WO2014044142A1 - Trousse de détection de mycobactérium et son procédé d'utilisation - Google Patents

Trousse de détection de mycobactérium et son procédé d'utilisation Download PDF

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Publication number
WO2014044142A1
WO2014044142A1 PCT/CN2013/083310 CN2013083310W WO2014044142A1 WO 2014044142 A1 WO2014044142 A1 WO 2014044142A1 CN 2013083310 W CN2013083310 W CN 2013083310W WO 2014044142 A1 WO2014044142 A1 WO 2014044142A1
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Prior art keywords
primer
reaction
mycobacterium
detection kit
mmol
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PCT/CN2013/083310
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English (en)
Chinese (zh)
Inventor
曹以诚
茅莉娜
陈振柳
杜正平
王静
吕冰凌
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广州华峰生物科技有限公司
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Publication of WO2014044142A1 publication Critical patent/WO2014044142A1/fr

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    • 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
    • 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
    • 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
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention relates to a biological detection reagent, and in particular to a Mycobacterium detection kit and a method of using the same. Background technique
  • Mycobacterium is a kind of slender, slightly curved bacillus with a tendency to grow branches, hence the name. There are many species of this genus, which can be divided into three groups: Mycobacterium tuberculosis complex, non-tuberculous mycobacteria and Mycobacterium leprae.
  • M. tuberculosis commonly known as Mycobacterium tuberculosis or tuberculosis, is the causative agent of tuberculosis. Tuberculosis is a major infectious disease that threatens human and animal health.
  • the World Health Organization (WHO) has issued a global TB control strategy and has designated March 24th as the World Tuberculosis Day.
  • Mycobacterium tuberculosis complex Corresponding to the Mycobacterium tuberculosis complex is a variety of non-tuberculous mycobacteria, hundreds of species have been found, widely found in soil, environment, and animals. According to the fourth national tuberculosis epidemiological sampling survey in 2000, there were 4.51 million active tuberculosis patients and 1.96 million patients with pulmonary tuberculosis. Among the positive cultures of mycobacteria, Mycobacterium tuberculosis accounted for 86.4%, Bovine Mycobacterium tuberculosis accounted for 2.5%, and nontuberculous mycobacteria accounted for 11.1%.
  • non-tuberculous mycobacteria In the third national tuberculosis epidemic in 1990, only non-tuberculous mycobacteria accounted for only 4.9 percent. This shows that the proportion of non-tuberculous mycobacteria is significantly higher than before.
  • Non-tuberculous mycobacteria patients are resistant to most first-line anti-tuberculosis drugs, and treatment with current standard chemotherapy regimens is ineffective.
  • Traditional mycobacterial strain identification and drug sensitivity test methods are based on culture, which is cumbersome and time-consuming. It takes 1-2 months to meet the needs of clinically effective early chemotherapy, making non-tuberculous mycobacteria patients long-term. Regular chemotherapy is not effective, the course of treatment is prolonged, and patients become refractory and retreated. Bacteria may spread locally.
  • PCR polymerase chain reaction
  • Fluorescence real-time quantitative polymerase chain reaction (real time PCR) technology solves the problem of cross-contamination and integrates the operation process, but it requires more complicated quantitative measurement instruments, so it is not suitable for rapid on-site detection. . Moreover, the cost of fluorescent probes in real-time quantitative polymerase chain reaction PCR technology is higher, which makes it more difficult to promote and apply. Immunological detection technology is fast and inexpensive, but requires high-quality and high-stability monoclonal antibodies. Otherwise, the accuracy is not enough. At present, it can only be an auxiliary detection method. Therefore, the timely application of the latest achievements in the development of biotechnology is of great significance to meet the continuous improvement of the requirements for detection of pathogenic microorganisms.
  • An object of the present invention is to provide a Mycobacterium detection kit which is more comprehensive, has high specificity, and has a low detection rate, which solves the deficiencies of the prior art.
  • a Mycobacterium detection kit wherein the Mycobacterium detection kit comprises a hsp65 gene of Mycobacterium as a target gene, based on a loop-mediated constant temperature expansion
  • Two pairs of primers designed by the technique: inner primer FIP/BIP and outer primer F3/B3, the two pairs of primers are
  • Primer F3 ( SEQ ID NO 1 )
  • External primer F3 ( SEQ ID NO 5 )
  • External primer B3 ( SEQ ID NO 6 )
  • V generation ⁇ A / C / G, R represents A / G.
  • the present invention is directed to a primer designed for the hsp65 gene of the genus Mycobacterium, which is a 65 kDa heat shock protein gene, which is highly conserved and widely present in each branching rod.
  • the primers are capable of amplifying and detecting all the species of the Mycobacterium tuberculosis complex and the non-tuberculous mycobacteria, including: M. tuberculosis, M. variabilis ( ⁇ Microti ), M. africanum, M. bovis, Mycobacterium kansii
  • the Mycobacterium test kit further comprises a DNA polymerase, a reaction solution, a stabilizing solution, a sample pretreatment solution, a color developing solution, and a positive control solution.
  • the polymerase enzyme concentration 4-10 ⁇ / ⁇ ⁇ ;
  • the reaction solution contains: 1.6 ⁇ 2mmol / L dNTP, 20 ⁇ 25mmol / L Tris-HCl, 10 ⁇ 12.5mmol / L KC1, 10 ⁇ 12.5mmol / L (NH 4 ) 2 S0 4 , 8 ⁇ lOmmol / L MgS0 4 , 0.1 ⁇ 0.125 vol% TritonX-100, 0.8 ⁇ lmol / L betaine;
  • the sample pretreatment liquid comprises: 10 - 20 mmol / L H 8.0 Tris-HCl, 1 ⁇ 2 mmol / L EDTA and 1 ⁇ 1.2 vol% Triton X-100;
  • the color developing solution is SYBR Green I or Eva Green
  • the stabilizing liquid is paraffin oil
  • the positive control is BCG genomic DNA
  • the internal primers FIP/BIP are each 0.2 to 0.25 mol/L, and the external primers F3/B3 are each 1.2 to 2.0 mol/L.
  • the fo DNA polymerase enzyme concentration 8 U / L;
  • the reaction solution contains: 2 mmol/L dNTP, 25 mmol/L Tris-HCl, 12.5 mmol/L KC1, 12.5 mmol/L (NH 4 ) 2 S0 4 , 10 mmol/L MgS0 4 , 0.125 vol% Triton X-100, Lmol/L betaine;
  • the sample pretreatment liquid comprises: 20 mmol/L Tris-HC1, pH 8.0, 2 mmol/L EDTA and 1.2% by volume Triton X-100;
  • the color developing solution is SYBR Green I;
  • the concentration of the internal primer FIP/BIP is 0.2 mol/L
  • the Mycobacterium detection kit further comprises a reaction tube, wherein the reaction tube is composed of a tube body and a tube cover, and the lower part of the tube body cavity A longitudinally extending partition is provided which divides it into two cavities A and B.
  • the two cavities A and B are respectively provided with a working liquid or a color developing liquid, and the upper layers of the liquid in both cavities are sealed by the stabilizing liquid.
  • the working fluid is a mixture of a reaction solution and a fo DNA polymerase.
  • Loop-mediated constant temperature amplification is a rapid, sensitive and accurate method for nucleic acid detection. Its amplification efficiency is super-excellent and manifests itself in two aspects: (1) Sensitivity is higher than PCR by an order of magnitude difference; (2) The amount of DNA of the amplified product is also much higher than that of the PCR product, which can reach several but too sensitive and the amount of the product is huge, making LAMP extremely susceptible to contamination, especially after the LAMP amplification reaction requires a reaction tube opening. Adding a developer to the cap is prone to aerosol contamination. Aerosol contamination can lead to high false positive rates and contaminate other samples, contaminate the detection area, and is difficult to remove.
  • the reaction tube of the invention effectively separates the color developing liquid from the working liquid through the design of the partition plate and the stabilizing liquid, which not only ensures relatively complete sealing during storage and transportation, but also can be opened without opening the tube cover. Achieve color reaction, greatly reducing aerosol pollution.
  • the invention also provides a method for using a Mycobacterium detection kit, the method comprising the following steps:
  • step (2) adding the precipitate of step (1) to the sample pretreatment liquid, mixing and hooking, inactivation of the boiling water bath, cooling on ice, centrifugation at high speed, and the supernatant is the sample template DNA;
  • the inner primer FIP/BIP and the outer primer F3/B3 in the step (3) are two pairs of primers designed based on the hsp65 gene of Mycobacterium and designed based on loop-mediated constant temperature amplification technology.
  • the reaction conditions of the constant temperature reaction are a temperature of 63 to 65 ° C and a reaction time of 45 to 90 min.
  • the method for rapidly detecting mycobacteria based on loop-mediated isothermal amplication of DNA (LAMP) according to the present invention is to use fo DNA polymerase and two pairs designed according to target gene sequences.
  • LAMP method is a kind of tube, fast Rapid, highly specific gene amplification method. Comparing the thermostatic gene amplification technology with PCR technology (including fluorescence real-time quantitative PCR), it can be found that the technology is equivalent to or superior to PCR technology in terms of sensitivity, specificity and detection range, and does not depend on any specialization.
  • the instrumentation can achieve high-throughput rapid detection in the field, and the detection cost is much lower than that of the real-time PCR technology.
  • the national standard is based on microbial isolation culture and morphological identification, combined with biochemical analysis and serological typing.
  • the preliminary identification takes 2 to 3 days, and the identification report takes 10 to 15 days.
  • the detection reagent of the present invention is used.
  • the box takes only 2 hours.
  • the color developing solution is added to the reaction system of the present invention, and the identification result is more intuitive and clear.
  • the invention has the following beneficial effects: 1.
  • the detection kit of the invention can amplify the reaction only by a constant temperature, does not require special reagents and equipment, and has low detection cost; 2.
  • the gene of the invention uses six segments, four primers, to determine whether the target substance is present or not depending on whether it is amplified, and thus has high specificity; 3.
  • the detection kit of the present invention is rapid and efficient in amplification, The amplification can be completed in 1 hour, and the yield is high; 4.
  • the detection kit of the invention has high sensitivity, and the amplification template only needs 10 copies or less; 5.
  • the gene rapid diagnostic kit of the invention identifies the cartridge, from The pyrophosphate ion precipitated by dNTP is combined with Mg 2+ in the reaction solution to produce a by-product, magnesium pyrophosphate precipitate, which can be identified by visual observation, and the color positive liquid has a significant difference in coloration after the addition of the color developing solution.
  • the detection kit of the present invention is more accurate in detecting Mycobacterium 7.
  • the special reaction tube is used in the detection kit of the invention, which reduces the possibility of aerosol contamination and is convenient to operate.
  • LAMP loop-mediated isothermal amplification, loop-mediated isothermal amplification
  • dNTP deoxyribonucleoside triphosphate, deoxynucleoside triacate
  • Bst enzyme Bst DNA polymerase (large fragment)
  • Bst DNA polymerase large fragment
  • EDTA ethylenediamine tetraacetic acid, ethylenediaminetetraacetic acid
  • DNA deoxyribonucleic acid, deoxyribonucleic acid
  • Triton X-100 Polyethylene glycol octyl phenyl ether
  • Hsp65 65 kDa heat shock protein
  • reaction solution contained 2 mmol/L dNTP, 25 mmol/L Tris-CK 12.5 mmol/L KC1, 12.5 mmol/L (NH 4 ) 2 S0 4 , 10 mmol/L MgS0 4 , 0.125 vol% TritonX -100, lmol/L betaine, internal primer FIP/BIP 0.2 mol/L and external primer F3/B3 each 0.25 mol/L, placed in a container;
  • the sample pretreatment liquid contains 20 mmol/L Tris-HC1 (pH 8.0), 2 mmol/L EDTA and 1.2% by volume Triton X-100, and placed in a container;
  • the preparation process is as follows:
  • the liquid prepared in the above steps (2) to (4) is aseptically packaged, and the concentration is determined according to the experiment, and the sample quality inspection is performed;
  • reaction solution contains 1.6 mmol/L dNTP, 20 mmol/L Tris-Cl, 1 Ommol/L KCl, 1 Ommol/L (NH 4 ) 2 S0 4 , 8 mmol/L MgS0 4 , 0.1 volume % TritonX-100, 0.8 mol/L betaine, internal primer FIP/BIP 0.2 mol/L and external primer F3/B3 each 0.25 mol/L, placed in the valley;
  • Preparing sample pretreatment liquid contains 10 mmol/L Tris-HCl (pH 8.0), mmol/L EDTA and 1.0% by volume Triton X-100, and placed in a container;
  • Extracting the positive control extracting the BCG genomic DNA and placing it in a container;
  • primers in the step (1) are:
  • External primer F3 ( SEQ ID NO 5 )
  • External primer B3 ( SEQ ID NO 6 )
  • primers in the step (1) are:
  • the invention adopts 29 strains, mainly from the American Standard Biological Collection Center and the China National Institute for the Control of Pharmaceutical and Biological Products. See Table 1 for details.
  • M.chelonae ATCC 14472
  • M.fortuitum ATCC 6841
  • M. gordonae ATCC 14470
  • M.marinum ATCC 927
  • M.smegmatis ATCC
  • M.asiaticum ATCC 25276
  • M.xenopi ATCC 19250
  • strains of Staphylococcus aureus Shigella, Vibrio parahaemolyticus, Salmonella, mononuclear hyperplasia Listeria monocytogenes, Yersinia enterocolitica, and one strain of Streptococcus hemolyticus.
  • reaction system in 20 ( ⁇ L reaction tube: 22 L of reaction solution and primer, 0.5 ⁇ (4U) of fo DNA polymerase, 30 ⁇ of stable solution, 2.5 L of template DNA.
  • the M. bovis BCG was extracted from the nucleic acid, and the LAMP assay was performed by serially diluting to 10 ⁇ 1() with a 10-fold ratio of TE. 8. The repeatability test specificity test and sensitivity test were repeated twice. Second, the results 1, specificity test
  • M.ulcerans ATCC 19423
  • M.shimoidei ATCC 27962
  • the concentration of the bacterial stock solution was 1.26 ⁇ 10 6 CFU/mL, and the fourth dilution was detected by the LAMP method, which was 1.26 ⁇ 10 2 CFU/mL.
  • Example 6 Mycobacterium detection kit using a reaction tube
  • the Mycobacterium detection kit of the present embodiment has the same reagents and primers as in Example 1, and the reagent cartridge further includes a reaction tube, and the reaction tube is composed of a tube body and The tube cover is composed of two parts, and the lower part of the inner cavity of the tube is provided with a longitudinally extending partition which divides the two cavities into A and B, wherein: the cavity A is filled with 22.0 ⁇ The LAMP reaction solution and 0.5 ⁇ M of Bst DNA polymerase, the upper layer of the liquid is sealed by paraffin; the cavity B contains 2.0 ⁇ M of LAMP reaction coloring solution, and the upper liquid layer is also sealed by paraffin, and the reaction tube is stored at -20 ° C.
  • the reaction tube is used as a container, and the LAMP test is performed on the Mycobacterium, and the positive control group and the negative control group are provided at the same time, and the following steps are specifically included:
  • the three reaction tubes prepared above are taken, and the pipettes are respectively added to the negative cells.
  • the control sample, the sample to be tested and the positive control sample each are 2.5 ⁇ .
  • the tip of the sample penetrates the protective layer, and the sample is added into the cavity of the reaction tube.
  • the tube cap is tightly covered and marked, and moved to the reaction zone.
  • the above reaction tubes were all reacted at 65 ° C for 1 h.
  • reaction tube was inverted and shaken once, and then placed upright once, and the working solution and the color developing solution were thoroughly mixed and observed. If the reaction tube is green as the positive control tube, it is positive, and if the reaction tube is orange as the negative control tube, it is negative.
  • the color developing solution and the working fluid are in a sealed state, and no mutual leakage occurs.
  • the inverted turbulent operation causes the coloring liquid and the working fluid to be mixed, and the coloration result is clear.

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  • Chemical & Material Sciences (AREA)
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Abstract

L'invention concerne une trousse de détection de mycobactérium, la trousse comprenant deux paires d'amorces conçues par l'utilisation du gène hsp65 de mycobactérium en tant que gène cible sur la base d'une technologie d'amplification isothermique à médiation par une boucle : une amorce interne FIP/BIP et une amorce externe F3/B3. L'invention concerne également un procédé d'utilisation de la trousse de détection de mycobactérium.
PCT/CN2013/083310 2012-09-19 2013-09-11 Trousse de détection de mycobactérium et son procédé d'utilisation WO2014044142A1 (fr)

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CN201210352230.5A CN102827944B (zh) 2012-09-19 2012-09-19 分枝杆菌属检测试剂盒及其使用方法

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CN102827944B (zh) * 2012-09-19 2014-07-23 广州华峰生物科技有限公司 分枝杆菌属检测试剂盒及其使用方法
CN103088139B (zh) * 2013-01-28 2015-04-22 清华大学 用于检测分枝杆菌的引物对和标准品以及它们的应用
CN116287324B (zh) * 2022-09-20 2023-09-22 北京理工大学 一种检测空间芽孢杆菌的引物、试剂盒及应用

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CN101157954A (zh) * 2007-09-13 2008-04-09 中华人民共和国徐州出入境检验检疫局 基于环介导等温扩增技术的结核分枝杆菌核酸筛查方法
CN101935693B (zh) * 2010-01-18 2012-03-28 广州华峰生物科技有限公司 结核分枝杆菌检测试剂盒及其使用方法
CN101942508B (zh) * 2010-05-18 2012-11-14 广州华峰生物科技有限公司 大肠杆菌检测试剂盒及其使用方法

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CN103451309B (zh) 2015-11-18

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