WO2017147729A1 - Procédé d'extraction sans instrument de l'adn total à partir d'un échantillon de levure après amplification d'acides nucléiques - Google Patents

Procédé d'extraction sans instrument de l'adn total à partir d'un échantillon de levure après amplification d'acides nucléiques Download PDF

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WO2017147729A1
WO2017147729A1 PCT/CN2016/000651 CN2016000651W WO2017147729A1 WO 2017147729 A1 WO2017147729 A1 WO 2017147729A1 CN 2016000651 W CN2016000651 W CN 2016000651W WO 2017147729 A1 WO2017147729 A1 WO 2017147729A1
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nucleic acid
yeast
total dna
lysate
acid amplification
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PCT/CN2016/000651
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English (en)
Chinese (zh)
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陈敏
方文捷
潘炜华
廖万清
尤其敏
洪南
刘加
孙刚
张蕾
李颖芳
姜伟伟
赵海霞
李娟�
吴俊琪
俞世冲
张辛颖
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中国人民解放军第二军医大学
杭州优思达生物技术有限公司
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Publication of WO2017147729A1 publication Critical patent/WO2017147729A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • 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

Definitions

  • the invention relates to the field of molecular biology research of fungi, in particular to a method for extracting total DNA of yeast-like fungi for nucleic acid amplification.
  • Yeast-like pathogenic fungi include both Cryptococcus. neoformans, Cryptococcus.gattii, Candida albicans, and some thermomorphic two-dimensional fungi (Thermally Dimorphic Fungi).
  • Histoplasma capsulatum is in the form of hyphae at normal temperature (22-28 ° C), and exhibits yeast morphology at 37 ° C in human body. Due to the great similarity between the above-mentioned yeast and bifidobacteria, it is difficult for the inspectors who have no systematic deep pathogenic mycology to be diagnosed by morphology, and the spectrum of fungal infections is changing. The difference is large, and the empirical antifungal treatment based on pathogen diagnosis is limited. Direct microscopy usually does not have high sensitivity and there are certain false positives and false negatives. Fungal cultures often take a long time (one week to one month), and the clinical emergency treatments compete for time, and often miss the best opportunity for diagnosis.
  • Serological testing is an important method for detecting clinical invasive fungal infections.
  • molecular biology detection methods are not easy to culture pathogenic fungi for rapid detection and identification, antifungal drug resistance detection and host tissue fluid directness due to their high sensitivity, high specificity and short detection time. Rapid detection brings hope, is a new trend in the diagnosis of fungal infectious diseases in the future, and has great development prospects.
  • the early diagnosis of an infectious disease determines the choice of treatment strategy and the prognosis of the patient.
  • the study found that, in some cases, no matter what pathogenic bacteria invasive infection, the delay in diagnosis of one hour increased the mortality rate by 7%.
  • early diagnosis can reduce the cost of subsequent treatment and reduce the economic burden of patients.
  • the emergence of molecular diagnostics has met the early requirements of infectious diseases.
  • the general test takes only 2 hours to get the test results, while the routine pathogen test (such as pathogen culture) takes several days.
  • the mainstream market for molecular diagnostics is now a high-end population, mainly used in large medical centers or central laboratories in rich countries or regions. Generally, the test needs to be equipped with large-scale automated instruments, such as centrifuges, cell disruptors, etc. for nucleic acid extraction, PCR equipment for nucleic acid amplification, and additional training personnel, so operating costs and testing costs are also required. Correspondingly higher.
  • instrument-free nucleic acid diagnosis is divided into two steps, namely, clinical sample nucleic acid extraction and subsequent nucleic acid constant temperature amplification detection.
  • the cell wall of yeast is composed of chitin, dextran, mannan, and glycoprotein. It is very strong and thick and difficult to remove by simple physical or chemical methods.
  • instrument-free fungal DNA extraction technology in the absence of power supply equipment, looking for cheap, high-speed, non-toxic, convenient cell wall breaking strategy, and the best match with instrument-free extraction equipment, is the most critical Technical difficulties.
  • the object of the present invention is to provide an instrument-free extraction method for total DNA of yeast-like fungi for nucleic acid amplification, which combines chemical lysis and enzymatic lysis into two yeast breaking methods.
  • an instrument-free extraction device such as a syringe filter and a nucleic acid extraction syringe
  • DNA extraction is completed in 1.5 to 3 hours, and it is the only efficient extraction method for yeast-free instrument DNA.
  • an apparatus for extracting total DNA of a yeast-like fungus for nucleic acid amplification and a material and an apparatus for the extraction method of the present invention: fungal lysate, washing solution, absolute ethanol, proteinase K , suction device (such as medical syringe), filter device (syringe filter), vacuum cup (or other insulation device, such as water bath, etc.), instrument-free nucleic acid extraction syringe (Hangzhou Yousida Biotechnology Co., Ltd.), centrifuge tube .
  • the yeast-like fungi include yeasts such as Cryptococcus, Candida, Rhodotorula, and Trichosporon, and yeast forms of bidirectional fungi (such as histoplasma).
  • the method for extracting the yeast-free total DNA of the yeast-like fungus for nucleic acid amplification comprises the following steps:
  • the yeast-like fungal liquid described herein may be a yeast culture bacterial liquid or a pre-treated clinical liquefaction sample.
  • the pre-treated clinical liquefaction sample is a clinical sample which contains a yeast-like fungus, a human body tissue or other microorganisms (such as bacteria), is subjected to liquefaction pretreatment, and the liquefaction pretreatment process is performed because the yeast cell wall is strong. Human tissue or other microbial cells decompose, but the yeast cells are still completely preserved.
  • the specific step of the liquefaction pretreatment comprises: first adding a sodium hydroxide solution to the biological sample, and then adding the lysate for cleavage to obtain a cleavage reaction solution; the lysate containing strontium salt, ethylenediaminetetraacetic acid Sodium, surfactants, nucleic acid adjunctive agents and buffers.
  • the concentration of the sodium hydroxide solution is 1 to 4 wt%, preferably 3 to 4 wt%, and most preferably 4 wt%; and the lysate contains 4-8 mol/L of cerium salt (the cerium salt is selected from guanidine thiocyanate or guanidine hydrochloride), Ethylenediaminetetraacetic acid disodium 10 ⁇ 30mmol/L, glycogen 0.015 ⁇ 0.15mmol/L, trishydroxymethylaminomethane 20 ⁇ 80mmol/L, polyethylene glycol octylphenyl ether 0.6 ⁇ 1.4v/v% More preferably, the lysate contains 5-7 mol/L of guanidinium thiocyanate, 15-25 mmol/L of disodium ethylenediaminetetraacetate, 0.03-0.12 mmol/L of glycogen, and 40-60 mmol of trishydroxymethylaminomethane. /L, polyethylene glycol octyl
  • the suction device is connected to the filtering device (pore size less than 1 ⁇ m, preferably 0.22 ⁇ m), the filtrate is pushed out, and the yeast cells are left in the filtering device for subsequent instrument-free nucleic acid extraction.
  • the filtrate can be used for bacterial and human tissue nucleic acid extraction and nucleic acid amplification.
  • the suction device sucks the strontium salt lysate through the filtering device.
  • the pumping process can be repeated to ensure that most of the cells on the filter are resuspended.
  • proteinase K is added.
  • the final working concentration is 50-100 ⁇ g/ml, preferably 100 ⁇ g/ml.
  • the incubation temperature is 55 to 65 ° C, preferably 58 ° C, and the incubation time is 15 minutes to 48 hours, preferably 2 hours.
  • nucleic acid precipitant such as adding 1/10 volume of sodium citrate lysate, or other commercial nucleic acid precipitant
  • mixing and adding 2 volumes of 95%-100% concentration of ethanol preferably 100% anhydrous
  • the ethanol is allowed to stand for 15-30 minutes, preferably 20 minutes.
  • the concentration of guanidinium isothiocyanate in the cerium salt lysate in the step C is 2.0-8.0 M, preferably 3.0 M.
  • strontium salt lysate The specific formula of the strontium salt lysate is:
  • Each 1000ml of lysate contains:
  • the pH was adjusted to 6.53 with hydrochloric acid, the solution was transferred to a storage container, labeled, and stored at room temperature protected from light.
  • Proteinase K is used as a lyase, rather than a specific fungal cell wall lyase such as snail enzyme or Zymolase.
  • the reasons are as follows: 1 Proteinase K is a broad-spectrum and highly active serine protease, which is mainly used to cleave the serine site of cellular proteins. This site is widely present in both prokaryotes and eukaryotes. Therefore, different fungal cell wall lyases other than snail enzymes, Zymolase enzymes can only selectively act on the cell wall of certain sensitive fungi. Due to the widespread presence of serine sites, if the cleavage site is fully exposed, proteinase K is Most fungi have a good lysis effect.
  • Proteinase K is active in a wide pH range (pH 4-12.5) with an optimal activity pH of 8.0.
  • 3 Proteinase K not only does not inactivate in some chemical lysates, but has a better lysis effect.
  • These chemical cracking systems include: sodium dodecyl sulfate (SDS), strontium salts, urea, creatine, Triton X-100, Tween 20, citrate, iodoacetic acid, tetraethyl oxalate (EDTA) )Wait.
  • SDS sodium dodecyl sulfate
  • strontium salts strontium salts
  • urea creatine
  • Triton X-100 Triton X-100
  • Tween 20 citrate
  • iodoacetic acid tetraethyl oxalate (EDTA)
  • the lysine thiocyanate lysate is used in the present invention for the following reasons: 1 Protease K has the best lysis effect. 2 The lysate can be inactivated in a high temperature water bath (above 90 ° C), and the cracking effect is good. 3 The nucleic acid extraction syringe performs well in the guanidinium lysate system.
  • the method firstly inactivates the yeast at a high temperature with a chemical lysis system to reduce the potential threat of the pathogenic microorganism to the operator under the condition of lack of protection at the site; high temperature and chemical corrosion also partially destroy the yeast cell wall and the capsule, The proteinase K cleavage site is exposed and the cell membrane is enzymatically decomposed to expose the nucleic acid.
  • the specific advantages of the method of the present invention are: short time (less than 3 hours); no irritating odor (other commercial kits mostly use mercaptoethanol, phenol, chloroform, isoamyl alcohol, etc., endangering operators health); the extraction rate (to give approximately 108 bacteria extract 700ng / ⁇ l, equivalent to the most commercial kit); high sensitivity (i.e., an extremely small amount of sample can be extracted yeast, in particular can be successfully extracted a total of 10 Yeast (1ml concentration of 10 1 /ml bacterial liquid), and verified by real-time quantitative PCR, there is potential for clinical promotion).
  • the invention does not require large instruments or even power supply equipment. Only warm water (55 to 65 ° C), hot water or boiling water (90 ° C or higher), thermos cup, nucleic acid extraction syringe, medical syringe, syringe filter device, etc. are required.
  • the method of the invention is applicable to most yeast-like fungi. Pyrolysis, chemical cleavage and proteinase K cleavage are not selective for different yeasts, and the extraction results between different bacteria are consistent and stable.
  • the method of the invention can be used for the extraction of trace yeasts and the extraction is successful by RT-PCR.
  • the syringe is connected to a 0.22 ⁇ m syringe filter, the filtrate is discarded, and the yeast cells are left inside the filter.
  • the amplification system is:
  • the method of the present invention can be used for the extraction of trace fungi and the extraction is successful by RT-PCR, see Figure 1.

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Abstract

L'invention concerne un procédé d'extraction sans instrument de l'ADN total à partir d'un échantillon de levure après amplification d'acides nucléiques. Le procédé intègre un procédé de chimiolyse et un procédé d'enzymolyse et utilise un dispositif d'aspiration, un dispositif de filtration et une seringue d'extraction d'acides nucléiques. L'invention peut accomplir un procédé d'extraction d'ADN en une durée entre 1,5 heure et 3 heures sans utiliser de dispositif électrique.
PCT/CN2016/000651 2016-03-01 2016-11-24 Procédé d'extraction sans instrument de l'adn total à partir d'un échantillon de levure après amplification d'acides nucléiques WO2017147729A1 (fr)

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WO2021042323A1 (fr) * 2019-09-05 2021-03-11 中国石油大学(北京) Procédé d'extraction d'acide désoxyribonucléique génomique d'huile lourde, et kit et application associée
CN113210024A (zh) * 2021-06-03 2021-08-06 北京中科生仪科技有限公司 基于pcr的连续进液装置
CN114231652A (zh) * 2021-12-06 2022-03-25 中国水稻研究所 一种酵母菌落pcr试剂盒及其应用

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CN105624152B (zh) * 2016-03-01 2019-02-19 中国人民解放军第二军医大学 一种用于核酸扩增的酵母样真菌总dna无仪器提取方法
CN110669673A (zh) * 2018-07-02 2020-01-10 王虎 一种酿酒酵母est3端粒酶激活因子提取的方法
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CN110241007A (zh) * 2019-07-09 2019-09-17 津熙医学科技(上海)有限公司 一种柱式核酸提取系统及其应用提取方法
CN112195177B (zh) * 2020-10-28 2021-08-06 上海慕柏生物医学科技有限公司 一种核酸提取方法及试剂盒
CN113265397A (zh) * 2021-06-01 2021-08-17 上海捷瑞生物工程有限公司 一种用于酵母基因组提取的细胞裂解液、试剂盒和方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021042323A1 (fr) * 2019-09-05 2021-03-11 中国石油大学(北京) Procédé d'extraction d'acide désoxyribonucléique génomique d'huile lourde, et kit et application associée
CN113210024A (zh) * 2021-06-03 2021-08-06 北京中科生仪科技有限公司 基于pcr的连续进液装置
CN113210024B (zh) * 2021-06-03 2022-04-08 北京中科生仪科技有限公司 基于pcr的连续进液装置
CN114231652A (zh) * 2021-12-06 2022-03-25 中国水稻研究所 一种酵母菌落pcr试剂盒及其应用
CN114231652B (zh) * 2021-12-06 2023-08-01 中国水稻研究所 一种酵母菌落pcr试剂盒及其应用

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