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CN-122012685-A - Multiplex primer combination and method for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug resistance genes thereof

CN122012685ACN 122012685 ACN122012685 ACN 122012685ACN-122012685-A

Abstract

The application relates to the technical field of detection, and provides a multiplex primer combination and a method for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug resistance genes thereof, wherein the nucleic acid sequence of the primer combination comprises sequences shown in SEQ ID NO. 1-SEQ ID NO. 380; the method comprises the steps of extracting nucleic acid, extracting DNA and RNA in a sample as templates, carrying out reverse transcription to convert the RNA into the DNA, carrying out multi-target amplification, configuring multi-PCR amplification, connecting joints, purifying a library, sequencing by an upper machine and analyzing data. The application has more comprehensive and accurate pathogen identification, contains 5 mycobacterium tuberculosis, 34 nontuberculous mycobacteria and 55 common respiratory tract pathogens, fully covers 2000 anti-tuberculosis first and second line drug resistance gene loci used clinically, can comprehensively detect all drug resistance gene loci recommended by WHO and guidelines, and has high sensitivity and high specificity.

Inventors

  • ZHOU YU
  • SU XIAOYU
  • GUO ZIWEI
  • XIONG CHANYU
  • CHEN GUANGJIONG
  • QIU BING

Assignees

  • 四川省医学科学院·四川省人民医院
  • 成都艾珂瑞科技有限公司

Dates

Publication Date
20260512
Application Date
20260313

Claims (9)

  1. 1. A multiplex primer combination for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug resistance genes thereof is characterized in that the nucleic acid sequence of the primer combination comprises sequences shown in SEQ ID NO. 1-SEQ ID NO. 380.
  2. 2. The multiplex primer combination for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug-resistant genes thereof according to claim 1, wherein the primer combination comprises 111 total primer pairs for identifying mycobacterium, the nucleic acid sequence of which is SEQ ID NO: 49-270, 55 total primer pairs for identifying respiratory tract pathogenic bacteria, the nucleic acid sequence of which is SEQ ID NO:271~SEQ ID NO:380, and 24 total primer pairs for detecting drug-resistant genes, and the nucleic acid sequence of which is SEQ ID NO: 1-48.
  3. 3. A method for the identification of mycobacteria and respiratory pathogens and detection of their drug resistance genes in an isolated sample for detecting a target nucleic acid in an isolated sample, comprising the steps of: Step S1, extracting nucleic acid, and extracting DNA and RNA in a sample as templates; Step S2, reverse transcription, namely reverse transcription of RNA into DNA; step S3, multiplex targeting amplification, wherein the multiplex targeting amplification and the multiplex primer combination of claim 1 or 2 are configured into multiplex PCR amplification; step S4, joint connection and library purification; and S5, sequencing and data analysis on the machine.
  4. 4. A method for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug resistance genes in an isolated sample according to claim 3 is characterized in that the specific process of the step S1 is that an equal volume of liquefying reagent is added into the sample, vortex vibration and mixing are carried out, standing is carried out for 10min at room temperature, the sample is fully liquefied, a full-automatic magnetic bead method is used for extracting nucleic acid, and then a Qubit fluorometer and a Qubit DNA detection kit matched with the Qubit fluorometer are used for measuring the DNA concentration of the nucleic acid.
  5. 5. A method for identifying Mycobacteria and respiratory pathogens and detecting drug-resistant genes thereof in an isolated sample according to claim 3, wherein in step S2, a reaction system is constructed, and each reaction amount is 5ul of sample nucleic acid, 2ul of dNTP mixture, 1ul of oligo (dT) 1ul,RNase Inhibitor, 1ul of reverse transcriptase, 10ul of nuclease-free water, and the reaction temperature is 42 ℃, the reaction time is 60min, the temperature is reduced to 4 ℃ and the subsequent use is waited.
  6. 6. A method for identifying Mycobacteria and respiratory pathogens and detecting their drug-resistance genes in an isolated sample according to claim 3, wherein step S3 comprises the steps of: step Q1, preparing a multiplex amplification system, wherein each reaction comprises 10 ul of PCR reaction liquid, 4 ul of primer mix, 1 ul of PCR enzyme and 5 ul of sample nucleic acid; The amplification reaction conditions are that a thermal cover is set to 105 ℃ and the volume is 20uL, the thermal cover is subjected to (1) 95 ℃ for 10min, (2) 95 ℃ for 30s, (3) 62 ℃ for 90s, (4) 70 ℃ for 90s, and the thermal cover is circularly subjected to (2) 30 times of (3) and (4), (5) 70 ℃ for 10min, and (6) the thermal cover is cooled to 4 ℃ and is waited for subsequent use; And step Q3, purifying the amplified product by using a nucleic acid purification kit.
  7. 7. The method for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting their drug resistance genes in an isolated sample according to claim 6, wherein the specific steps of step Q3 are as follows: 3.1 Purifying the magnetic beads and balancing at room temperature for 30min; 3.2 Vortex to make magnetic beads fully suspended, adding 75uL of purified magnetic beads into amplified products, and fully oscillating and uniformly mixing; 3.3 Incubating for 5min at room temperature to enable DNA to be fully combined on the magnetic beads, placing the reaction tube on a magnetic rack, and carefully sucking the supernatant by a pipette after the solution is clarified; 3.4 The PCR tube is always placed on a magnetic frame, 200uL of 80% ethanol is added into the reaction tube, the reaction tube is kept stand for 30 seconds, and a pipettor carefully absorbs and discards the supernatant; 3.5 Repeating the step 3.4 once; 3.6 Sucking out residual cleaning liquid by using a 10uL pipetting tip; 3.7 The reaction tube is always arranged on the magnetic frame, and is aired for 2min after being opened at an empty temperature; 3.8 Adding 35uL of eluent into the tube, shaking and uniformly mixing to suspend the magnetic beads, standing for 5min, instantly centrifuging, placing the reaction tube on a magnetic rack for 5min, and transferring 35uL of supernatant into a new PCR tube after the solution is clarified for subsequent experiments.
  8. 8. A method for identifying Mycobacteria and respiratory pathogens and detecting drug-resistant genes thereof in an isolated sample according to claim 3, wherein in the step S4, the reaction system of the linker ligation is 16 uL of the linker ligation buffer, 10 uL of ligase, 7 uL of NF water, 2 uL of 3 'linker and 2 uL of 5' linker, the library of the previous step is 45 uL, and the reaction temperature is 25 ℃ for 15min, and the temperature is reduced to 4 ℃.
  9. 9. A method for the identification of mycobacteria and respiratory pathogens and the detection of their drug resistance genes in an isolated sample according to claim 3, wherein in step S5, DNA sequence detection is performed using Ullumina NextSeq and a sequencing reaction universal kit.

Description

Multiplex primer combination and method for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug resistance genes thereof Technical Field The invention relates to the technical field of detection, in particular to a multiplex primer combination and a multiplex primer method for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug resistance genes thereof. Background Mycobacteria and respiratory pathogens are both relatively common pathogens. Existing methods for detecting mycobacteria mainly comprise traditional bacterial culture methods, microscopy methods and molecular biology methods. Bacterial culture is the gold standard for mycobacterial detection, but is complex to operate and takes a long time, often requiring weeks or even months to obtain results. Molecular biological methods, such as PCR melting curve methods and gene chip techniques, can provide highly sensitive and specific detection results in a shorter time, but still have some limitations. For example, a single PCR assay cannot identify multiple mycobacteria simultaneously and cannot provide information on the site of drug resistance. Genomic and transcriptomic studies have been greatly driven with the development of high throughput sequencing technologies. High throughput sequencing techniques are capable of deep sequencing a large number of DNA samples in a short period of time, thereby providing comprehensive genomic information. This provides new possibilities for pathogen identification and drug resistance detection. Current mainstream detection techniques: The traditional method, bacterial culture, has long time consumption (2-8 weeks for culture) and low sensitivity, and can not meet the clinical rapid diagnosis requirement. The fluorescent quantitative PCR/PCR melting curve method is characterized in that only a single target (such as a single strain or a single drug resistance gene) can be detected at one time, multiple times of detection are needed, and the operation is complicated and the cost is high. Gene-Xpert-Mycobacterium tuberculosis complex can be identified, but only a single drug, rifampin-resistance (rpoB Gene), is detected. PCR inverse dot hybridization-Gene chip-Mycobacterium tuberculosis complex can be identified, but only drug-resistant genes of several first-line drugs (rifampicin, isoniazid, etc.) commonly used can be detected. Disadvantages of current mainstream detection techniques: The traditional method, bacterial culture, has long time consumption (2-8 weeks for culture) and low sensitivity, and can not meet the clinical rapid diagnosis requirement. The fluorescent quantitative PCR/PCR melting curve method can detect only a single target (such as a single strain or a single drug resistance gene) at a time and needs to detect for multiple times. The common two-line drug resistance genes in clinic cannot be comprehensively detected. Gene-Xpert-Mycobacterium tuberculosis complex can be identified, but only a single drug, rifampin-resistance (rpoB Gene), is detected. The common two-line drug resistance genes in clinic cannot be comprehensively detected. PCR inverse dot hybridization-Gene chip-Mycobacterium tuberculosis complex can be identified, but only drug-resistant genes of several first-line drugs (rifampicin, isoniazid, etc.) commonly used can be detected. The common two-line drug resistance genes in clinic cannot be comprehensively detected. Disclosure of Invention The invention aims to develop a high-flux, high-sensitivity and low-cost detection technology which is used for identifying mycobacterium strains and respiratory tract pathogenic bacteria in an isolated sample and comprehensively screening drug-resistant genes, and provides detection information of the pathogenic and drug-resistant genes in the sample for clinic. In order to achieve the above objects, the present invention provides a multiplex primer combination and a method for identification of Mycobacteria and respiratory pathogens and detection of drug resistance genes thereof, to improve the above problems. The application is specifically as follows: The multiplex primer combination proposed in the first aspect is specifically: A multiplex primer combination for identifying mycobacterium and respiratory tract pathogenic bacteria and detecting drug resistance genes thereof, wherein the nucleic acid sequence of the primer combination comprises sequences shown in SEQ ID NO. 1-SEQ ID NO. 380. The primer combination comprises 111 total primer pairs for identifying mycobacteria, the nucleic acid sequence of the primer pair is SEQ ID NO. 49-SEQ ID NO. 270, 55 total primer pairs for identifying respiratory tract pathogenic bacteria, the nucleic acid sequence of the primer pair is SEQ ID NO. 271~SEQ ID NO:380, 24 total primer pairs for drug resistance genes, and the nucleic acid sequence of the primer pair is SEQ ID NO. 1-SEQ ID NO. 48. The second aspect proposes a specific detection