WO2024120270A1 - 检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法 - Google Patents

检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法 Download PDF

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WO2024120270A1
WO2024120270A1 PCT/CN2023/134919 CN2023134919W WO2024120270A1 WO 2024120270 A1 WO2024120270 A1 WO 2024120270A1 CN 2023134919 W CN2023134919 W CN 2023134919W WO 2024120270 A1 WO2024120270 A1 WO 2024120270A1
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mycobacterium tuberculosis
nucleic acid
primer
kit
tuberculous
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PCT/CN2023/134919
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French (fr)
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蒋析文
徐小解
蒋少龙
马晓晶
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广州达安基因股份有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the technical field of tuberculosis and non-tuberculous mycobacteria nucleic acid detection, and in particular to a primer probe set, a kit and a method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria.
  • Tuberculosis is a disease caused by the Mycobacterium tuberculosis complex (MTBC).
  • MTBC Mycobacterium tuberculosis complex
  • the spread of the disease seriously threatens human life and health, and is listed by the WHO as a major human disease similar to ADIS and malaria.
  • the Mycobacterium tuberculosis complex includes Mycobacterium tuberculosis (MTB), Mycobacterium bovis, Mycobacterium africanum, and Mycobacterium microti. Except for Mycobacterium microti, all of them can cause disease to humans.
  • Nontuberculous mycobacteria is a general term for a large class of mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae. It is an opportunistic pathogen that is widely present in the natural environment. Common nontuberculous mycobacteria in clinical practice include avian, intracellular, turtle, abscess, fortuitous, Kansas, toad, scrofula, Marseille and light yellow mycobacterium. The clinical manifestations caused by nontuberculous mycobacteria are similar to those of tuberculosis. In the absence of sterile species identification results, they are often misdiagnosed as tuberculosis.
  • the identification method of tuberculosis and non-tuberculosis mainly relies on culture method combined with p-nitrobenzoic acid selective test, followed by MPB64 antigen detection method, which is widely used.
  • MPB64 antigen detection method which is widely used.
  • time-of-flight mass spectrometry fluorescence PCR technology
  • PCR reverse dot hybridization method PCR direct sequencing method
  • Culture method combined with p-nitrobenzoic acid selective test The current traditional clinical isolation and identification method is to first culture to determine mycobacterial infection, and then use p-nitrobenzoic acid selective culture medium to distinguish NTM from MTB (most NTM can grow, but MTB cannot). This method is not only cumbersome and time-consuming, but also expensive.
  • MPB64 antigen detection MPB64 antigen is one of the main secretory proteins of Mycobacterium tuberculosis complex when it grows in liquid culture medium. When the mycobacterium culture is positive, if MPB64 antigen is detected in the culture filtrate, it is judged as Mycobacterium tuberculosis, otherwise it is presumed to be NTM. This method requires culture and is time-consuming; some bovine Mycobacterium tuberculosis subspecies do not secrete MPB64 antigen during culture, and the test results are false negatives. Mycobacterium marinum can secrete trace amounts of MPB64 antigen and the test results are weakly positive. It is specific. In addition, this method cannot detect samples with mixed tuberculosis and non-tuberculosis infections.
  • Time-of-flight mass spectrometry Each mycobacterium has different protein components. By analyzing the characteristic protein spectra obtained during vacuum ionization of these protein components with different mass/nuclear ratios, mycobacteria can be identified to the species level. This method has the advantages of high resolution and accuracy, but it has high requirements for sample purity, high instrumentation, and high cost.
  • Fluorescence PCR technology This method is fast and low-cost.
  • the only fluorescent PCR kit that has obtained a registration certificate for distinguishing tuberculosis and non-tuberculous mycobacteria is the Mycobacterium Nucleic Acid Detection Kit (PCR-fluorescent probe method) from Chengdu Bio Jingxin Biotechnology Co., Ltd.
  • this kit cannot identify mixed infections of tuberculosis and non-tuberculous mycobacteria.
  • PCR-reverse dot hybridization method The "Mycobacterium Species Identification Gene Detection Kit (PCR-reverse dot hybridization method)" produced by Yaneng Biotechnology (Shenzhen) Co., Ltd. is currently the only kit on the market in China that uses PCR-reverse dot hybridization to detect Mycobacterium tuberculosis and non-tuberculous mycobacteria. This kit can distinguish and detect Mycobacterium tuberculosis and 21 types of non-tuberculous mycobacteria through the complementary pairing effect between bases.
  • this method is relatively complicated to operate. After the amplification is completed, a lot of manual operations are required. It usually takes 5 hours to give the results. The cost is high and it is not conducive to widespread promotion.
  • Chip method The "Mycobacterium Species Identification Kit (DNA Microarray Chip Method)" produced by Chengdu Bio Jingxin Biotechnology Co., Ltd. is currently the only kit on the market in China that uses the DNA microarray chip method to detect Mycobacterium tuberculosis and non-tuberculous mycobacteria. This kit also uses the complementary pairing effect between bases to achieve the distinction between Mycobacterium tuberculosis and 17 types of non-tuberculous mycobacteria. After amplification, it also requires a lot of manual operations and takes 5 hours to give results. It is also expensive and not conducive to promotion.
  • Direct DNA sequencing of bacterial genome is to use PCR to amplify the gene to be tested, purify or clone the product, take the DNA fragment and directly sequence it, compare the measured sequence in the database, and then get the species with the closest relationship.
  • DNA sequencing has been used as the "gold standard" for bacterial nucleic acid detection.
  • the disadvantages are that the operation is complicated, time-consuming, costly, and prone to cross-contamination.
  • the current rapid and low-cost detection methods have low specificity and cannot detect mixed infections of tuberculosis and non-tuberculosis.
  • the detection methods that can distinguish and identify Mycobacterium tuberculosis from non-tuberculous mycobacteria are expensive or complicated to operate and cannot be widely promoted.
  • the purpose of the embodiments of the present application is to provide a primer probe set, a kit and a method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria, which have high specificity, can detect mixed infection of tuberculosis and non-tuberculosis, and can distinguish and identify Mycobacterium tuberculosis from non-tuberculous mycobacteria.
  • the present application provides a primer probe set for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria, which adopts the following technical solution:
  • a primer probe set for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria comprising:
  • An upstream primer targeting the conserved region of a Mycobacterium tuberculosis complex-specific gene the nucleic acid sequence of which is shown in SEQ ID NO: 1;
  • the downstream primers for the conserved region of the Mycobacterium tuberculosis complex-specific gene have a nucleic acid sequence such as As shown in SEQ ID NO: 2;
  • An upstream primer targeting a conserved region of a non-tuberculous mycobacterium-specific gene the nucleic acid sequence of which is shown in SEQ ID NO: 3;
  • a downstream primer targeting the conserved region of a non-tuberculous mycobacterium-specific gene the nucleic acid sequence of which is shown in SEQ ID NO: 4;
  • the upstream primer for the internal standard gene has a nucleic acid sequence as shown in SEQ ID NO: 5;
  • the downstream primer for the internal standard gene has a nucleic acid sequence as shown in SEQ ID NO: 6;
  • a probe targeting a conserved region of a Mycobacterium tuberculosis complex-specific gene the nucleic acid sequence of which is shown in SEQ ID NO: 7;
  • a probe targeting a conserved region of a non-tuberculous mycobacterium-specific gene the nucleic acid sequence of which is shown in SEQ ID NO: 8;
  • the nucleic acid sequence of the probe targeting the internal standard gene is shown in SEQ ID NO: 9.
  • the present application also provides a kit for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria, which adopts the following technical solution:
  • a kit for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria comprising MN PCR reaction solution A, wherein the MN PCR reaction solution A is prepared from a mixed solution of the primer probe set described in any one of claims 1 to 5.
  • the present application also provides a method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria using the above kit, which adopts the following technical solution:
  • a method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria comprising the following steps:
  • nucleic acid extraction operations on the pretreated sputum sample and the MN negative control product respectively to obtain nucleic acid of the sample to be tested and nucleic acid of the MN negative control product respectively;
  • the nucleic acid of the sample to be tested, the nucleic acid of the MN negative quality control product and the MN positive quality control product are added into different PCR reaction tubes respectively, and placed into the instrument reaction tank for PCR amplification operation to obtain the amplification result;
  • the amplification results are analyzed to obtain detection results.
  • the present application optimizes primers and probes for the conserved regions of Mycobacterium tuberculosis-specific genes and the conserved regions of non-tuberculous mycobacteria-specific genes.
  • the present application has high specificity, can detect mixed infection of tuberculosis and non-tuberculosis, can distinguish and identify Mycobacterium tuberculosis from non-tuberculous mycobacteria, is simple to operate, short in time, low in cost and low in pollution.
  • FIG1 is a flow chart of a method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria according to the present application;
  • FIG2 is a graph showing the PCR results of the tuberculosis detection (FAM) channel according to the present application.
  • FIG3 is a graph showing the PCR results for detecting the non-tuberculosis (Texas Red) channel according to the present application.
  • FIG4 is a graph showing the PCR results of a triple mixed sample according to the present application.
  • FIG5 is a graph showing a specific sample amplification curve according to the present application.
  • FIG6 is a diagram showing the detection effect of accuracy samples Z1 to Z3 according to the present application.
  • FIG7 is a diagram showing the detection effect of accuracy samples Z4 to Z18 according to the present application.
  • FIG8 is a diagram showing the detection effect of national reference products P1 to P15 according to the present application.
  • FIG9 is a diagram showing the detection effect of national reference products N1 to N10 according to the present application.
  • FIG10 is a graph showing amplification curves of sensitivity samples L1 to L3 according to the present application.
  • FIG11 is an amplification curve diagram of sensitivity samples L4 to L28 according to the present application.
  • FIG. 12 is a graph showing amplification curves of national reference products S1 to S4 according to the present application.
  • the present application provides a primer probe set, a kit and a method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria.
  • the present application is based on multiplex fluorescence PCR technology and can rapidly and qualitatively detect the Mycobacterium tuberculosis complex-specific gene conserved regions and non-tuberculous mycobacteria-specific gene conserved regions in human sputum samples of tuberculosis patients in vitro.
  • the present application provides a primer probe set for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria.
  • the primer probe group includes: an upstream primer for the conserved region of the specific gene of the Mycobacterium tuberculosis complex group, and its nucleic acid sequence is shown in SEQ ID NO.1; a downstream primer for the conserved region of the specific gene of the Mycobacterium tuberculosis complex group, and its nucleic acid sequence is shown in SEQ ID NO.2; an upstream primer for the conserved region of the specific gene of non-tuberculous mycobacteria, and its nucleic acid sequence is shown in SEQ ID NO.3; a downstream primer for the conserved region of the specific gene of non-tuberculous mycobacteria, and its nucleic acid sequence is shown in SEQ ID NO.4; an upstream primer for the internal standard gene (RHOG gene), and its nucleic acid sequence is shown in SEQ ID NO.5; a downstream primer for the internal standard gene (RHOG gene), and its nucleic acid sequence is shown in SEQ ID NO.6; a probe for the conserved region
  • nucleic acid sequence of the probe targeting the conserved region of the Mycobacterium tuberculosis complex-specific gene (SEQ ID NO: 7) is labeled with FAM at the 5’ end and BHQ1 at the 3’ end;
  • nucleic acid sequence of the probe targeting the conserved region of the non-tuberculosis mycobacterium-specific gene (SEQ ID NO: 8) is labeled with Texas Red at the 5’ end and BHQ2 at the 3’ end;
  • nucleic acid sequence of the probe targeting the internal standard gene SEQ ID NO: 9) is labeled with CY5 at the 5’ end and BHQ1 at the 3’ end.
  • the final concentration of the upstream primer for the conserved region of the Mycobacterium tuberculosis complex-specific gene is 0.6 ⁇ mol/L; the final concentration of the downstream primer for the conserved region of the Mycobacterium tuberculosis complex-specific gene is 0.6 ⁇ mol/L; the final concentration of the upstream primer for the conserved region of the non-tuberculous mycobacterium-specific gene is 0.6 ⁇ mol/L; the final concentration of the downstream primer for the conserved region of the non-tuberculous mycobacterium-specific gene is 0.6 ⁇ mol/L; the final concentration of the upstream primer for the internal standard gene is 0.3 ⁇ mol/L; the final concentration of the downstream primer for the internal standard gene is 0.3 ⁇ mol/L; the final concentration of the probe for the conserved region of the Mycobacterium tuberculosis complex-specific gene is 0.3 ⁇ mol/L; the final concentration of the probe for the conserved region of the non-tuberculous mycobacterium-specific gene is
  • the Cy5 channel (internal standard channel) may be negative due to the competition of the system.
  • the kit prepared by the above-mentioned specific primers and probes can quickly and qualitatively detect Mycobacterium tuberculosis and non-tuberculous mycobacteria based on the PCR platform, providing effective technical guidance for the auxiliary diagnosis of clinical differentiation and detection of Mycobacterium tuberculosis and non-tuberculous mycobacteria.
  • the present application also provides a kit for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria, including MN PCR reaction solution A, which is prepared by a mixture of the above-mentioned primer probe group.
  • the components for preparing the MN PCR reaction solution A also include Buffer and purified water.
  • the buffer of the MN PCR reaction solution A is 5 ⁇ Buffer, and the components are shown in Table 3:
  • the kit also includes MN PCR reaction solution B, MN negative quality control and MN positive quality control.
  • the MN PCR reaction solution B includes hot start Taq enzyme, UDG enzyme and dNTPs; wherein the dNTPs include dUTP, dATP, dGTP and dCTP.
  • the components of the MN negative quality control and the MN positive quality control are shown in Table 4:
  • MN negative quality control products include internal standard fragments
  • MN positive quality control products include Mycobacterium tuberculosis target fragments, nontuberculous mycobacteria target fragments and internal standard fragments.
  • the kit described in the present application is applicable to sputum samples of patients suspected of being infected with Mycobacterium tuberculosis and/or non-tuberculous mycobacteria.
  • test results of the pending samples can only be determined when the test results of the negative control show that there is no obvious amplification curve in the FAM and Texas Red detection pathways, the CY5 channel has an obvious amplification curve, and the Ct value is ⁇ 38; the test results of the positive control show that both the FAM and Texas Red detection pathways have obvious amplification curves, and the Ct value is ⁇ 38.
  • the relevant anti-pollution component uracil DNA glycosylase, i.e. UDG/UNG
  • UDG/UNG uracil DNA glycosylase
  • the present application also provides a method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria, as shown in FIG1 , which is a flow chart of the method for detecting Mycobacterium tuberculosis and non-tuberculous mycobacteria according to the present application, and the specific steps are as follows:
  • S2 performing nucleic acid extraction operations on the pretreated sputum sample and the MN negative control product, respectively, to obtain nucleic acid of the sample to be tested and nucleic acid of the MN negative control product;
  • the sample to be tested can be a sputum sample; extract the sputum sample into a 50mL centrifuge tube, add 4 times the volume of 4% NaOH to the sputum, shake well, and place it at room temperature for about 30 minutes to liquefy; take 0.5mL to a 1.5mL centrifuge tube, add 0.5mL 4% NaOH and place it at room temperature for 10 minutes to fully liquefy it (no obvious solid matter and no delamination when aspirated means complete liquefaction). Centrifuge at 13,000rpm for 5 minutes, discard the supernatant, collect the lower sediment for standby use, and obtain the pretreated sputum sample.
  • the nucleic acid extraction or purification reagent (Yuesui Medical No. 20181263) produced by Guangzhou Daan Gene Co., Ltd. is used to extract nucleic acid from the treated sample to obtain the nucleic acid of the sample.
  • the specific operation steps can be found in the instructions of the reagent.
  • the MN negative control product in this kit needs to participate in the extraction to obtain the nucleic acid of the MN negative control product.
  • the preparation of the PCR reaction system specifically includes: taking out MN PCR reaction solution A, MN PCR reaction solution B, MN negative quality control product, and MN positive quality control product from the kit, wherein MN PCR reaction solution A includes the above-mentioned primer probe set.
  • composition of the PCR reaction system is shown in Table 6.
  • the results are automatically saved after the reaction is completed.
  • adjust the Start value, End value and Threshold value of the Baseline (users can adjust according to actual conditions.
  • the Start value can be set between 3 and 15, and the End value can be set between 5 and 20.
  • the Threshold Value In the Log spectrum window, set the Threshold Value to 1/10 of the highest fluorescence value of the MN positive quality control product, so that the threshold line is in the exponential phase of the amplification curve, and the amplification curve of the negative quality control product is flat or below the threshold line). Click Analysis to automatically obtain the analysis results.
  • This kit uses one-step multiplex real-time fluorescent PCR technology, with the conserved gene region of the tuberculosis-specific mycobacterium and the conserved gene region of the non-tuberculosis mycobacterium as the target region, and designs specific primers and fluorescent probes to distinguish tuberculosis and non-tuberculosis mycobacteria through fluorescent PCR amplification.
  • Specific primers and probes combined with hot-start Taq enzyme and other components, form nucleic acid amplification reagents, use fluorescent PCR instrument for PCR amplification, and detect fluorescent signals to detect unknown samples.
  • the internal standard sequence is a conserved gene sequence of the human genome, which is used to monitor the nucleic acid extraction process and PCR amplification process, and can reduce the occurrence of false negative results.
  • the present application provides a method for rapid in vitro qualitative detection of nucleic acids of tuberculosis-like mycobacteria and non-tuberculosis-like mycobacteria in sputum samples of patients suspected of tuberculosis and non-tuberculosis mycobacteria, a primer probe set, and a kit including the primer probe set.
  • the specific primer probes are optimized, and the specific gene conserved regions of tuberculosis-like mycobacteria and non-tuberculosis-like mycobacteria are respectively screened for primers and probes.
  • Table 8 The beneficial effects of the present application compared with the prior art are shown in Table 8:
  • the only registered Mycobacterium nucleic acid detection kit in China that uses fluorescent qPCR technology is the "Mycobacterium Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method)" (National Medical Device Registration No. 20173401341) produced by Chengdu Bio Jingxin Biotechnology Co., Ltd.
  • this kit cannot detect mixed infections of tuberculosis and non-tuberculous mycobacteria. As the number of clinical cases of non-tuberculous mycobacterium infection increases, the number of cases of mixed infection is also increasing, and the clinical application of this reagent is gradually restricted.
  • the present application designs probes for the conserved regions of tuberculosis and non-tuberculosis mycobacteria-specific genes respectively, and can simultaneously achieve differentiated detection of tuberculosis and non-tuberculosis.
  • the test results of the present application can provide doctors with more accurate medication guidance and have a broader application scenario.
  • the present invention is simple to operate, time-saving, low-cost, and less polluting, and can distinguish and identify tuberculosis from non-tuberculosis.
  • the present kit is worthy of promotion and application.
  • Figure 2 is a graph showing the PCR results of the tuberculosis (FAM) channel detected by the present application.
  • Figure 3 is a graph showing the PCR results of the non-tuberculosis (Texas Red) channel detected by the present application. Note: At present, the verification of 25 non-tuberculosis mycobacterium-specific gene plasmids has been completed, including: bird, land, schrenck, Kansas, Asia, scrofula, Gordon, turtle, abscess, occasional, grass, monkey, toad, stomach, Malmo, intracellular, non-color-producing sea, ulcer, Surga, smegma, sea, light yellow, pig, hemophilus, and Marseilles Mycobacterium.
  • Figure 4 is a graph showing the PCR results of the triple mixed sample of the present application.
  • the tuberculosis and non-tuberculosis branched nucleic acid detection method based on multiplex fluorescence PCR technology of the present application is a simple and rapid PCR detection technology. Its main detection principle is that during PCR annealing, the Taqman probe specifically binds to the target sequence. During the extension stage, the Taqman probe is hydrolyzed by the Taq enzyme with 5'-3' exonuclease activity, thereby separating the fluorescent reporter group from the fluorescent quencher group and releasing Fluorescence signal, the accumulation of signal is positively correlated with the number of PCR products.
  • the change of fluorescence signal is detected by the instrument and displayed as an exponential amplification curve, and different targets are displayed in different wavelengths of fluorescence channels, so as to distinguish and detect multiple targets.
  • This kit designs primers and probes based on the conservative regions of the Mycobacterium tuberculosis complex group and non-tuberculous mycobacterium-specific genes. When selecting the conservative regions of non-tuberculous mycobacterium-specific genes, the Mycobacterium tuberculosis complex group and Mycobacterium leprae genes that are highly homologous to them are avoided.
  • tuberculosis When distinguishing tuberculosis from non-tuberculosis, there is no mutual interference, avoiding the occurrence of false positive results; at the same time, human housekeeping genes are selected as internal reference genes, which can monitor the collection of test samples and the process of nucleic acid extraction, avoiding the occurrence of false negative results.
  • Three target probes are labeled with fluorescent dyes of different wavelengths to detect three target DNAs in one tube reaction.
  • This kit is used to detect samples numbered T1 to T14 and N11 to N15 in the "National Reference for Mycobacterium tuberculosis PCR Detection Kit".
  • the nucleic acid extraction or purification reagent produced by Guangzhou Daan Gene (Guangzhou Medical Equipment No. 20181263) is used to extract nucleic acid from the above samples and MN negative quality control products.
  • Guangzhou Daan Gene Guangzhou Medical Equipment No. 20181263
  • FIG5 is a graph showing a specific sample amplification curve of the present application.
  • This kit is used to test the accuracy of samples Z1 to Z18 and samples numbered P1 to P15 and N1 to N10 in the "National Reference for Mycobacterium tuberculosis PCR Detection Kit".
  • the nucleic acid extraction or purification reagent produced by Guangzhou Daan Gene Co., Ltd. (Guangzhou Suixibei 20181263) is used to extract nucleic acid from the above samples and MN negative quality control products.
  • Guangzhou Daan Gene Co., Ltd. Guangzhou Suixibei 20181263
  • Figure 6 is a diagram of the detection effect of the accuracy samples Z1 to Z3 of the present application
  • Figure 7 is a diagram of the detection effect of the accuracy samples Z4 to Z18 of the present application.
  • Detection effect diagram Figure 8 is the detection effect diagram of the present application for national reference products P1 ⁇ P15
  • Figure 9 is the detection effect diagram of the present application for national reference products N1 ⁇ N10.
  • This kit is used to detect the sensitivity samples L1 to L28 "National Reference Materials for Mycobacterium tuberculosis PCR Detection Kit” S1 to S4 samples.
  • the nucleic acid extraction or purification reagents produced by Guangzhou Daan Gene Co., Ltd. (Guangzhou Suixibei 20181263) are used to extract nucleic acid from the above samples and MN negative quality control products.
  • the reagent instructions Take 10 ⁇ L of each extracted nucleic acid sample and add it to the PCR reaction tube to which the above PCR reaction solution has been added, so that the total volume of each PCR reaction solution is 30 ⁇ L; cover the eight-tube cap tightly, mix thoroughly, and centrifuge at high speed for 10 seconds.
  • Figure 10 is an amplification curve diagram of sensitivity samples L1 to L3 according to the present application
  • Figure 11 is an amplification curve diagram of sensitivity samples L4 to L28 according to the present application
  • Figure 12 is an amplification curve diagram of national reference products S1 to S4 according to the present application.
  • This kit was used to detect 50 clinical positive samples and 20 negative samples, and the sequencing method was used for verification.
  • the amplified PCR products were directly sequenced by the standard Sanger DNA sequencing method (Shanghai Biotechnology Co., Ltd.), and the consistency of the two methods was analyzed.
  • the nucleic acid extraction or purification reagent produced by Guangzhou Daan Gene Co., Ltd. (Guangzhou Medical Equipment No. 20181263) was used to extract nucleic acids from the above 70 clinical samples and MN negative quality control products. The specific operation steps are detailed in the reagent instructions.
  • the specificity of the kit is good, the accuracy is 100%, the sensitivity of Mycobacterium tuberculosis detection is 1 ⁇ 10 2 bacteria/mL, the sensitivity of non-tuberculous mycobacteria detection is 1 ⁇ 10 3 bacteria/mL, and the consistency rate with the sequencing method when detecting clinical samples is 100%, indicating that the detection performance of the kit of this application meets the requirements.
  • the present application designs several pairs of primers and several probes for the conserved regions of Mycobacterium tuberculosis complex-specific genes and the conserved regions of non-tuberculous mycobacteria-specific genes, hoping to obtain primer sets and detection probes with good amplification effect, high sensitivity and high accuracy.
  • primer-probe combinations Due to differences in primer specificity, inconsistent annealing temperatures, and primer dimers, different primer-probe combinations have a significant impact on the sensitivity of reagent detection, and it is difficult to obtain better PCR amplification primers and probe sequences.
  • control primer pair is used for detection
  • nucleotide sequence is shown in Table 16, and the other detection steps and conditions are the same as those in the above embodiment:
  • the sensitivity test was performed according to the method in "VI. Sensitivity test of the kit of this application" above. The test results showed that the control primers and probes had poor amplification curves for low-concentration nucleic acid samples.

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Abstract

属于结核与非结核分枝杆菌核酸检测技术领域，涉及检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法，引物探针组包括:针对结核分枝杆菌复合群特异性基因保守区的上游引物；针对结核分枝杆菌复合群特异性基因保守区的下游引物；针对非结核分枝杆菌特异性基因保守区的上游引物；针对非结核分枝杆菌特异性基因保守区的下游引物；针对内标基因的上游引物；针对内标基因的下游引物；针对结核分枝杆菌复合群特异性基因保守区的探针；针对非结核分枝杆菌特异性基因保守区的探针；针对内标基因的探针，特异性高，能够检出结核与非结核混合感染且能够区分鉴定结核分枝杆菌与非结核分枝杆菌。

Description

检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法
本申请要求于2023年3月24日提交中国专利局、申请号为202310304020.7，发明名称为“检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法”以及于2022年12月6日提交中国专利局、申请号为202211559443.5，发明名称为“检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及结核与非结核分枝杆菌核酸检测技术领域，尤其涉及检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法。
背景技术
结核病是由于结核分枝杆菌复合群(Mycobacterium tuberculosis complex，MTBC)引起的疾病，该病的传播严重威胁人类的生命健康，被WHO列为与ADIS、疟疾同类的人类重大疾病。结核分枝杆菌复合群包括结核分枝杆菌(MTB)、牛结核分枝杆菌、非洲分枝杆菌和田鼠分枝杆菌，除田鼠分枝杆菌外均可对人致病。
非结核分枝杆菌(Nontuberculous Mycobacterium，NTM)是结核分枝杆菌复合群和麻风分枝杆菌以外的一大类分枝杆菌的总称，是机会性致病菌，广泛存在于自然环境中。临床上常见非结核分枝杆菌包括鸟、胞内、龟、脓肿、偶然、堪萨斯、蟾蜍、瘰疬、马赛以及浅黄分枝杆菌等。非结核分枝杆菌引起的临床表现与结核病相似，在无菌种鉴定结果的情况下，经常被误诊为结核病，且由于其形态与抗酸性与结核分枝杆菌极为相似、临床上极难分辨，传统的先培养确定分枝杆菌感染后再进行对硝基苯甲酸选择性培养基来区分NTM与MTB(绝大多数NTM可以生长，而MTB则不能生长)的分离鉴定方法，不仅耗时长，而且价格昂贵。多数NTM对抗结核药物耐药，导致一般临床治疗结核病和NTM病的用药方案存在较大差异，若发生结核与非结核混合感染，治疗方式则更加复杂。快速检测区分结核病和非结核分枝杆菌感染具有重要的临床意义，能够让病人获得及时、有效的药物治疗，降低发病率和死亡率。
目前，对结核与非结核的鉴定方法主要以培养法结合对硝基苯甲酸选择性试验为主，其次MPB64抗原检测法应用较为广泛。随着分子生物学技术的发展，越来越多的技术用于结核与非结核分枝杆菌的诊断，包括飞行时间质谱、荧光PCR技术、PCR反向点杂交法、PCR直接测序法等，但是目前这些技术或者产品在应用上均存在缺陷。
1、培养法结合对硝基苯甲酸选择性试验：目前临床传统的分离鉴定方法，先培养确定分枝杆菌感染后，再使用对硝基苯甲酸选择性培养基来区分NTM与MTB(绝大多数NTM可以生长，而MTB则不能生长)，该方法不仅操作繁琐、耗时长，而且价格昂贵。
2、MPB64抗原检测：MPB64抗原是结核分枝杆菌复合群在液体培养基中生长时主要的分泌蛋白之一，当分枝杆菌培养阳性时，培养滤液中检测到MPB64抗原则判定为结核分枝杆菌，否则推定为NTM。此方法需要培养，耗时较长；一些牛结核分枝杆菌亚种由于在培养时不分泌MPB64抗原而检测结果为假阴性，海分枝杆菌可分泌微量的MPB64抗原而检测结果呈弱阳性，特异 性稍差。另外，该方法无法检出结核与非结核混合感染的样本。
3、飞行时间质谱：每种分枝杆菌内部的蛋白成分不同，通过分析这些不同质/核比的蛋白成分在真空电离过程中获得的特征性的蛋白谱，可以鉴别分枝杆菌至种水平。该方法具有分辨率高、准确的优点，但是该方法对样本的纯净度要求较高，仪器要求高，成本昂贵。
4、荧光PCR技术：该方法检测快速、成本较低，目前已经获注册证的能够对结核和非结核分枝杆菌进行区分的荧光PCR试剂盒，仅成都博奥晶芯生物科技有限公司的分枝杆菌核酸检测试剂盒(PCR-荧光探针法)，但是该试剂盒无法对结核和非结核分枝杆菌混合感染进行鉴定。
5、PCR-反向点杂交法：亚能生物技术(深圳)有限公司生产的“分枝杆菌菌种鉴定基因检测试剂盒(PCR-反向点杂交法)”是目前国内唯一上市的利用PCR-反向点杂交法检测结核分枝杆菌和非结核分枝杆菌的试剂盒，该试剂盒通过碱基之间的互补配对作用，实现对结核分枝杆菌和21种非结核分枝杆菌的区分检测，但是该方法操作较为复杂，在扩增完成后，需要大量的手工操作，一般需要5h才能够给出结果，成本较高，不利于广泛推广。
6、芯片法：成都博奥晶芯生物科技有限公司生产的“分枝杆菌菌种鉴定试剂盒(DNA微阵列芯片法)”是目前国内唯一上市的利用DNA微阵列芯片法检测结核分枝杆菌和非结核分枝杆菌的试剂盒，该试剂盒是也是利用碱基之间的互补配对作用，实现对结核分枝杆菌和17种非结核分枝杆菌的区分检测，在扩增完成后，同样的需要大量的手工操作，需要5h才能够给出结果，且成本昂贵，不利于推广。
7、熔解曲线法：厦门致善生物科技股份有限公司生产的“分枝杆菌鉴定试剂盒(荧光PCR熔解曲线法)”通过荧光通道与熔解温度的二维标记，能够鉴定19种分枝杆菌，但是该试剂盒需要先扩增再进行熔解曲线分析，耗时也较长，且单管中成分较多成本昂贵，不利于大规模推广。
8、DNA直接测序法：细菌基因组DNA直接测序法(Direct Sequencing,DS)是利用PCR扩增待测基因，产物纯化或克隆化，取其DNA片段直接测序，将测得序列在数据库中进行比对，即可得到其与亲缘关系最为相近的物种，目前已把DNA测序作为细菌核酸检测的“金标准”。但是缺点是操作复杂、耗时长、成本高，且容易发生交叉污染。
综上所述，目前快速且成本低的检测方法特异性不高，不能够检出结核与非结核混合感染，能够区分鉴定结核分枝杆菌与非结核分枝杆菌的检测方法却价格昂贵或操作复杂，无法大范围推广。
发明内容
本申请实施例的目的在于提出一种检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法，特异性高，能够检出结核与非结核混合感染且能够区分鉴定结核分枝杆菌与非结核分枝杆菌。
为了解决上述技术问题，本申请实施例提供一种检测结核分枝杆菌与非结核分枝杆菌的引物探针组，采用了如下所述的技术方案：
一种检测结核分枝杆菌与非结核分枝杆菌的引物探针组，所述引物探针组包括:
针对结核分枝杆菌复合群特异性基因保守区的上游引物，其核酸序列如SEQ ID NO：1所示；
针对结核分枝杆菌复合群特异性基因保守区的下游引物，其核酸序列如 SEQ ID NO：2所示；
针对非结核分枝杆菌特异性基因保守区的上游引物，其核酸序列如SEQ ID NO：3所示；
针对非结核分枝杆菌特异性基因保守区的下游引物，其核酸序列如SEQ ID NO：4所示；
针对内标基因的上游引物，其核酸序列如SEQ ID NO：5所示；
针对内标基因的下游引物，其核酸序列如SEQ ID NO：6所示；
针对结核分枝杆菌复合群特异性基因保守区的探针，其核酸序列如SEQ ID NO：7所示；
针对非结核分枝杆菌特异性基因保守区的探针，其核酸序列如SEQ ID NO：8所示；
针对内标基因的探针，其核酸序列如SEQ ID NO：9所示。
为了解决上述技术问题，本申请实施例还提供一种检测结核分枝杆菌与非结核分枝杆菌的试剂盒，采用了如下所述的技术方案：
一种检测结核分枝杆菌与非结核分枝杆菌的试剂盒，包括MN PCR反应液A，所述MN PCR反应液A由权利要求1至5任意一项所述引物探针组的混合液配制得到。
为了解决上述技术问题，本申请实施例还提供一种应用如上所述的试剂盒检测结核分枝杆菌与非结核分枝杆菌的方法，采用了如下所述的技术方案：
一种检测结核分枝杆菌与非结核分枝杆菌的方法，包括如下步骤：
抽取痰液样本至离心管中，按4倍的体积加入4％NaOH，摇匀后室温放置至液化，取0.5mL液化后的痰液样本至离心管中，加入0.5mL 4％NaOH室温放置至液化完全，离心，保留沉淀，获得预处理后的痰液样本；
分别对所述预处理后的痰液样本和MN阴性质控品进行核酸提取操作，分别获得待测样本核酸和MN阴性质控品的核酸；
混合所述试剂盒中的MN PCR反应液A和MN PCR反应液B，获得PCR反应液，并将所述PCR反应液分装至PCR反应管中；
将所述待测样本核酸、MN阴性质控品的核酸和MN阳性质控品分别加入不同的所述PCR反应管中，放入仪器反应槽内进行PCR扩增操作，获得扩增结果；
分析所述扩增结果，得到检测结果。
与现有技术相比，本申请实施例主要有以下有益效果：
本申请优化了针对结核分枝杆菌类特异性基因保守区以及非结核分枝杆菌类特异性基因保守区的引物和探针，本申请特异性高、能够检出结核与非结核混合感染且能够区分鉴定结核分枝杆菌与非结核分枝杆菌、操作简单、耗时短、成本低污染小。
附图说明
为了更清楚地说明本申请中的方案，下面将对本申请实施例描述中所需要使用的附图作一个简单介绍，显而易见地，下面描述中的附图是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1是根据本申请的检测结核分枝杆菌与非结核分枝杆菌的方法的流程图；
图2是根据本申请的检测结核(FAM)通道的PCR结果图；
图3是根据本申请的检测非结核(Texas Red)通道的PCR结果图；
图4是根据本申请的三重混合样本的PCR结果图；
图5是根据本申请的特异性样本扩增曲线图；
图6是根据本申请的对准确性样本Z1～Z3的检出效果图；
图7是根据本申请的对准确性样本Z4～Z18的检出效果图；
图8是根据本申请对国家参考品P1～P15的检出效果图；
图9是根据本申请对国家参考品N1～N10的检出效果图；
图10是根据本申请的灵敏度样本L1～L3扩增曲线图；
图11是根据本申请的灵敏度样本L4～L28扩增曲线图；
图12是根据本申请的国家参考品S1～S4扩增曲线图。
具体实施方式
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同；本文中在申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请；本申请的说明书和权利要求书及上述附图说明中的术语“包括”和“具有”以及它们的任何变形，意图在于覆盖不排他的包含。本申请的说明书和权利要求书或上述附图中的术语“第一”、“第二”等是用于区别不同对象，而不是用于描述特定顺序。
在本文中提及“实施例”意味着，结合实施例描述的特定特征、结构或特性可以包含在本申请的至少一个实施例中。在说明书中的各个位置出现该短语并不一定均是指相同的实施例，也不是与其它实施例互斥的独立的或备选的实施例。本领域技术人员显式地和隐式地理解的是，本文所描述的实施例可以与其它实施例相结合。
以下的实施例便于更好地理解本申请，但并不限定本申请。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的实验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。
为了使本技术领域的人员更好地理解本申请方案，下面将结合附图，对本申请实施例中的技术方案进行清楚、完整地描述。
本申请提供一种检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法。本申请基于多重荧光PCR技术，能够体外快速定性检测结核病患者的人痰液样本中的结核分枝杆菌复合群特异性基因保守区与非结核分枝杆菌特异性基因保守区。
一、本申请提供一种检测结核分枝杆菌与非结核分枝杆菌的引物探针组。
所述引物探针组包括:针对结核分枝杆菌复合群特异性基因保守区的上游引物，其核酸序列如SEQ ID NO.1所示；针对结核分枝杆菌复合群特异性基因保守区的下游引物，其核酸序列如SEQ ID NO.2所示；针对非结核分枝杆菌特异性基因保守区的上游引物，其核酸序列如SEQ ID NO.3所示；针对非结核分枝杆菌特异性基因保守区的下游引物，其核酸序列如SEQ ID NO.4所示；针对内标基因(RHOG基因)的上游引物，其核酸序列如SEQ ID NO.5所示；针对内标基因(RHOG基因)的下游引物，其核酸序列如SEQ ID NO.6所示；针对结核分枝杆菌复合群特异性基因保守区的探针，其核酸序列如SEQ ID NO.7所示；针对非结核分枝杆菌特异性基因保守区的探针，其核酸序列如SEQ ID NO.8所示；针对内标基因(RHOG基因)的探针，其核酸序列如SEQ ID NO.9所示。具体如表1所示：
表1检测引物和探针序列
其中，所述针对结核分枝杆菌复合群特异性基因保守区的探针的核酸序列(SEQ ID NO：7)的5’端标记有FAM，3’端标记有BHQ1；所述针对非结核分枝杆菌特异性基因保守区的探针(SEQ ID NO：8)的核酸序列的5’端标记有Texas Red，3’端标记有BHQ2；所述针对内标基因的探针(SEQ ID NO：9)的核酸序列的5’端标记有CY5，3’端标记有BHQ1。
各引物和探针在反应中的终浓度如下：
所述针对结核分枝杆菌复合群特异性基因保守区的上游引物的终浓度为0.6μmol/L；所述针对结核分枝杆菌复合群特异性基因保守区的下游引物的终浓度为0.6μmol/L；所述针对非结核分枝杆菌特异性基因保守区的上游引物的终浓度为0.6μmol/L；所述针对非结核分枝杆菌特异性基因保守区的下游引物的终浓度为0.6μmol/L；所述针对内标基因的上游引物的终浓度为0.3μmol/L；所述针对内标基因的下游引物的终浓度为0.3μmol/L；所述针对结核分枝杆菌复合群特异性基因保守区的探针的终浓度为0.3μmol/L；所述针对非结核分枝杆菌特异性基因保守区的探针的终浓度为0.3μmol/L；所述针对内标基因的探针的终浓度为0.2μmol/L。
上述PCR引物和标有不同类型荧光基团的探针可分别用于对结核分枝杆菌复合群、非结核分枝杆菌特异性基因保守区以及内标基因的检测。在实际检测过程中结果判定如表2所示：
表2实际检测结果判定

FAM和VIC检测通道为阳性时，由于体系的竞争关系，Cy5通道(内标通道)结果可能为阴性。上述特异性引物和探针所制备的试剂盒，基于PCR平台可快速定性检测结核分枝杆菌、非结核分枝杆菌，为临床区分检测结核分枝杆菌和非结核分枝杆菌的辅助诊断提供有效的技术指导。
二、本申请还提供一种检测结核分枝杆菌与非结核分枝杆菌的试剂盒，包括MN PCR反应液A，该MN PCR反应液A由上述的引物探针组的混合液配制得到。
配制所述MN PCR反应液A的组分还包括Buffer和纯化水。
所述MN PCR反应液A的Buffer为5×Buffer，成分如表3所示：
表3 5×Buffer
所述试剂盒还包括MN PCR反应液B、MN阴性质控品和MN阳性质控品，所述MN PCR反应液B包括热启动Taq酶、UDG酶和dNTPs；其中，所述dNTPs包括dUTP、dATP、dGTP和dCTP。MN阴性质控品和MN阳性质控品包括成分如表4所示：
表4 MN阴性质控品和MN阳性质控品

MN阴性质控品包括内标片段，MN阳性质控品包括结核分枝杆菌目的片段、非结核分枝杆菌目的片段以及内标片段。
本申请所述试剂盒适用样本为疑似感染结核分枝杆菌和/或非结核分枝杆菌患者的痰液样本。
本申请所述试剂盒用于判定检测有效性的标准为：每次实验均需检测阴性质控品和阳性质控品，当阴性质控品检测结果为FAM、Texas Red两个检测通路无明显扩增曲线，CY5通道有明显扩增曲线，且Ct值≤38；阳性质控品检测结果为FAM、Texas Red两个检测通路均有明显扩增曲线，且Ct值≤38时，方可进行待见样本检测结果的判定。
本申请的分型检测结核分枝杆菌和非结核分枝杆菌试剂盒的组成成分、包装及数量(以24人份/盒为例)，如表5所示：
表5试剂盒的组成成分、包装及数量
本申请试剂盒中添加了相关的防污染组分(尿嘧啶DNA糖基化酶，即UDG/UNG)。其作用机理是选择性水解断裂含有dU的双链或者单链DNA中的尿嘧啶糖苷键，形成的有缺失碱基的DNA链，在碱基介质以及高温下会进一步水解断裂，从而消除产物污染。
三、本申请还提供一种检测结核分枝杆菌与非结核分枝杆菌的方法，如图1所示，图1为根据本申请的检测结核分枝杆菌与非结核分枝杆菌的方法的流程图，具体步骤如下：
S1：抽取痰液样本至离心管中，按4倍的体积加入4％NaOH，摇匀后室温放置至液化，取0.5mL液化后的痰液样本至离心管中，加入0.5mL 4％NaOH室温放置至液化完全，离心，保留沉淀，获得预处理后的痰液样本；
S2：分别对所述预处理后的痰液样本和MN阴性质控品进行核酸提取操作，分别获得待测样本核酸和MN阴性质控品的核酸；
S3：混合所述试剂盒中的MN PCR反应液A和MN PCR反应液B，获得PCR反应液，并将所述PCR反应液分装至PCR反应管中；
S4：将所述待测样本核酸、MN阴性质控品的核酸和MN阳性质控品分别加入不同的所述PCR反应管中，放入仪器反应槽内进行PCR扩增操作，获得扩增结果；
S5：分析所述扩增结果，得到检测结果。
具体步骤如下：
1.处理待测样本
所述待测样本可为痰液样本；抽取痰液样本至50mL离心管中，痰液中加入4倍体积的4％NaOH，摇匀，室温下放置30分钟左右液化；取0.5mL至1.5mL离心管中，再加入0.5mL 4％NaOH室温放置10分钟，使其充分液化(无明显固状物并且吸出时无脱丝现象即为液化完全)。13,000rpm离心5分钟，弃上清，收集下层沉淀待用，获得预处理后的痰液样本。
2.核酸提取
采用广州达安基因股份有限公司生产的核酸提取或纯化试剂(粤穗械备20181263号)对处理后的待测样本进行核酸提取，获得待测样本核酸，具体的操作步骤可详见该试剂的说明书，本试剂盒中的MN阴性质控品需参与提取，获得MN阴性质控品的核酸。
3.PCR反应体系制备
配制PCR反应体系具体包括：取出试剂盒中的MN PCR反应液A、MN PCR反应液B、MN阴性质控品、MN阳性质控品，其中，MN PCR反应液A包括上述的引物探针组。
室温融化上述试剂，将MN PCR反应液A和MN PCR反应液B混匀后离心10秒，分装至八连管的PCR反应管中。向不同的所述PCR反应管中加入步骤2中获得的所述待测样本核酸、MN阴性质控品的核酸和MN阳性质控品各10μL，得到PCR反应体系。
具体的：取步骤2中获得的所述待测样本核酸各10μL、MN阴性质控品的核酸10μL、MN阳性质控品10μL，分别加入至所述PCR反应管中，使得每个PCR反应体系的总体积为30μL；盖紧八连管管盖，充分混匀，高速离心10秒。
PCR反应体系构成如表6所示。
表6 PCR反应体系
4、PCR扩增
1)将所述PCR反应体系放入仪器反应槽内，并记录放置顺序。
2)荧光通道选择：
选择FAM通道检测结核分枝杆菌特异性基因保守区；选择Texas Red通道检测非结核分枝杆菌特异性基因保守区；选择Cy5通道检测内标通道；选择参比荧光(Passive Reference)为None(ABI7500需)。设置循环条件如表7所示，反应体系体积设置为30μL。设置完成后，保存文件，运行程序。
表7循环条件
5.结果分析
反应结束后自动保存结果，根据分析后图像调节Baseline的Start值、End值以及Threshold值(用户可根据实际情况自行调整，Start值可以在3～15、End值可设在5～20，在Log图谱窗口设置Threshold的Value值设置为MN阳性质控品最高荧光值的1/10，使阈值线位于扩增曲线指数期，阴性质控品的扩增曲线平直或低于阈值线)，点击Analysis自动获得分析结果。
6.本申请的PCR检测原理如下：
本试剂盒利用一步法多重实时荧光PCR技术，分别以待检结核类分枝杆菌特异性基因保守区与非结核类分枝杆菌特异性基因保守区为靶区域，设计特异性引物及荧光探针，通过荧光PCR扩增以区分结核分枝杆菌和非结核分枝杆菌。特异性的引物和探针，配以热启动Taq酶等成分组成核酸扩增试剂，使用荧光PCR仪进行PCR扩增，并检测荧光信号，实现对未知样本的检测。内标序列为人类基因组保守基因序列，用于对核酸提取过程及PCR扩增过程的监控，可减少假阴性结果的出现。
与现有技术相比，本申请的有益效果在于：
本申请提供了一种体外快速定性检测疑似结核病和非结核分枝杆菌病患者痰液样本中结核类分枝杆菌与非结核类分枝杆菌的核酸的方法、引物探针组及包括该引物探针组的试剂盒。优化了特异性引物探针，分别针对结核分枝杆菌类特异性基因保守区以及非结核分枝杆菌类特异性基因保守区引探进行筛选。本申请与现有技术相比有益效果如表8所示：
表8本申请与现有技术相比

目前利用荧光qPCR技术、国内唯一获注册证的分枝杆菌核酸检测试剂盒为成都博奥晶芯生物科技有限公司的“分枝杆菌核酸检测试剂盒(PCR-荧光探针法)”(国械注准20173401341)，但是该试剂盒无法检出结核和非结核分枝杆菌的混合感染，随着非结核分枝杆菌感染的临床病例越来越多，混合感染的病例也在增加，该试剂的临床应用也逐渐受到限制。
本申请与成都博奥晶芯生物科技有限公司的分枝杆菌核酸检测试剂盒(PCR-荧光探针法)相比：本申请分别针对结核和非结核分枝杆菌特异性基因保守区设计引探，能够同时对于结核与非结核实现区分检测，在结核非结核混合感染病例日益增加的今天，本申请的测试结果能够更加准确地为医生提供用药指导，有着更加广阔的应用情景。
总体上来说，本申请操作简单、耗时短、成本低、污染小，且能够区分鉴定结核与非结核。在非结核分枝杆菌日渐增多的大背景下，本试剂盒值得推广应用。
图2是本申请检测结核(FAM)通道的PCR结果图。图3是本申请检测非结核(Texas Red)通道的PCR结果图。注：目前已完成对于25种非结核分枝杆菌特异性基因质粒的验证，其中包括：鸟、土地、施氏、堪萨斯、亚洲、瘰疬、戈登、龟、脓肿、偶发、草、猿猴、蟾蜍、胃、玛尔摩、胞内、不产色海、溃疡、苏尔加、耻垢、海、浅黄、猪、嗜血、马赛分枝杆菌。图4是本申请三重混合样本的PCR结果图。
本申请的基于多重荧光PCR技术的结核和非结核分枝核酸检测方法是一种简单快速的PCR检测技术，其主要检测原理是在PCR退火时，Taqman探针特异性地结合到靶序列上，在延伸阶段时Taqman探针会被具有5’-3’外切酶活性的Taq酶水解，从而使得荧光报告基团与荧光淬灭基团分离，并释放 荧光信号，信号的积累与PCR产物的数量成正相关，荧光信号的变化被仪器检测，并以指数扩增曲线展现出来，而不同靶标间以不同的波长的荧光通道展现，以此来对多个靶标进行区分检测。本试剂盒根据结核分枝杆菌复合群与非结核分枝杆菌特异性基因的保守区进行引物和探针的设计，其中非结核分枝杆菌特异性基因在挑选保守区时，避开了与其高度同源的结核分枝杆菌复核群以及麻风分枝杆菌基因，在对结核与非结核进行区分检测时，不存在相互之间的干扰，避免了假阳性结果的出现；同时选择人管家基因作为内参基因，可对于检测样本的采集及核酸提取过程进行监控，避免了假阴性结果的出现。以不同波长的荧光素标记三种靶标探针，实现一管反应检测三种靶标DNA。
四、本申请的试剂盒特异性检测
用本试剂盒检测编号为T1～T14和“结核分枝杆菌PCR检测试剂盒用国家参考品”中编号为N11～N15的样本，采用广州达安基因生产的核酸提取或纯化试剂(粤穗械备20181263号)对以上样本和MN阴性质控品进行核酸提取，具体的操作步骤详见试剂说明书。取提取好的样本核酸、MN阴性质控品的核酸和MN阳性质控品各10μL，加入至已添加上述PCR反应液的PCR反应管中，使每管PCR反应液的总体积为30μL；盖紧八连管管盖，充分混匀，高速离心10秒。
将PCR反应管放入仪器反应槽内，并记录放置顺序。选择FAM通道检测结核分枝杆菌特异性基因保守区；选择Texas Red通道检测非结核分枝杆菌特异性基因保守区；选择Cy5通道检测内标通道；选择参比荧光(Passive Reference)为None(ABI7500需)。设置循环条件如下表所示，反应体系体积设置为30μL，设置完成后，保存文件，运行程序。
检测结果如图5和表9和表10所示，图5为本申请的特异性样本扩增曲线图。
表9特异性样本T1～T14检测结果

表10国家参考品N11～N15检测结果
注：“+”表示检测结果阳性，“-”表示检测结果阴性。
五、本申请试剂盒准确性检测
用本试剂盒检测准确性样本Z1～Z18和“结核分枝杆菌PCR检测试剂盒用国家参考品”中编号为P1～P15和N1～N10的样本，采用广州达安基因股份有限公司生产的核酸提取或纯化试剂(粤穗械备20181263号)对以上样本和MN阴性质控品进行核酸提取，具体的操作步骤详见试剂说明书。取提取好的核酸样本各10μL，加入至已添加上述PCR反应液的PCR反应管中，使每管PCR反应液的总体积为30μL；盖紧八连管管盖，充分混匀，高速离心10秒。
将PCR反应管放入仪器反应槽内，并记录放置顺序。选择FAM通道检测结核分枝杆菌特异性基因保守区；选择Texas Red通道检测非结核分枝杆菌特异性基因保守区；选择Cy5通道检测内标通道；选择参比荧光(Passive Reference)为None(ABI7500需)。设置循环条件如表7所示，反应体系体积设置为30μL，设置完成后，保存文件，运行程序。
检测结果如图6至图9以及表11和表12所示。其中，图6为本申请的对准确性样本Z1～Z3的检出效果图；图7为本申请的对准确性样本Z4～Z18的 检出效果图；图8为本申请的对国家参考品P1～P15的检出效果图；图9为本申请的对国家参考品N1～N10的检出效果图。
表11准确性样本Z1～Z18检测结果
表12国家参考品检测结果

注：“+”表示检测结果阳性，“-”表示检测结果阴性。
六、本申请试剂盒灵敏度检测
用本试剂盒检测灵敏度样本L1～L28“结核分枝杆菌PCR检测试剂盒用国家参考品”中的S1～S4样本，采用广州达安基因股份有限公司生产的核酸提取或纯化试剂(粤穗械备20181263号)对以上样本和MN阴性质控品进行核酸提取，具体的操作步骤详见试剂说明书。取提取好的核酸样本各10μL，加入至已添加上述PCR反应液的PCR反应管中，使每管PCR反应液的总体积为30μL；盖紧八连管管盖，充分混匀，高速离心10秒。
将PCR反应管放入仪器反应槽内，并记录放置顺序。选择FAM通道检测结核分枝杆菌特异性基因保守区；选择Texas Red通道检测非结核分枝杆菌特异性基因保守区；选择Cy5通道检测内标通道；选择参比荧光(Passive Reference)为None(ABI7500需)。设置循环条件如表7所示，反应体系体积设置为30μL，设置完成后，保存文件，运行程序。
检测结果如表13和表14以及图10至图12所示。其中，图10是根据本申请的灵敏度样本L1～L3扩增曲线图；图11是根据本申请的灵敏度样本L4～L28扩增曲线图；图12是根据本申请的国家参考品S1～S4扩增曲线图。
表13灵敏度样本L1～L28检测结果

注：“+”表示检测结果阳性，“-”表示检测结果阴性。
表14国家参考品检测结果

注：“+”表示检测结果阳性，“-”表示检测结果阴性。
七、临床样本的检测
用本试剂盒对50例临床阳性样本和20例阴性样本进行检测，并使用测序法进行验证，扩增得到的PCR产物通过标准SangerDNA测序方法进行直接DNA序列测定(生工生物工程(上海)股份有限公司)，分析两种方法的一致性。采用广州达安基因股份有限公司生产的核酸提取或纯化试剂(粤穗械备20181263号)对以上70例临床样本和MN阴性质控品进行核酸提取，具体的操作步骤详见试剂说明书。取提取好的样本核酸、MN阴性质控品的核酸和MN阳性质控品各10μL，加入至已添加上述PCR反应液的PCR反应管中，使每管PCR反应液的总体积为30μL；盖紧八连管管盖，充分混匀，高速离心10秒。
将PCR反应管放入仪器反应槽内，并记录放置顺序。选择FAM通道检测结核分枝杆菌特异性基因保守区；选择Texas Red通道检测非结核分枝杆菌特异性基因保守区；选择Cy5通道检测内标通道；选择参比荧光(Passive Reference)为None(ABI7500需)。设置循环条件如表7所示，反应体系体积设置为30μL，设置完成后，保存文件，运行程序。
检测结果以及测序结果如表15所示。
表15临床样本检测结果表



根据上述结果，本试剂盒的特异性好，准确性为100％，结核分枝杆菌检测的灵敏度为1×102个菌/mL，非结核分枝杆菌检测灵敏度为1×103个菌/mL，在检测临床样本时与测序法的符合率为100％，表明本申请试剂盒的检测性能符合要求。
八、对比例1
本申请针对结核分枝杆菌复合群特异性基因保守区与非结核分枝杆菌特异性基因保守区域设计了数对引物和数条探针，期望能够获得扩增效果好，灵敏度高，准确率高的引物组和检测探针。
由于引物特异性差异、退火温度不一致、以及引物二聚体等原因，不同引物探针组合对于试剂检测灵敏度影响较大，很难获得较优的PCR扩增引物以及探针序列。本申请人通过大量的实验，对设计的引物和探针进行优化选择并验证，最终确定了可以用于本试剂盒所采用的引物、探针序列及其组合。
实验中发现，即使在已经基本确定针对各目标核酸的引物对和探针序列的情况下，不同引物对组合进行多重扩增的效果也存在显著差异。
例如，使用如下的对照引物对进行检测，核苷酸序列见表16，其它检测步骤和条件同上述实施例：
表16对照引物探针
按照上述“六、本申请试剂盒灵敏度检测”的方法进行灵敏度检测。检测结果表明对照的引物和探针对低浓度核酸样本检测扩增曲线较差。
以上描述公开了本申请的产品、用途、使用方法及优点，应当指出的是，上述实施例仅为了清楚说明本申请所举的实例，并不能用来限定本专利的权利要求。对于本领域的技术人员，在未背离本申请精神实质与原理的前提下，可以做出一定的改进与替换，均在本申请要求保护的范围之内。
应该理解的是，虽然附图的流程图中的各个步骤按照箭头的指示依次显示，但是这些步骤并不是必然按照箭头指示的顺序依次执行。除非本文中有明确的说明，这些步骤的执行并没有严格的顺序限制，其可以以其他的顺序执行。而且，附图的流程图中的至少一部分步骤可以包括多个子步骤或者多个阶段，这些子步骤或者阶段并不必然是在同一时刻执行完成，而是可以在不同的时刻执行，其执行顺序也不必然是依次进行，而是可以与其他步骤或 者其他步骤的子步骤或者阶段的至少一部分轮流或者交替地执行。
显然，以上所描述的实施例仅仅是本申请一部分实施例，而不是全部的实施例，附图中给出了本申请的较佳实施例，但并不限制本申请的专利范围。本申请可以以许多不同的形式来实现，相反地，提供这些实施例的目的是使对本申请的公开内容的理解更加透彻全面。尽管参照前述实施例对本申请进行了详细的说明，对于本领域的技术人员来而言，其依然可以对前述各具体实施方式所记载的技术方案进行修改，或者对其中部分技术特征进行等效替换。凡是利用本申请说明书及附图内容所做的等效结构，直接或间接运用在其他相关的技术领域，均同理在本申请专利保护范围之内。

Claims (20)

  1. 一种检测结核分枝杆菌与非结核分枝杆菌的引物探针组，所述引物探针组包括:
    针对结核分枝杆菌复合群特异性基因保守区的上游引物，其核酸序列如SEQ ID NO：1所示；
    针对结核分枝杆菌复合群特异性基因保守区的下游引物，其核酸序列如SEQ ID NO：2所示；
    针对非结核分枝杆菌特异性基因保守区的上游引物，其核酸序列如SEQ ID NO：3所示；
    针对非结核分枝杆菌特异性基因保守区的下游引物，其核酸序列如SEQ ID NO：4所示；
    针对内标基因的上游引物，其核酸序列如SEQ ID NO：5所示；
    针对内标基因的下游引物，其核酸序列如SEQ ID NO：6所示；
    针对结核分枝杆菌复合群特异性基因保守区的探针，其核酸序列如SEQ ID NO：7所示；
    针对非结核分枝杆菌特异性基因保守区的探针，其核酸序列如SEQ ID NO：8所示；
    针对内标基因的探针，其核酸序列如SEQ ID NO：9所示。
  2. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，SEQ ID NO：7的核酸序列的5’端标记有FAM，3’端标记有BHQ1。
  3. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，SEQ ID NO：8的核酸序列的5’端标记有Texas Red，3’端标记有BHQ2。
  4. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，SEQ ID NO：9的核酸序列的5’端标记有CY5，3’端标记有BHQ1。
  5. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，所述针对结核分枝杆菌复合群特异性基因保守区的上游引物的终浓度为0.6μmol/L；
    所述针对结核分枝杆菌复合群特异性基因保守区的下游引物的终浓度为0.6μmol/L。
  6. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，所述针对非结核分枝杆菌特异性基因保守区的上游引物的终浓度为0.6μmol/L；
    所述针对非结核分枝杆菌特异性基因保守区的下游引物的终浓度为0.6μmol/L。
  7. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，所述针对内标基因的上游引物的终浓度为0.3μmol/L；
    所述针对内标基因的下游引物的终浓度为0.3μmol/L。
  8. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，所述针对结核分枝杆菌复合群特异性基因保守区的探针的终浓度为0.3μmol/L。
  9. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，所述针对非结核分枝杆菌特异性基因保守区的探针的终浓度为0.3μmol/L。
  10. 根据权利要求1所述的检测结核分枝杆菌与非结核分枝杆菌的引物探针组，其中，所述针对内标基因的探针的终浓度为0.2μmol/L。
  11. 一种检测结核分枝杆菌与非结核分枝杆菌的试剂盒，包括MN PCR反应液A，所述MN PCR反应液A由权利要求1至10任意一项所述引物探针组的混合液配制得到。
  12. 根据权利要求11所述的试剂盒，其中，配制所述MN PCR反应液A的组分还包括Buffer和纯化水。
  13. 根据权利要求12所述的试剂盒，其中，所述Buffer为5×Buffer。
  14. 根据权利要求12所述的试剂盒，其中，所述Buffer包括(NH4)2SO4、Tris-HCl、MgCl2和Tween-20。
  15. 根据权利要求11所述的试剂盒，其中，所述试剂盒还包括MN阴性质控品和MN阳性质控品，其中，所述MN阴性质控品包括内标片段，所述MN阳性质控品包括结核分枝杆菌目的片段、非结核分枝杆菌目的片段以及内标片段。
  16. 根据权利要求11所述的试剂盒，其中，所述试剂盒还包括MN PCR反应液B，所述MN PCR反应液B包括热启动Taq酶、UDG酶和dNTPs；
    其中，所述dNTPs包括dUTP、dATP、dGTP和dCTP。
  17. 一种应用如权利要求11至16任意一项所述的试剂盒检测结核分枝杆菌与非结核分枝杆菌的方法，包括如下步骤：
    抽取痰液样本至离心管中，按4倍的体积加入4％NaOH，摇匀后室温放置至液化，取0.5mL液化后的痰液样本至离心管中，加入0.5mL 4％NaOH室温放置至液化完全，离心，保留沉淀，获得预处理后的痰液样本；
    分别对所述预处理后的痰液样本和MN阴性质控品进行核酸提取操作，分别获得待测样本核酸和MN阴性质控品的核酸；
    混合所述试剂盒中的MN PCR反应液A和MN PCR反应液B，获得PCR反应液，并将所述PCR反应液分装至PCR反应管中；
    将所述待测样本核酸、MN阴性质控品的核酸和MN阳性质控品分别加入不同的所述PCR反应管中，放入仪器反应槽内进行PCR扩增操作，获得扩增结果；
    分析所述扩增结果，得到检测结果。
  18. 根据权利要求17所述的应用，其中，所述方法还包括以下步骤：
    抽取痰液样本至50mL离心管中，痰液中加入4倍体积的4％NaOH，摇匀，室温下放置30分钟液化；
    取0.5mL至1.5mL离心管中，再加入0.5mL 4％NaOH室温放置10分钟，使其充分液化；
    离心5分钟，弃上清，收集下层沉淀待用，获得预处理后的痰液样本。
  19. 根据权利要求18所述的应用，其中，所述离心的转速为13,000rpm。
  20. 根据权利要求17所述的应用，其中，所述PCR扩增的循环条件如下：
    第一阶段、循环一次：温度为50℃，时间2min；
    第二阶段、循环一次：温度为95℃，时间15min；
    第三阶段、循环四十次：温度为94℃，时间15s；温度为55℃，时间45s，并收集荧光。
PCT/CN2023/134919 2022-12-06 2023-11-29 检测结核分枝杆菌与非结核分枝杆菌的引物探针组、试剂盒及方法 WO2024120270A1 (zh)

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