WO2017117697A1 - Quick method for extracting total dna of yeast-like fungi for nucleic acid amplification - Google Patents

Abstract

Provided is a quick method for extracting total DNA of yeast-like fungi for nucleic acid amplification. The method combines the freeze-thaw method, the chemical cleavage method and the mechanical wall-breaking method together in a certain order.

Description

Method for rapid extraction of total DNA of yeast-like fungi for nucleic acid amplification Technical field

The invention relates to the field of molecular biology research of fungi, in particular to a method for rapidly 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. For example, according to conservative estimates, there are 250,000 new cases of invasive candidiasis in medically advanced areas, causing more than 50,000 deaths, and the prevalence of the population is 2-14 per 100,000 people. Seventy-nine years of surveillance data from 49 hospitals in the United States from 1995 to 2002 showed that Candida sepsis ranked fourth (8-10%) in nosocomial infectious sepsis, and the mortality rate ranked first, and its incidence rate was in most areas. Increase or remain stable year by year. In another example, according to related epidemiological studies, there are approximately 957,900 new cases of cryptococcal meningitis in the HIV (+) population, resulting in 624,700 deaths within three months of illness. According to a survey by Chen Yu-Chong, a total of 8769 cases of cryptococcosis were reported in China from 1985 to 2010, and they showed an increasing trend year by year. At the same time, some emerging yeast infections are on the rise. Yeast-Like Fungal Pathogens are important in various invasive fungal infections.

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 extraction of DNA from yeast-like fungi is the basis for subsequent clinical rapid molecular diagnostics. In the DNA extraction of yeast-like fungi, bacterial cell wall breaking is its most critical technical difficulty. Unlike other microorganisms, the cell wall of fungi is composed of chitin, dextran, mannan, and glycoprotein. It is very strong and thick and difficult to remove by simple physical or chemical methods. Although there are a variety of commercially available kits and classic laboratory methods, there are a number of drawbacks as follows: 1. The kit is expensive, and the YeaStar ^TM Genomic DNA Kit yeast extraction kit is used as an example to extract nearly 25 yuan per sample, zymo The ZR Fungal/Bacterial DNA Kit extraction kit extracts nearly 20 yuan per sample. 2. The extraction speed is slow. Since most of the commercially available kits and laboratory methods have added the digestion steps of proteinase K and snail enzyme, the extraction time process is made. It takes several hours, such as the Ezup Column Yeast Genomic DNA Purification Kit, which takes more than four hours for the enzymatic digestion step (proteinase K for one hour, snail enzyme for three hours); 3. Transport preservation is difficult due to protease activity The transportation conditions are sensitive, and the kit has certain requirements for transportation and preservation. 4. Toxic and harmful substances, such as benzyl chloride in the benzyl chloride extraction method is a carcinogen. The phenol chloroform in the classic phenol chloride extraction method also stimulates the human respiratory tract. The thiol ethanol commonly used in the kit is highly toxic. There is a strong stench. 5, most commercial kits and existing extraction methods in the laboratory, for a small amount of fungal extraction is poor, so lack of potential for clinical diagnosis. The drawbacks of the above various extraction methods increase the extraction time and impair the health of the operator, while its high cost limits the application of DNA-based molecular diagnostic techniques in the laboratory and clinical applications. Therefore, it is still a major difficulty in the industry to obtain high-concentration and high-quality fungal DNA inexpensively and quickly. There is a need for a cheap, high-speed, non-toxic, and convenient transport of fungal DNA, which is convenient for scientific research and clinical work.

For fungal DNA extraction, the main methods of breaking the wall can be divided into four categories.

1. Thermic lysis: The combination of a water bath or a heating block by a low temperature medium such as liquid nitrogen or dry ice causes a sharp change in temperature, which damages the cell wall. Temperature changes are not very harmful to DNA, and repeated freezing and thawing based on liquid nitrogen and water bath is a more classical step in extracting fungal DNA;

2. Enzymatic lysis: Degradation of fungal cell walls by proteinase K, snail enzyme, etc., but this process usually takes several hours, which leads to YeaStar ^TM Genomic DNA Kit, Takara Dr.

(from Yeast) The main reason for the slower speed of kits such as High Recovery and Ezup Column Yeast Genomic DNA Purification Kit;

3. Chemical lysis: The lysate containing urea, strontium salt, strong acid, strong alkali and the like as the main component is used to lyse the fungal cell wall, but the simple chemical cleavage has a limited effect on the fungal cell wall and the extraction efficiency is not high. ;

4. Mechanical lysis: The use of micro-glass beads, microwaves, mortars and pestles that produce micropores by pickling, damages the fungal cell wall by mechanical force. The method has a reliable wall breaking effect, but the mechanical degradation of the wall also leads to DNA degradation, and the destruction of the cell wall to obtain the DNA and DNA breakage consumption is a contradiction under the method. For example While the glass beads continue to hit the cell wall, it also causes sustained degradation of the hydrated DNA already present in the extract.

Summary of the invention

The object of the present invention is to provide a method for rapidly extracting total DNA of yeast-like fungi for nucleic acid amplification, which comprises thermic lysis, chemical lysis and mechanical lysis. Ingeniously integrated to complete DNA extraction in 10 to 20 minutes, it is by far the fastest and inexpensive method for the extraction of inexpensive, non-toxic fungal DNA, called Thermic-Chemical-Mechanical Method.

In a first aspect of the present invention, there is provided a method for rapidly extracting total DNA of a yeast-like fungus for nucleic acid amplification, a material and an apparatus required for the extraction method of the present invention: a fungal lysate, a washing solution, and a pickled glass bead (200 μm) Left and right); anhydrous ethanol; high speed centrifuge; cell disruptor, liquid nitrogen, heating block or water bath, spin column.

The yeast-like fungi include: Cryptococcus, Candida, Rhodotorula, and yeast morphology of bidirectional fungi (such as histoplasma).

The method for rapidly extracting total DNA of a yeast-like fungus for nucleic acid amplification comprises the following steps:

A. Add 150-200 mg of pickled glass microbeads to a 2 ml EP tube and add fungal solid colonies;

*If it is a liquid or fungal infection tissue liquefaction specimen (the fungal cell wall is relatively strong, can not be liquefied with tissue in a short time with ordinary reagents), add bacteria liquid or clinical liquefaction sample (up to 1.8ml) after adding glass beads, high speed Centrifuge at 12000 rpm/min for 2 minutes, and pipette as far as possible to absorb the supernatant (because the cells are deposited under the glass beads after centrifugation, the cells are not easily adsorbed with the supernatant);

B, adding 300 μl of strontium salt lysate;

C, metal bath (also known as dry heater) heated at 105 ° C for 90 seconds (if the room temperature is 25 ° C, the temperature of the lysate reaches approximately 70-80 ° C);

D, quickly put into liquid nitrogen freezing (about 30 seconds);

E, quickly placed in a metal bath heated at 105 ° C for 150 seconds (the temperature of the lysate reaches approximately 40-50 ° C);

F, the tissue breaker is broken at 55Hz for 60 seconds;

G, centrifugation at 12000 rpm/min for one minute;

H, take 200μl supernatant to add 1.5ml EP tube, add 20μl cleaning solution (washing solution formula: anhydrous ethanol 90% volume + 3M NaAC 10% volume, pH = 5.2, adjust pH with HAc), mix thoroughly;

I, add 560μl of absolute ethanol, gently mix;

J, take 750 μl of the mixture into a nucleic acid adsorption column, centrifuge at 10,000 rpm / min for one minute, discard the centrate;

K, add 500 μl eluate (double distilled water or TE buffer), centrifuge at 10,000 rpm / min for one minute, discard the centrifuge liquid;

L, repeat step K;

M, nucleic acid adsorption column 10,000 rpm / min idle for 2-3 minutes;

N, remove the adsorption column from the collection tube, put in a new 1.5ml EP tube, add 60μl double distilled water or TE buffer at the center of the adsorption column adsorption membrane;

O, the DNA solution was collected by centrifugation for 30 seconds at 10,000 rpm/min.

The concentration of guanidinium isothiocyanate in the cerium salt lysate in step B is from 4.0 to 5.5 M, preferably 4.7 M.

The specific formula of the strontium salt lysate is:

Each 1000ml of lysate contains:

Tris) 0.047mol

Disodium edetate (EDTA-2Na) 0.020mol

Barium isothiocyanate 4.0-5.5mol (optimum value 4.7mol)

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.

Preferably, after the step B, a working concentration of proteinase K is added, and the mixture is digested at 56 ° C for one hour, followed by a subsequent liquid nitrogen freeze-thaw and glass bead hitting step.

The advantages of the invention are:

1. Advantages: fast and sensitive (10c.f.u./ml), non-toxic and cheap.

2. The extract formulation used in the present invention is relatively common, but the concentration of the strontium salt lysate is about 4.7 M, which can achieve a strong cracking effect, and can melt faster after freezing the liquid nitrogen.

3. The present invention focuses on the ingenious combination of several extraction methods (chemical cracking, temperature change, mechanical), using the shortest time (fastest compared to all reported fungal DNA extraction methods), achieving the maximum extraction effect. . There are other combinations (see below), but the extraction effect is not as good as the method of the present invention.

4. The specific advantages of the method of the present invention are: short time (only 15 minutes); no irritating odor (other kits use mercaptoethanol, phenol, chloroform, isoamyl alcohol, etc., which endangers the health of the operator); The extraction yield is high (10 ⁸ bacteria extraction is nearly 1000 ng / μl, the effect is equivalent to the current best kit, better than most kits); high sensitivity (that is, the ability to extract a very small sample of fungi, specifically A total of 10 yeasts (1 ml concentration of 10 ¹ /ml bacterial solution) were successfully extracted and verified by real-time quantitative PCR, which has potential for clinical promotion.

5. The extraction method of the present invention is a combination of several fungal cracking methods, and the combination thereof is ingenious in that: before mechanical breaking, a relatively vigorous liquid nitrogen metal bath is used for repeated pre-treatment, firstly, the sample temperature is used. Through the metal bath (105 ° C) rapid rise for 2 minutes (about 75 degrees or so, at higher temperatures, the strontium salt lysate will severely corrode the fungal cell wall and cell membrane), and then quickly put in liquid nitrogen (slow while freezing, can speed up Freezing speed, liquid nitrogen freezing on the one hand reduces the integrity of the cell wall, on the other hand can make the water in the fungal cytoplasm rapidly form ice crystals, puncture the cell membrane and cell wall), take samples from liquid nitrogen after about 30 seconds, and put the metal again. In the bath (105 degrees), within three minutes, the sample is thawed and rises to about 50-60 degrees. At this time, the fungal cell wall has been greatly weakened by the above process. The sample was quickly placed in the tissue disrupter (in the sample tube and at the beginning of the test, the pickled micro glass beads with sharp micropores on the surface were added. Under the action of the crusher, the glass beads quickly hit the fungus cell wall and finished. Broken wall), the breaking frequency is 55Hz, and the time is 60 seconds. In contrast: ZR Fungal/Bacterial DNA Kits also use glass beads to strike, but since the kit does not perform sample pretreatment, the hitting time takes five minutes, and the obtained bacteria are only the ten obtained by the method of the present invention. About one point. The reason for this difference is that the glass beads required to break the wall have a longer attack time because there is no prior pretreatment. On the one hand, the glass beads break the cell wall to release DNA, and on the other hand, the fast moving glass beads act on the release for a long time. DNA, leading to massive degradation of DNA. Another method compared to CTAB + glass beads + proteinase K method. The general procedure of this method is a sample of CTAB buffer with weak cleavage effect. First, the 45S55HZ glass bead is broken, then the proteinase K56 is used for one hour, and then the 45S is broken again. The DNA obtained by this method is of high quality, but the method takes a long time and takes about four hours.

6. Cheap: The method of the invention does not use the higher-priced enzyme to digest the fungal cell wall, and the lysate selects a relatively common sputum salt lysate, and the subsequent cleaning solution and eluent are relatively conventional. It is worth mentioning that, since the sputum salt lysate does not affect the activity of the proteinase of the fungal cell wall, proteinase K, if you want to achieve higher extraction, you can add it after freezing and thawing liquid nitrogen. Proteinase K, digested for one hour, and then hit with glass beads.

7. The method of the present invention can be applied to most yeast-like fungi, because temperature changes, mechanical wall breaking, and chemical cleavage are similar to different strains, and are relatively stable, but different enzymes have different effects on different fungi. For example, the Yeast lytic enzyme of the YeaStar ^TM Genomic DNA Kit is better for Aspergills fumigatus, Aspergills nidulans, Aspergills nivens var. aureus, Candida albicans, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, etc., but for cell wall structure for yeast The lytic enzyme is not sensitive to the strain and the kit cannot be extracted.

DRAWINGS

Figure 1. The method of the invention can be used for the extraction of trace fungi 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:

Five fungi were selected for DNA extraction:

Table 1 The extraction effect of this method on a large number of fungi

The above strains with a total amount of 10 ⁷ -10 ¹ were extracted by DNA using TCM method, and the results of real-time fluorescent quantitative PCR (RT-PCR) using NS5 and NS6 fungal universal primers were all 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

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.

Example 2: Comparison with other extraction methods

A comparison of the method of the present invention with other fungal extraction kits is shown in Table 2, and a comparison with other methods reported in the literature is shown in Table 3.

Table 2 Comparison with other fungal extraction kits

Table 3 Comparison with methods reported in the literature

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 rapidly extracting total DNA of a yeast-like fungus for nucleic acid amplification, comprising the steps of:

   A. Add 150-200mg of acid-washed glass beads to a 2ml EP tube and add fungal solid colonies; or add 150-200mg of acid-washed glass beads to a 2ml EP tube, add bacteria or fungal infection tissue liquefaction specimens, and centrifuge at high speed. 12,000 rpm / min 2 minutes, pipette as much as possible to absorb the supernatant;

   B, adding 300 μl of strontium salt lysate;

   C, the metal bath is heated at 105 ° C until the temperature of the lysate reaches 70-80 ° C;

   D, quickly put into liquid nitrogen freezing for 30 seconds;

   E, quickly placed in a metal bath heated at 105 ° C until the temperature of the lysate reaches 40-50 ° C;

   F, the tissue breaker is broken at 55Hz for 60 seconds;

   G, centrifugation at 12000 rpm/min for one minute;

   H, take 200μl supernatant to add 1.5ml EP tube, add 20μl cleaning solution, and mix well;

   I, add 560μl of absolute ethanol, gently mix;

   J, take 750 μl of the mixture into a nucleic acid adsorption column, centrifuge at 10,000 rpm / min for one minute, discard the centrate;

   K, add 500 μl of eluate, centrifuge at 10,000 rpm / min for one minute, discard the centrate;

   L, repeat step K;

   M, nucleic acid adsorption column 10,000 rpm / min idle for 2-3 minutes;

   N, remove the adsorption column from the collection tube, put in a new 1.5ml EP tube, add 60μl double distilled water or TE buffer at the center of the adsorption column adsorption membrane;

   O, the DNA solution was collected by centrifugation for 30 seconds at 10,000 rpm/min.
2. The method for rapidly extracting total DNA of a yeast-like fungus 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 rapidly extracting total DNA of yeast-like fungi for nucleic acid amplification according to claim 2, wherein the yeast comprises Cryptococcus, Candida, Rhodotorula, and Trichosporon, said two-way The yeast morphology of the fungus includes histoplasma.
4. The method for rapidly 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 B is 4.0-5.5 mol. .
5. The method for rapidly extracting total DNA of a yeast-like fungus for nucleic acid amplification according to claim 4, wherein the bismuth salt lysate in the step B is formulated to contain: trishydroxyl per 1000 ml of the lysate. Base aminomethane 0.047mol, disodium edetate 0.020mol, guanidinium isothiocyanate 4.0-5.5mol, Triton X-10011.3ml, The water was made up to 1000 ml, and the pH was adjusted to 6.53 with hydrochloric acid.
6. The method for rapidly extracting total DNA of a yeast-like fungus for nucleic acid amplification according to claim 4, wherein the concentration of guanidinium isothiocyanate in the cerium salt lysate is 4.7M.
7. The method for rapidly extracting total DNA of a yeast-like fungus for nucleic acid amplification according to claim 1, wherein the cleaning liquid in the step H is: adding 90% by volume of absolute ethanol to 10% by volume of 3M. NaAC, the pH of the cleaning solution was 5.2, and the pH was adjusted with HAc.
8. The method according to claim 1, wherein the eluate in the step K is double distilled water or TE buffer.
9. The method for rapidly extracting total DNA of a yeast-like fungus for nucleic acid amplification according to claim 1, wherein after the step B, a working concentration of proteinase K is added, and the mixture is digested at 56 ° C for one hour, and then followed. Liquid nitrogen freeze-thaw and glass bead hitting steps.

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