent preparation (reagent preparation area)

[0084] Prepare two wells of extension primer (Ext P), extension buffer, ddNTP solution, and extension enzyme. Thaw at room temperature, shake well, and centrifuge at 2000 rpm for 10 seconds. Calculate the number of reaction reagents needed and prepare one for each well.

[0085] Each test reaction system was prepared as follows:

[0086]

[0087] 7) Sample addition (nucleic acid amplification area)

[0088] Add 2 μL of the above extension reaction solution to the SAP product in step 5 in a certain order, and cover the reaction tube tightly.

[0089] Place the reaction tubes in a certain order on the PCR instrument and perform extension amplification according to the following procedure:

[0090]

[0091] *Note: The extension product can be stored at 4°C overnight without further experimental operation.

[0092] 8) Mass spectrometry detection using a DP-TOF time-of-flight mass spectrometry detection system (amplification analysis area)

[0093] Standard operations were performed according to the DP-TOF operating instructions, and the results were analyzed.

[0094] Figure 1 This is the result of primer set 1 detection for sample 1. The figure shows the peaks detected at all 10 sites using primer set 1. The DP-TOF nucleic acid mass spectrometer detection software automatically determines the base type at each site based on the peaks of the extension products in each well.

[0095] Figure 2 This is the result of primer set 2 testing for sample 1. The figure shows the peaks detected by primer set 2 at all 13 sites. The DP-TOF nucleic acid mass spectrometer detection software automatically determines the base type at each site based on the peaks of the extension products in each well.

[0096] Example 4:

[0097] The detection method of the present invention was used to test the accuracy of second-line tuberculosis drug resistance detection.

[0098] 56 pulmonary tuberculosis-positive samples were selected and second-line drug-resistant gene loci were detected for the pulmonary tuberculosis-positive samples using Example 3. The results are shown in the following table.

[0099]

[0100] like Figure 3 As shown in the results, it can be seen that the resistance rate to any one of fluoroquinolones, linezolid, cycloserine, and clofazimine is 14%; clofazimine-sensitive sample No. 1, fluoroquinolone-resistant sample No. 2, cycloserine-resistant sample No. 3, and linezolid-resistant sample No. 4 were selected for Sanger sequencing to accurately test the method of the present invention.

[0101] Example 5:

[0102] Sensitivity testing of second-line tuberculosis drug resistance detection using the detection method of the present invention

[0103] In this study, two tuberculosis-positive DNA samples obtained from Huzhou Central Hospital were selected for serial dilution. The starting DNA concentration was determined using a Qubit spectrophotometer. The starting DNA loading amounts were 0.5 ng / μl, 0.25 ng / μl, 0.1 ng / μl, 0.05 ng / μl, 0.025 μl, 0.01, and 0.001 ng / μl, respectively. Subsequent PCR, digestion, extension, and mass spectrometry were performed. The statistical results of the two samples are shown in the table below:

[0104]

[0105]

[0106]

[0107] It can be seen that when the DNA concentration is above 0.025 ng / ul, all sites can be detected; when the concentration is 0.01 ng / ul, only a few sites are detected, and when the concentration is 0.001 ng / ul, all sites are not detected. This indicates that when the sample concentration is not less than 0.025 ng / ul, all sites can be detected, that is, the minimum detection limit is 0.025 ng / ul, and the sensitivity is excellent.

[0108] Example 6:

[0109] The detection method of the present invention is used to test the minimum detection ratio of second-line tuberculosis drug resistance

[0110] The pure wild type (WT) sample and the fluoroquinolone resistant mutant (Mutant) sample measured in Example 4 were selected as control samples and quantified to 10 4 The samples were mixed in a gradient ratio, and the detection method of the present invention was used to perform the experimental operation according to Example 3. The statistical results are shown in the following table:

[0111]

[0112] Figure 4 The results are as follows: 100% wild type, 20% mutant, 5% mutant, and 100% mutant of gyrA94_2EV1. Conclusion: The detection method of the present invention can detect drug-resistant mutations with a frequency as low as 5%.

[0113] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A rapid detection primer combination based on the second-line drug-resistant mutation site of Mycobacterium tuberculosis, characterized in that: Including PCR amplification primer sets 1 and 2 and single base extension primer sets 1 and 2; The PCR amplification primer set 1 includes five first-group 1st-PCR primers and five first-group 2st-PCR primers, the nucleotide sequences of the first-group 1st-PCR primers are shown in SEQ ID NOs. 1 to 5, and the nucleotide sequences of the first-group 2st-PCR primers are shown in SEQ ID NOs. 15 to 19; The PCR amplification primer set 2 includes 9 second group 1st-PCR primers and 9 second group 2st-PCR primers, the nucleotide sequences of the second group 1st-PCR primers are shown in SEQ ID NOs. 6 to 14, and the nucleotide sequences of the second group 2st-PCR primers are shown in SEQ ID NOs. 20 to 28; The single-base extension primer set 1 includes 10 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 29 to 38; The single-base extension primer set 2 includes 13 extension primers, and the nucleotide sequences of the primers are shown in SEQ ID NOs. 39 to 51.

2. Use of the primer combination according to claim 1 in preparing a rapid detection product for second-line drug-resistant mutation sites in Mycobacterium tuberculosis, wherein the second-line drug resistance is fluoroquinolone, linezolid, cycloserine and clofazimine.

3. A kit comprising the primer combination according to claim 1.

4. The kit according to claim 3, wherein The kit is a kit for detecting second-line drug-resistant mutation sites of Mycobacterium tuberculosis, and the second-line drug resistance sites are fluoroquinolones, linezolid, cycloserine and clofazimine.

5. The kit according to claim 3, wherein It also contains reagents required for PCR amplification reaction, SAP reaction and / or single base extension reaction.

6. The kit according to claim 3, wherein Also included are the reagents required for time-of-flight mass spectrometry detection.

7. The kit according to claim 3, wherein Also included are the reagents needed to extract DNA from the sample.

8. The kit according to claim 5, wherein The reagents required for the PCR amplification reaction include a PCR reaction mixture and a PCR enzyme mixture.

9. The kit according to claim 5, wherein The reagents required for the SAP reaction include a SAP reaction mixture and a SAP enzyme mixture.

10. The kit according to claim 5, wherein The reagents required for the single base extension reaction include an extension reaction mixture and an extension enzyme mixture.

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