WO2017147729A1 - Method for instrument-free extraction of total dna from yeast sample after nucleic acid amplification - Google Patents

Abstract

Provided is a method for instrument-free extraction of total DNA from an yeast sample after nucleic acid amplification. The method integrates a chemolysis method and an enzymolysis method, and uses a suction device, a filtering device, and a nucleic acid extraction syringe. The invention can complete a DNA extraction process between 1.5 hours to 3 hours without using any powered device.

Description

Yeast-like fungus total DNA extraction method for nucleic acid amplification Technical field

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.

Background technique

In recent decades, tremendous advances in the field of biomedicine have contributed to a significant increase in human disease prevention and control capabilities. However, with the widespread use of hematopoietic stem and solid organ transplants, tumor chemotherapy, immunosuppressive agents, broad-spectrum antibiotics, various catheterization techniques, and the continued spread of HIV worldwide, the incidence and mortality of various invasive fungal infections There is a significant upward trend in the world. Yeast-like pathogenic fungi include both Cryptococcus. neoformans, Cryptococcus.gattii, Candida albicans, and some thermomorphic two-dimensional fungi (Thermally Dimorphic Fungi). For example, 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. However, there are many influencing factors, and there are different degrees of false positive and false negative results. Compared with traditional methods, 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%. At the same time, 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.

Compared with developed countries or regions, developing countries have always been the hardest hit by infectious diseases. The incidence rate is high, the mortality rate is high, and the treatment costs are difficult to bear. However, in China and many developing countries and regions in the world, it is difficult for primary medical facilities to provide large-scale diagnostic instruments, and it is impossible to afford the operation and maintenance of large-scale diagnostic instruments; patients cannot afford high inspection costs. Therefore, a large number of scientific research results in the early diagnosis direction, especially molecular diagnostic techniques, cannot benefit the population, and these diagnostic techniques are exactly what most people need. At the same time, the inconvenience of large-scale instruments has limited the application of molecular diagnostic techniques in field investigations. Therefore, the lack of diagnostic and therapeutic means has led to the spread of infectious diseases and increased malignant epidemics.

However, the emergence of pathogen-free "instrumentless detection technology" (no instrumental nucleic acid extraction, constant temperature amplification technology), set up advanced pathogen molecular diagnostic technology and bridges in poor areas, providing a new way for infectious disease prevention and control. Since there is no need for large medical instruments or even power supply equipment, instrument-free molecular diagnostic technology can be popularized in poor and remote areas, and has the potential to greatly improve the status of prevention and treatment of infectious diseases. Instrument-free nucleic acid diagnosis is divided into two steps, namely, clinical sample nucleic acid extraction and subsequent nucleic acid constant temperature amplification detection. There have been a large number of technological breakthroughs in constant temperature amplification, such as the cross-primer amplification technology (CPA) developed by Hangzhou Yousida Biotechnology Co., Ltd. and the ring-mediated isothermal expansion developed by Japan's Rongyan Biotechnology Co., Ltd. LAMP, etc., has been able to achieve "disease detection by only one cup of warm water." As described in Chinese invention patent ZL 200810084367.0 and Chinese invention patent application 201510410272.3, bacterial nucleic acid extraction of bacteria has been achieved, but the patent does not provide a nucleic acid extraction method for yeast, especially that can be matched with an instrument-free extraction instrument. Wall method. Unlike other microorganisms, 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. In the development of 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.

Summary of the invention

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. In combination with 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.

In a first aspect of the present invention, there is provided 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:

A. Extract the yeast-like fungus liquid with a suction device.

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 phenyl ether 0.8 ~ 1.2v / v%.

B. The suction device is connected to the filtering device (pore size less than 1 μm, preferably 0.22 μm), the filtrate is pushed out, and the yeast cells are left in the filtering device for subsequent instrument-free nucleic acid extraction.

If the clinical sample is processed, since the human tissue and the bacterial cells have completed the rupture in the sample liquefaction pretreatment stage, the filtrate can be used for bacterial and human tissue nucleic acid extraction and nucleic acid amplification.

C. 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.

D. Remove the filtration device, push out the yeast-lysate mixture, and incubate at 90-100 ° C for 10-15 minutes, preferably 15 minutes.

E. After cooling, proteinase K is added. The final working concentration is 50-100 μg/ml, preferably 100 μg/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.

F. Connect a new filter device and push the lysate to the EP tube.

G, adding a 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.

H. Slowly inhale the mixture with a nucleic acid extraction syringe, and slowly push out the liquid to complete the nucleic acid adsorption.

I. Purify the impurities such as proteins on the filter by using the nucleic acid cleaning solution. Washing solution formula: anhydrous ethanol 90% volume + 3M NaAC 10% volume, pH = 5.2, pH value was adjusted with HAc.

J. Complete the nucleic acid elution with a nucleic acid eluent (double distilled water or TE buffer).

The concentration of guanidinium isothiocyanate in the cerium salt lysate in the step C is 2.0-8.0 M, preferably 3.0 M.

The specific formula of the strontium salt lysate is:

Each 1000ml of lysate contains:

Tris) 0.047mol

Disodium edetate (EDTA-2Na) 0.020mol

Guanidine isothiocyanate 2.0-8.0 mol (optimum value 3.0 mol)

Triton X-100 11.3ml

Make up 1000ml with water

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.

The invention is characterized by:

1. 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. 2 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. According to the corresponding research report, the above system enhances its action by cleavage of the site of action of proteinase K. Proteinase K works best in the guanidinium lysate as shown in the table below. Therefore, the present invention selects a lysate based on guanidinium isothiocyanate.

Table 1 Activity of proteinase K in different buffers

2. 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.

3. 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.

4. 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 / μl, 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).

5. 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.

6. 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.

DRAWINGS

Figure 1. The method of the invention can be used for the extraction of trace yeasts and the extraction is successful by RT-PCR.

detailed description

The specific embodiments provided by the present invention will be described in detail below with reference to the embodiments.

Example 1: Extraction of DNA effect:

Specific preferred extraction methods:

A. Extract 2 ml of uniform yeast culture solution with a 3 ml syringe at a concentration of 10 ⁸ -10 ¹ /ml.

B. The syringe is connected to a 0.22 μm syringe filter, the filtrate is discarded, and the yeast cells are left inside the filter.

C. Syringe inhaled 1 ml of bismuth salt lysate (see formula below), the pumping process can be repeated to ensure that most of the cells on the filter can be resuspended.

D. Remove the filter, push the cell-lysate into the EP tube, and place it in a thermos cup with hot water above 95 °C for 15 minutes.

E. Cool at room temperature for 10 minutes, inject a suitable volume of protease solution into the syringe to a concentration of 100 μg/ml, and put it in a 58-degree vacuum flask for 2 hours.

F. Connect a new filter and push the lysate to a 5ml EP tube.

G. Add 100 μl of sodium acetate (about 1/10 lysate volume), mix well, add 2 mL of absolute ethanol, and let stand for 20 minutes.

H, Yousta nucleic acid extraction syringe inhaled the whole mixture, and then slowly pushed away, the nucleic acid adsorption membrane in the front of the syringe completes the nucleic acid adsorption.

I. Inhale 2ml of cleaning solution and slowly push it away.

J. Slowly push the syringe piston 5 times to fully remove the residual liquid in the membrane.

K. Remove the nucleic acid-rich fraction from the front end of the nucleic acid extraction syringe and mount it on a 1 ml syringe. The action is gentle during operation, and the anterior segment of the nucleic acid enricher is kept inclined downward to prevent the nucleic acid affinity membrane from falling off.

L. Pipette 50 μl of the eluate and let stand for one minute to eject the eluted nucleic acid template into a 0.6 ml centrifuge tube for amplification detection.

Five yeasts were selected for DNA extraction.

The above strains with a total amount of 10 ⁶ -10 ¹ were extracted by DNA without instrumental method, and the results of real-time quantitative PCR (RT-PCR) using NS5 and NS6 fungal universal primers were successfully amplified. No amplification was seen without template control (NTC).

Primer sequence:

The amplification system is:

Premix Ex Taq ^TM II (Tli RNaseH Plus) (takara) 12.5μl

NS5 1μl

NS6 1μl

DNA template 10.5μl

25μl in total

Amplification conditions

95 ° C 30 seconds -{95 ° C 5 seconds -55 ° C 30 seconds -72 ° C 60 seconds} a total of 40 cycles -95 ° C 5 seconds -60 ° C 30 seconds

Table 2 The extraction effect of this method on a large amount of yeast

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.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments, and those skilled in the art can make various equivalents without departing from the inventive spirit of the present invention. Modifications or substitutions of the invention are intended to be included within the scope of the appended claims.

Claims (9)

1. A method for extracting total DNA of yeast-like fungi for nucleic acid amplification without instrument, comprising the steps of:

   A, using a suction device to extract yeast-like fungal liquid;

   B. The suction device is connected to the filtering device to push out the filtrate, and the yeast cells are left in the filtering device for subsequent nucleic acid extraction without instrument;

   C. The suction device sucks the strontium salt lysate through the filtering device;

   D, remove the filter device, push the yeast-lysate mixture, and put it in 90-100 ° C for 10-15 minutes;

   E. After cooling, proteinase K is added; the final working concentration is 50-100 μg/ml; the incubation temperature is 55 to 65 ° C; and the incubation time is 15 minutes to 48 hours;

   F. Connect a new filter device and push out the lysate to the EP tube;

   G, adding 1/10 volume of the nucleic acid precipitant of the lysate, mixing, adding 2 volumes of 95%-100% concentration ethanol, and letting stand for 15-30 minutes;

   H, slowly inhaling the mixture with a nucleic acid extraction syringe, slowly ejecting the liquid to complete the nucleic acid adsorption;

   I. Using a nucleic acid cleaning solution to clean impurities such as proteins on the filter membrane;

   J. Complete the nucleic acid elution with the nucleic acid eluent.
2. The method for extracting yeast-like fungi total DNA from a nucleic acid for nucleic acid amplification according to claim 1, wherein the yeast-like fungus comprises a yeast form of a yeast and a two-way fungus.
3. The method for extracting yeast-like fungi total DNA without nucleic acid for nucleic acid amplification according to claim 1, wherein the yeast comprises Cryptococcus, Candida, Rhodotorula, and Trichosporon, said The yeast morphology of the two-way fungus includes histoplasma.
4. The method for extracting yeast-like fungi total DNA from a nucleic acid for nucleic acid amplification according to claim 1, wherein the filter membrane has a pore size of less than 1 μm.
5. The method for extracting total DNA of yeast-like fungi for nucleic acid amplification according to claim 1, wherein the concentration of guanidinium isothiocyanate in the cerium salt lysate in step C is 2.0-8.0. M.
6. The method for extracting a yeast-like fungus total DNA from a nucleic acid for nucleic acid amplification according to claim 5, wherein the bismuth salt lysate in the step C is formulated as: per 1000 ml of the lysate, comprising: three Hydroxymethylaminomethane 0.047 mol, disodium edetate 0.020 mol, guanidinium isothiocyanate 2.0-8.0 mol, Triton X-100 11.3 ml, 1000 ml with water, adjusted to pH 6.53 with hydrochloric acid.
7. The method for extracting yeast-like fungus total DNA for nucleic acid amplification according to claim 5, wherein the concentration of guanidinium isothiocyanate in the cerium salt lysate is 3.0M.
8. The method for extracting total DNA of yeast-like fungi for nucleic acid amplification according to claim 1, wherein The washing liquid in the step I is as follows: 90% by volume of absolute ethanol is added to 10% by volume of 3M NaAC, and the pH of the washing liquid is 5.2, and the pH is adjusted by HAc.
9. The method for extracting yeast-like fungi total DNA from a nucleic acid for nucleic acid amplification according to claim 1, wherein the eluent in the step J is double distilled water or TE buffer.

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