CN111394347A - Preparation method of candida-free nucleic acid sample suitable for PCR (polymerase chain reaction) experiment - Google Patents
Preparation method of candida-free nucleic acid sample suitable for PCR (polymerase chain reaction) experiment Download PDFInfo
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Abstract
A process for preparing the candida nucleic acid sample without extracting includes such steps as mixing yeast lyase, helicase and β -mercaptoethanol, adding physiological saline to obtain a mixture (100 microns L), enzymolyzing by putting candida specimen in eppendorf tube, washing with PBS, centrifugal separation, adding 100 microns L, water bath heating for cracking, thermal cracking by putting eppendorf tube in metal bath, heating for cracking, enzymolyzing and thermal cracking, low-temp high-speed centrifugal separation at 10000 rpm for 10min, and extracting DNA.
Description
Technical Field
The invention relates to a preparation method of a candida nucleic acid sample without extraction, which is suitable for a PCR experiment and belongs to the technical field of biology.
Background
Humans are increasingly challenged by Invasive Candidiasis (IC), with an increasing incidence of IC in many areas, with mortality rates as high as 40% even when patients are treated with antifungal therapy. Especially candida otic infections, which have recently attracted global attention, have been listed by the united states CDC as an emergency threat. It is reported that Candida albicans infection reaches 587 cases, and nearly 50% of infected people die within 90 days. Early detection is key to reducing the fatality rate of IC.
The prior laboratory detection mainly adopts the traditional detection means comprising microbial culture and the serological detection technology which is developed more rapidly in recent years and becomes an important supplement of the traditional IC detection method, wherein the technologies mainly comprise a G test, a GM test, a GXM test (cryptococcus capsular polysaccharide), a Mn test (candida mannan), antibody detection of various genus-specific Ig M and Ig G, and the like.
Compared with the traditional method and the mass spectrum platform, the nucleic acid diagnosis represented by PCR and isothermal amplification brings hope for rapid detection and identification of difficultly-cultured pathogenic fungi, antifungal drug resistance detection and direct rapid detection of host tissue body fluid, and is a new trend of fungal infectious disease diagnosis in the future and has great development prospect.
Nucleic acid diagnosis is largely divided into two stages, clinical specimen DNA sample preparation and nucleic acid analysis. The quality and concentration of the clinical sample DNA preparation are the first guarantee of the nucleic acid amplification detection. Unlike bacteria, viruses, human cells, etc., the fungal cell wall is composed of chitin, glucan, mannan, glycoproteins, and is the most robust and massive, difficult to remove by simple physical or chemical methods. Therefore, the preparation difficulty of the fungal DNA sample is extremely high, the extraction has no standardized flow and industrial standard, and the method is a key research and development and industrial difficulty in the field of molecular diagnosis and is also a main reason for the delay of the diagnosis and development of fungal infection nucleic acid. In addition, DNA preparation is also one of the important reasons for the false positive of fungus detection, and researches show that 3.3 percent of false positive is caused by the DNA extraction process.
The cell wall of candida consists of chitin, glucan, cellulose, protein, glycoprotein and the like, different polysaccharide chains are mutually wound, the protein, lipoid and the like are embedded in the polysaccharide chains, so that the cell wall strength is very high, and the protein, lipoid and the like are the biggest obstacle to the full release of DNA during the preparation of candida samples.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a preparation method of a candida nucleic acid sample without extraction, which is suitable for a PCR experiment.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method of a candida exempta nucleic acid sample suitable for a PCR experiment, which is a mode combining enzyme cracking and thermal cracking, comprises the following steps:
a. preparing an enzyme lysate, namely mixing yeast lyase lyticase, helicase and β -mercaptoethanol, and adding physiological saline until the volume of the mixed solution is 100 mu L to obtain the enzyme lysate;
b. d, enzyme lysis, namely placing the candida specimen into an eppendorf tube, washing the candida specimen with PBS, centrifuging the candida specimen, removing supernatant, adding 100 mu L of the enzyme lysis solution prepared in the step a into the candida specimen, and heating the candida specimen in a water bath for lysis;
c. thermal cracking: and (3) placing the eppendorf tube which finishes the enzymatic cracking water bath in a metal bath, heating and cracking, carrying out enzymatic cracking and thermal cracking on the candida sample, carrying out low-temperature high-speed centrifugation at 10000 r/min for 10min, and obtaining the supernatant, namely the prepared candida DNA sample.
According to the preparation method of the candida nucleic acid sample suitable for PCR experiments, the adding amount of the yeast lyase lyticase is 10-50U/100 mu L, the adding amount of the helicase is 50-100 mu g/100 mu L, and the adding amount of the mercaptoethanol is 1 mu L/100 mu L.
According to the preparation method of the candida nucleic acid sample suitable for the PCR experiment, the temperature of the enzyme cracking water bath in the step b is 30-37 ℃, and the water bath time is 30-60 min.
In the preparation method of the extraction-free candida nucleic acid sample suitable for the PCR experiment, the metal bath pyrolysis temperature in the step c is 100 ℃, and the pyrolysis time is 5-10 min.
According to the preparation method of the candida exempta nucleic acid sample suitable for the PCR experiment, the normal saline is 0.9% of sodium chloride solution.
The invention has the beneficial effects that:
the invention optimizes the combination of lyase, namely yeast lyase lyticase and helicase are combined for use, so that the enzyme cracking efficiency is higher, the enzyme cracking process does not need to add components such as EDTA and sorbitol for inhibiting nucleic acid amplification reaction, the two enzymes act on different parts of candida, the lyticase mainly acts on the candida cell wall, the helicase is a mixed enzyme and contains more than 20 enzyme components such as cellulase, pectinase, amylase, protease and the like, the mixed use of the two enzymes can be used for digesting and damaging different parts of the candida cell wall in a targeted manner, so that the cell cracking is more complete, the cracking efficiency is improved, small-volume cracking (100 mu L) is adopted, the higher DNA concentration is kept in a cracked system, a prepared DNA sample can be directly applied to a PCR experiment, the DNA extraction process is omitted, the labor capacity and the experiment time are greatly reduced, the preparation time is shortened from 3h to 1-1.5h, the sensitivity of the PCR reaction is improved, a bacterial liquid with 1 spore/m L concentration can be sensitively detected for various strains, and the thermal cracking sensitivity of the candida is improved in the next step, and the activity of the thermal cracking liquid is further favorable for inactivating the next step.
Drawings
The invention will be further explained with reference to the drawings.
FIG. 1 is a real-time fluorescent quantitative PCR sensitivity analysis of Candida albicans samples prepared by the present invention;
FIG. 2 is a real-time fluorescent quantitative PCR sensitivity analysis of Candida glabrata samples prepared in the present invention;
FIG. 3 is a real-time fluorescence quantitative PCR sensitivity analysis of Candida parapsilosis samples prepared in the present invention;
FIG. 4 is a real-time fluorescent quantitative PCR sensitivity analysis of Candida krusei samples prepared in the present invention;
FIG. 5 is a real-time fluorescent quantitative PCR sensitivity analysis of Candida tropicalis samples prepared according to the present invention;
FIG. 6 shows the efficiency of the four different lyase enzymes to cleave Candida albicans SC5314 spores;
FIG. 7 shows the lysis efficiency of Candida albicans SC5314 spores by mixing snailase with L lyticase at different concentrations;
FIG. 8 shows the effect of the components of the Candida mixed enzyme lysate on PCR amplification;
FIG. 9 is a comparison of the lysis efficiency of Candida albicans before and after the removal of sorbitol and EDTA by the enzyme lysis system;
FIG. 10 shows the effect of pyrolysis at 100 ℃ in a metal bath.
In FIGS. 1 to 5, the amplification curves are 1 × 10 from left to right51 spore/m L specimen.
Detailed Description
The present invention will be further described with reference to the following examples.
① the preparation of enzyme lysate, mixing yeast lyase 10U, helicase 100 mug, β -mercaptoethanol 1 mug L, physiological saline (0.9% NaCl) supplementing to 100 mug L, the preparation method is to add the lysate to 100 mug LThe lysate can completely crack 1 × 105② enzyme cracking method for candida of one or more endospores, which includes placing candida specimen in 1.5m L eppendorf tube, washing with PBS, centrifuging, removing supernatant, adding 100 mu L of the above enzyme cracking liquid, placing in 35 ℃ water bath for 1 hour, ③ thermal cracking method, placing the eppendorf tube which completes enzyme cracking water bath in metal bath, 10 minutes at 100 ℃, carrying out enzyme cracking and thermal cracking on candida sample, centrifuging at 10000 r/min at low temperature and high speed for 10 minutes, obtaining supernatant which is prepared candida DNA sample, and directly using in PCR experiment.
Example 1
Preparation and amplification of Candida albicans DNA sample
Culturing Candida albicans in Saburgh culture medium, centrifuging, washing with PBS, removing supernatant, and gradually regulating bacteria concentration to 1 × 105Spores/m L, 1 × 104Spores/m L, 1 × 103Spores/m L, 1 × 102Spores/m L, 1 × 101And spores/m L and 1 spore/m L. the Candida albicans suspension with the adjusted concentration, 1m L, is added into an eppendorf tube with the concentration of 1.5m L, the supernatant is centrifuged, the enzyme lysate, 100 mu L, the 35 ℃ water bath is added for 1 hour, the 100 ℃ metal bath is used for 10 minutes, then, the high-speed centrifugation is carried out at the low temperature of 10000 r/min for 10 minutes, and the supernatant is absorbed for standby.
The nucleic acid amplification of the candida albicans adopts real-time fluorescent quantitative PCR, and the specific method comprises the following steps of synthesizing a candida albicans primer probe according to the prior art, specifically synthesizing by Shanghai bioengineering company, wherein a forward primer is 5'-GGG TTT GCT TGA AAGACG GTA-3', a reverse primer is 5'-TTG AAG ATA TAC GTG GTG GAC GTT A-3', a fluorescent probe is FAM-ACCTAA GCC ATT GTC AAA GCG ATC CCG-TAMRA, PCR amplification is carried out by using a Quant One Step qRT-PCR Kit (Beijing Tiangen organisms), and a 50 mu L amplification system is as follows:
the reaction conditions are carried out in two stages, wherein the first stage is a pre-denaturation stage, the temperature is 50 ℃ for 2min, the temperature is 95 ℃ for 5min, the second stage is a circulation stage, the temperature is 95 ℃ for 15S, annealing and extension are carried out for 60 ℃ for 1min, and the total number of cycles is 40, the result is shown in figure 1 by using AN instrument S L AN-96P Real time PCR system, and the prepared candida albicans nucleic acid sample can be directly applied to Real-time fluorescence quantitative PCR detection and can detect 1 spore/m L candida albicans bacterial liquid at the lowest.
Example 2
Preparation and amplification of Candida glabrata DNA samples
Culturing Candida glabrata in Saburgh culture medium, centrifuging, washing with PBS, removing supernatant, and gradually adjusting the concentration to 1 × 105Spores/m L, 1 × 104Spores/m L, 1 × 103Spores/m L, 1 × 102Spores/m L, 1 × 101Spore/m L and 1 spore/m L. the candida glabrata suspension with the adjusted concentration of 1m L is added into an eppendorf tube with the concentration of 1.5m L, the supernatant is centrifuged, the enzyme lysate of 100 mu L is added, the water bath at the temperature of 35 ℃ is carried out for 1 hour, the metal bath at the temperature of 100 ℃ is carried out for 10 minutes, then, the high-speed centrifugation is carried out at the low temperature of 10000 r/min for 10 minutes, and the supernatant is absorbed for standby.
The nucleic acid amplification of the candida glabrata adopts real-time fluorescent quantitative PCR, and the specific method comprises the following steps of synthesizing a candida glabrata primer probe according to the prior art, specifically synthesizing by Shanghai bioengineering company, wherein a forward primer is 5'-TTT CTC CTG CCT GCGCTT AA-3', a reverse primer is 5'-ACG CAC ACT CCC AGG TCT TT-3', a fluorescent probe is FAM-AGA ACA CCCACC AAC CGC GCA-TAMRA, PCR amplification is carried out by using a Quant One Step qRT-PCR Kit (Beijing Tiangen organisms), and a 50 mu L amplification system is as follows:
the reaction conditions are carried out in two stages, wherein the first stage is a pre-denaturation stage, the temperature is 50 ℃ for 2min, the temperature is 95 ℃ for 5min, the second stage is a circulation stage, the temperature is 95 ℃ for 15S, the annealing extension is 60 ℃ for 1min, and the total number of 40 cycles is 40, the result of AN instrument is S L AN-96P Real time PCR system, and is shown in figure 2.
Example 3
Preparation and amplification of candida parapsilosis DNA sample
Culturing Candida parapsilosis in Saburgh medium, centrifuging, washing with PBS, removing supernatant, and gradually adjusting bacteria concentration to 1 × 105Spores/m L, 1 × 104Spores/m L, 1 × 103Spores/m L, 1 × 102Spores/m L, 1 × 101Spore/m L and 1 spore/m L, the candida parapsilosis suspension with the regulated concentration of 1m L is added into an eppendorf tube with the concentration of 1.5m L, the supernatant is centrifuged, the enzyme lysate of 100 mu L is added, the water bath at the temperature of 35 ℃ is carried out for 1 hour, the metal bath at the temperature of 100 ℃ is carried out for 10 minutes, then, the high-speed centrifugation is carried out at the low temperature of 10000 r/min for 10 minutes, and the supernatant is absorbed for standby.
The nucleic acid amplification of the candida parapsilosis adopts real-time fluorescent quantitative PCR, and the specific method comprises the following steps of synthesizing a candida parapsilosis primer probe according to the prior art, specifically synthesizing the candida parapsilosis primer probe by Shanghai bioengineering company, wherein a forward primer is 5'-GGG TTT GGT GTTGAGCGA TAC-3', a reverse primer is 5'-GGA GTT TGT ACC AAT GAG TGG AAA-3', a fluorescent probe is FAM-CTC CGC CTT TCT TTC AAG CAA ACC CAG-TAMRA, PCR amplification is carried out by using a Quant One Step qRT-PCRKit kit (Beijing Tiangen organisms), and a 50 mu L amplification system is as follows:
the reaction conditions are carried out in two stages, wherein the first stage is a pre-denaturation stage, the temperature is 50 ℃ for 2min, the temperature is 95 ℃ for 5min, the second stage is a circulation stage, the temperature is 95 ℃ for 15S, annealing and extension are carried out for 60 ℃ for 1min, and the total number of cycles is 40, the result is shown in figure 3 by using AN instrument S L AN-96P Real time PCR system, and the prepared candida parapsilosis nucleic acid sample can be directly applied to Real-time fluorescence quantitative PCR detection and can detect the candida parapsilosis bacterial liquid of 1 spore/m L at the lowest.
Example 4
Preparation and amplification of Candida krusei DNA samples
Culturing Candida krusei in Saburgh medium, centrifuging, washing with PBS, removing supernatant, and gradually adjusting the concentration to 1 × 105Spores/m L, 1 × 104Spores/m L, 1 × 103Spores/m L, 1 × 102Spores/m L, 1 × 101Spore/m L and 1 spore/m L. the regulated concentration of the candida krusei suspension, 1m L, is added into an eppendorf tube with the diameter of 1.5m L, the supernatant is centrifuged, the enzyme lysate, 100 mu L, the 35 ℃ water bath is added for 1 hour, the 100 ℃ metal bath is used for 10 minutes, then, the high-speed centrifugation is carried out at the low temperature of 10000 r/min for 10 minutes, and the supernatant is absorbed for standby.
The nucleic acid amplification of the candida krusei adopts real-time fluorescent quantitative PCR, and the specific method comprises the following steps of synthesizing a candida krusei primer probe according to the prior art, specifically synthesizing by Shanghai bioengineering company, wherein a forward primer is 5'-GCT GCG ACT CGCCTG AA-3', a reverse primer is 5'-TTG TCT CGC AAC ACT CGC TCT-3', a fluorescent probe is FAM-CTA GTT CGCTCG GCC AGC TTC GCT-TAMRA, PCR amplification is carried out by using a Quant One Step qRT-PCR Kit (Beijing Tiangen organisms), and a 50 mu L amplification system is as follows:
the reaction conditions are carried out in two stages, wherein the first stage is a pre-denaturation stage, the temperature is 50 ℃ for 2min, the temperature is 95 ℃ for 5min, the second stage is a circulation stage, the temperature is 95 ℃ for 15S, annealing and extension are carried out for 60 ℃ for 1min, and the total number of cycles is 40, the result is shown in figure 4 by using AN instrument S L AN-96P Real time PCR system, and the prepared candida krusei nucleic acid sample can be directly applied to Real-time fluorescence quantitative PCR detection and can detect the candida krusei bacterial liquid with 1 spore/m L at the lowest.
Example 5
Preparation and amplification of candida tropicalis DNA samples
Culturing Candida tropicalis in Saburgh medium, centrifuging, washing with PBS, removing supernatant, and gradually regulating bacteria concentration to 1 × 105Spores/m L, 1 × 104Spores/m L, 1 × 103Spores/m L, 1 × 102Spores/m L, 1 × 101Spore/m L and spore/m L, and adding Candida tropicalis suspension with adjusted concentration of 1m LThe supernatant was centrifuged in an eppendorf tube of 1.5m L, and 100. mu. L of an enzyme lysate was added thereto, followed by 1 hour in 35 ℃ water bath and 10 minutes in 100 ℃ metal bath, followed by 10 minutes of low-temperature high-speed centrifugation at 10000 rpm, and the supernatant was aspirated for use.
The nucleic acid amplification of the candida tropicalis adopts real-time fluorescent quantitative PCR, and the specific method comprises the following steps of synthesizing a candida tropicalis primer probe according to the prior art, specifically synthesizing the candida tropicalis primer probe by Shanghai bioengineering company, wherein a forward primer is 5'-GCG GTA GGA GAATTG CGT T-3', a reverse primer is 5'-TCA TTA TGC CAA CAT CCT AGG TTT A-3', a fluorescent probe is FAM-CGC AGT CCT CAG TCT AGG CTG GCA G-TAMRA, PCR amplification is carried out by using a Quant One Step qRT-PCR Kit (Beijing Tiangen organisms), and a 50 mu L amplification system is as follows:
the reaction conditions are carried out in two stages, wherein the first stage is a pre-denaturation stage, the temperature is 50 ℃ for 2min, the temperature is 95 ℃ for 5min, the second stage is a circulation stage, the temperature is 95 ℃ for 15S, annealing and extension are carried out for 60 ℃ for 1min, and the total number of cycles is 40.
The selection and optimization of the lyase combination was tested:
four common Candida lyases, Snailase (sigma), L lyticase (sigma), dextranase (sigma) and Zymolyase (ZymoResearch Corp), were selected for comparison.
(I) preparation methods of four enzyme lysates:
1) snail enzyme lysate: helicase, Tris-HCl, EDTA-Na2And NaN3Dissolving in double distilled water, wherein the concentration of helicase is 13mg/m L, and the concentration of Tris-HCl is 10 mmol/L-Na2The concentration is 0.1 mmol/L3At a concentration of 0.1％。
2) L lyticase lysate L lyticase enzyme was dissolved in double distilled water at a concentration of 10U/. mu. L.
3) Dextranase lysate: dextranase and EDTA-Na2All dissolved in double distilled water, the dextranase concentration is 8% (8mg/100m L), EDTA-Na2The concentration was 0.1 mmol/L.
4) Zymolyase was dissolved in double distilled water at a concentration of 5U/. mu. L.
Preparation of candida mycosis liquid
The cultured Candida albicans SC5314 was washed with physiological saline (0.9% NaCl), resuspended, counted on a cell counting plate, and adjusted to a concentration of 4.7 × 107CFU/m L, spare.
Candida albicans SC5314 was lysed separately with four enzyme lysates at different concentrations:
1) and adding the helicase lysate into the 5 tubes of candida sediment (the enzyme content in a lysis system is respectively 0 mu g, 130 mu g, 650 mu g, 1040 mu g and 2080 mu g), supplementing the same system with 1m L by using physiological saline, and incubating for 5 hours at 35 ℃.
2) L lysate, namely putting the bacterial suspension 1m L in a high-pressure sterile EP tube, taking 5 tubes together, centrifuging at 12000rmp for 5min at low temperature and high speed, discarding the supernatant, and reserving the precipitate, adding 100 mu L sorbitol and 1% β -mercaptoethanol into the 5 tubes of candida precipitate respectively, adding different amounts of L lysate (so that the enzyme content in the lysate system is 0U, 100U, 200U, 500U and 800U respectively), supplementing the lysate to the same system 1m L by using physiological saline, and incubating for 5h at 30 ℃.
3) And (2) preparing the glucanase lysate, namely putting 1m L of the bacterial suspension into a high-pressure sterile EP tube, taking 5 tubes altogether, centrifuging at a low temperature of 12000rmp for 5min at a high speed, discarding the supernatant, reserving the precipitate, adding 100 mu L sorbitol and 1% β -mercaptoethanol into the 5 tubes of candida precipitate, adding different amounts of glucan lysate (ensuring that the enzyme content in a lysis system is 0U, 40U, 80U, 160U and 320U respectively), supplementing the same system with 1m L by using physiological saline, and incubating for 5h at 50 ℃.
4) Zymolyase lysate, namely putting the bacterial suspension 1m L in a high-pressure sterile EP tube, taking 5 tubes together, centrifuging at 12000rmp for 5min at low temperature and high speed, discarding the supernatant, and reserving the precipitate, adding 100 mu L sorbitol and 1% β -mercaptoethanol into the 5 tubes of Candida precipitates respectively, adding different amounts of Zymolyase lysate (the enzyme content in a lysis system is 0U, 5U, 20U, 50U and 100U respectively), supplementing the same system with 1m L by using physiological saline, and incubating for 5h at 30 ℃.
5) The lysis effect was observed with a microscope and the amount of the remaining bacteria was counted with a cell counting plate.
In FIG. 6, A is a graph of Candida albicans cleaved by helicase, B is a graph of Candida albicans cleaved by L yticase, C is a graph of Candida albicans cleaved by glucanase, and D is a graph of Candida albicans cleaved by Zymolyase, and the results show that the helicase and L yticase can cleave Candida albicans spores with better efficiency, the cleavage rate is gradually increased along with the increase of enzyme amount, and the glucanase and Zymolyase have no cleavage effect on the Candida albicans spores under the conditions set by experiments.
(III) combination of Snailase with L lyticase for Candida lysis, dose optimization test
Because the snail enzyme is used only, the larger enzyme amount is needed, the protein content in the lysate is too high, the subsequent PCR reaction is possibly influenced, and the L yticase is expensive, the experiment cost is greatly improved when the snail enzyme is used only, the popularization and the development of the experiment are not facilitated, so the defect of single use is overcome by mixing the two enzymes.
In order to obtain the optimal use amount of the two enzymes, the two enzymes are mixed according to the principle that the concentration is from low to high respectively to prepare enzyme mixed liquor with six concentrations from low to high, wherein, no lyase is added in a No. 0 tube, and the negative control is obtained.
No. 0 tube, 10 mu L sorbitol, 1 mu L mercaptoethanol, 89 mu L normal saline, total volume 100 mu L;
no. 2 tube, 50 μ g snailase +10U L lyticase, 10 μ L sorbitol, 1 μ L mercaptoethanol, adding physiological saline to 100 μ L;
no. 4 tube, 100 μ g snailase +50U L lyticase, 10 μ L sorbitol, 1 μ L mercaptoethanol, adding physiological saline to 100 μ L;
no. 5 tube, 150 μ g snailase +100U L lyticase, 10 μ L sorbitol, 1 μ L mercaptoethanol, adding physiological saline to 100 μ L;
the content of candida in most clinical samples is 1.0 × 105Under the actual condition within CFU/m L, the 6-tube enzyme lysate and 1.0 × 105CFU/m L Candida albicans SC5314 spore reaction, incubation for 1h at 30 ℃, observing the lysis effect by a microscope, and counting the residual bacterial amount by a cell counting plate, the result is shown in figure 7, the abscissa tube number in the figure represents the enzyme usage amount of different concentrations, according to the result shown in figure 7, the optimal usage amount of the helicase is 50-100 mu g, the optimal usage amount of L yticase is 10-50U, the mixed lyase liquid prepared by the enzyme content in the range is incubated for 1h at 30 ℃, the efficient lysis amount is 1.0 × 105Candida spores within CFU.
(IV) confirmation of the Effect of the Components of the enzymatic lysate on PCR amplification
As mentioned above, the main components of the mixed enzyme lysate include mixed enzyme (snailase + L lyticase), β -mercaptoethanol, sorbitol and EDTA, interference experiments are designed according to the above components, i.e., various components of the mixed enzyme lysate are added into the PCR reaction system respectively, and the influence of the components on the PCR amplification reaction is observed, the template used for amplification is DNA prepared by enzyme cleavage and purification through a yeast genome DNA extraction kit (Tiangen organism), and the components of each tube of the interference experiments are shown in the following table 1:
table 1 interference experiment tube compositions
The nucleic acid amplification adopts real-time fluorescent quantitative PCR, and the specific method comprises the following steps of synthesizing a candida albicans primer probe according to the prior art, specifically synthesizing by Shanghai bioengineering company, wherein a forward primer is 5'-GGG TTT GCT TGA AAG ACG GTA-3', a reverse primer is 5'-TTG AAG ATA TAC GTG GTG GAC GTT A-3', a fluorescent probe is FAM-ACC TAA GCC ATTGTC AAA GCG ATC CCG-TAMRA, PCR amplification uses a Quant One Step qRT-PCR Kit (Beijing Tiangen organisms), and a 50 mu L amplification system is as follows:
the reaction conditions were carried out in two stages, the first being a pre-denaturation stage at 50 ℃ for 2min and 95 ℃ for 5min, the second being a cycle stage at 95 ℃ for 15S, annealing and extension at 60 ℃ for 1min, for 40 cycles, the instrumentation was S L AN-96P Real TimePCR System.
Referring to FIG. 8, curve 1 is a positive control, curve 2 is a mixed enzyme (helicase and L yticase), curve 3 is β -mercaptoethanol, curve 4 is sorbitol, curve 5 is EDTA, curve 6 is a negative control, it can be seen from FIG. 8 that when the amount of liquid added into No. 1-5 interfering tubes is 10 μ L, amplification can be successfully completed in the positive control tube, the mixed enzyme interfering tube and the mercaptoethanol interfering tube, CT values of amplification curves are similar (17.36, 17.37 and 17.28, respectively), it can be seen that the added mixed enzyme and β -mercaptoethanol do not affect PCR reaction, and the sorbitol interfering tube and the EDTA interfering tube have no amplification curves, i.e., sorbitol and EDTA can inhibit PCR reaction.
In order to confirm whether the removal of sorbitol and EDTA has influence on the enzyme lysis system and compare the lysis effect of the enzyme lysis system on Candida albicans before and after the removal of sorbitol and EDTA, as shown in FIG. 9 by microscopic observation, A is a negative control (no lyase is added) in FIG. 9, B is a complete enzyme lysis system containing sorbitol and EDTA, and C is an enzyme lysis system for removing sorbitol and EDTA, the comparison shows that the enzyme lysis solution for removing sorbitol and EDTA components can successfully lyse Candida albicans, and compared with the previous lysis scheme, the Candida albicans spores can be lysed by two lysis schemes under the microscope (400 ×), and broken Candida albicans components are aggregated in large pieces and basically have no complete Candida albicans spores.
(V) Effect of thermal cracking
The cultured Candida albicans spores are taken, washed and evenly divided into two parts, centrifuged to remove supernatant, 100 mu L of optimized enzyme lysate (containing 10U of lyticase, 100 mu g of helicase and 1 mu L of β -mercaptoethanol) is respectively added into the precipitates, the precipitates are incubated for 1h at 30 ℃, then one part of the precipitates is subjected to 100 ℃ metal bath for 10min, the other part of the precipitates is not treated, the two parts of the specimens are centrifuged at low temperature and high speed for 5min at 12000rmp, 5 mu L of the centrifuged supernatant is taken, the Candida albicans PCR system is amplified, and the difference between two groups of CT values and amplification curves is observed, as shown in a graph 10, in the graph, a curve 1 is a PCR amplification curve of 10min after 100 ℃ metal bath treatment, and a curve 2 is a PCR amplification curve of an untreated sample, the cracked sample is treated by the 100 ℃ metal bath, the PCR amplification value (19.18) is slightly lower than that of the untreated sample (20.27), the metal bath high temperature cracking can enable the PCR method to be more sensitive, and the PCR amplification curve of the 100 ℃ metal bath treatment is a PCR amplification curve, and the PCR amplification value is higher than that the PCR amplification curve of the untreated sample can be generated when the Candida albicans can be generated by the.
Sequence listing
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Claims (5)
1. A preparation method of a candida exempt nucleic acid sample suitable for a PCR experiment is characterized by comprising the following steps: the preparation method is a mode combining enzyme cracking and thermal cracking, and comprises the following steps:
a. preparing an enzyme lysate, namely mixing yeast lyase lyticase, helicase and β -mercaptoethanol, and adding physiological saline until the volume of the mixed solution is 100 mu L to obtain the enzyme lysate;
b. d, enzyme lysis, namely placing the candida specimen into an eppendorf tube, washing the candida specimen with PBS, centrifuging the candida specimen, removing supernatant, adding 100 mu L of the enzyme lysis solution prepared in the step a into the candida specimen, and heating the candida specimen in a water bath for lysis;
c. thermal cracking: and (3) placing the eppendorf tube which finishes the enzymatic cracking water bath in a metal bath, heating and cracking, carrying out enzymatic cracking and thermal cracking on the candida sample, carrying out low-temperature high-speed centrifugation at 10000 r/min for 10min, and obtaining the supernatant, namely the prepared candida DNA sample.
2. The method for preparing candida albicans nucleic acid samples for PCR experiment as claimed in claim 1, wherein the yeast lyase lyticase is added in an amount of 10-50U/100 μ L, and the snailase is added in an amount of 50-100 μ g/100 μ L-mercaptoethanol is added in an amount of 1 μ L/100 μ L.
3. The method for preparing candida exempta nucleic acid sample for PCR experiment as claimed in claim 2, wherein: and the temperature of the enzyme cracking water bath in the step b is 30-37 ℃, and the water bath time is 30-60 min.
4. The method for preparing candida exempta nucleic acid sample for PCR experiment as claimed in claim 3, wherein: in the step c, the metal bath thermal cracking temperature is 100 ℃, and the thermal cracking time is 5-10 min.
5. The method for preparing candida exempta nucleic acid sample for PCR experiment as claimed in claim 3, wherein: the normal saline is 0.9% sodium chloride solution.
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