CN112342308A - Method for detecting zygosaccharomyces rouxii living cells in honey - Google Patents

Abstract

The invention relates to detection of live yeast cells by using a detection method combining nucleic acid dye enhanced azido propylpyridine bromide (PMAXX) with real-time PCR (real-time PCR), and particularly discloses a detection method of live cells of zygosaccharomyces rouxii in honey. The method greatly shortens the detection period of the zygosaccharomyces rouxii, provides a method with a relatively promising application prospect for the rapid detection of the living cells of the zygosaccharomyces rouxii in the honey, and provides technical support for the quality control of the honey product.

Description

Method for detecting zygosaccharomyces rouxii living cells in honey

Technical Field

The invention relates to detection of live yeast cells by using a detection method combining a nucleic acid dye PMAXX with real-time PCR (real-time PCR), in particular to a detection method of live cells of zygosaccharomyces rouxii in honey.

Background

Compared with other foods, honey has the properties of high sugar, high osmotic pressure and the like, so that the honey has a certain effect of inhibiting the growth of microorganisms, and the types and the number of the microorganisms in the honey are relatively small. However, yeasts, molds, and some bacteria capable of sporulation, which can survive in high sugar, low water activity environments, also often present problems for the honey industry. The main yeasts in honey are osmophilic yeast (osmophilic yeast) and osmotolerant yeast (osmolerant yeast), which have great influence on the quality of honey.

The source of yeast in honey is generally considered to be divided into two parts: some are from nectar, the bees themselves, the soil of the bee field, and the air of the bee nest, etc. The microorganisms are various in types and complex in sources, and are difficult to control in actual production. The second part is mainly introduced in the production and processing process after the raw honey is collected, and comprises air, operators, equipment, containers and the like which are contacted in the production process. This contamination can be avoided by a specification-critical operation.

Zygosaccharomyces rouxii (z. rouxii) is a high osmotic pressure resistant yeast commonly found in honey, and is often present in high-sugar high-salt food, and can adjust the permeability and fluidity of cell walls or cytoplasmic membranes, dynamic balance of cations, sugar transport, biosynthesis and accumulation pathways of glycerol and the like through a genetic pathway or a metabolic pathway so as to adapt to a high-sugar high-salt environment. Zygosaccharomyces rouxii can cause honey and its processed products to deteriorate, affecting the shelf life and quality stability of honey products.

Traditional yeast identification, including identification of saccharomyces rouxii in honey, generally adopts a traditional biochemical identification method. The method is long in time consumption and complicated in operation steps, generally needs 1-2 weeks to complete, can only detect culturable bacteria, and cannot meet the requirements of sensitive and accurate real-time quality safety supervision on honey and honey products in emergency. The development of molecular biology techniques provides a new alternative for the rapid identification of zygosaccharomyces isolated from honey. The real-time fluorescence PCR technology has the advantages of rapidness, sensitivity, strong specificity and the like, and is a molecular biological technology with great advantages. However, like most of the traditional nucleic acid detection methods, since DNA can be retained for a long time in dead cells, the identification method cannot distinguish dead bacteria from live bacteria, and false positive results are easy to occur, thereby interfering with the detection result.

Disclosure of Invention

The invention aims to establish a rapid detection method for zygosaccharomyces rouxii living cells by combining nucleic acid dye with real-time fluorescence PCR, greatly shortens the detection period of the zygosaccharomyces rouxii, provides a method with a relatively promising application prospect for rapid detection of the zygosaccharomyces rouxii living cells in honey, and provides technical support for quality control of honey products.

In order to achieve the purpose, the invention adopts the following technical means:

a method for detecting living cells of zygosaccharomyces rouxii in honey, which comprises the following steps: dividing a sample to be detected into a plurality of parts, centrifuging, collecting precipitates, adding sterile water into the precipitates to obtain a bacterial suspension, treating the bacterial suspension by using a nucleic acid dye, performing exposure treatment, extracting DNA, performing fluorescent quantitative PCR amplification to obtain a Ct value, and obtaining the concentration of the living cells of the zygosaccharomyces rouxii in the sample to be detected from an amplification curve.

Further, the nucleic acid dye is enhanced azido propyl bromide (PMAXX), and the addition concentration of the nucleic acid dye is in the range of 20.00-120.00 mu mol/L; when the cell concentration is 1.0X 10⁸CFU/mL, the concentration of the additive was 76.92. mu. mol/L.

Further, the detection method specifically comprises the following steps:

(1) treating a sample to be tested: dividing a sample to be detected into 4 parts, each 500 mu L of the sample to be detected, respectively filling the 4 parts into 2mL centrifuge tubes, adding 1mL of sterile water, fully mixing, washing, centrifuging (12000 r/min multiplied by 3 min), collecting precipitates, adding 500 mu L of sterile water, mixing uniformly, and dividing the mixture into an experimental group E and a control group C, wherein each group comprises two tubes;

(2) treatment of the control group: heat-treating the bacterial suspension in group C in a constant temperature mixing instrument at 90 deg.C for 20 min;

(3) nucleic acid dye PMAXX treatment: adding PMAXX solution with concentration of 2mmol/L into the bacterial suspension of group E and group C until the final concentration range of PMAXX is 20.00-120.00 mu mol/L, mixing, performing dark treatment for 10-30 min, transferring E, C two groups of centrifuge tubes onto ice, irradiating for 10min × 2 times by using 650w halogen lamp, centrifuging, and collecting precipitate;

(4) and (3) extracting DNA: extracting DNA of the precipitated thallus obtained in the step (3) by using a genome DNA extraction kit, detecting the concentration and quality of the extracted DNA, and adjusting the concentration of the DNA template to be consistent;

(5) fluorescent quantitative PCR amplification, wherein the total volume of an amplification system is 20 mu L, the amplification system contains 10 mu L of real-time fluorescent PCR reaction premixed solution, 0.2 mu mol/L of forward primer and reverse primer respectively, 0.1 mu mol/L of probe and 1-50ng of DNA template, and the amplification conditions are as follows: pre-denaturing at 95 ℃ for 10 mm, then performing 40 cycles of denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 60 seconds, repeating each sample for 2 times to obtain a Ct value, and finding out the concentration of the zygosaccharomyces rouxii living cells in the sample from a standard curve according to the Ct value.

Further, the method of selecting nucleic acid dyes and determining optimal concentrations is as follows:

(1) culturing the strain: inoculating the zygosaccharomyces rouxii standard strain on a malt extract powder agar (MEA) slant culture medium, and culturing for 36 hours in an incubator at 28 ℃;

(2) preparing a bacterial suspension: washing zygosaccharomyces rouxii on MEA slant culture medium with sterile water, adjusting thallus concentration to 1.0 × 10⁸CFU/mL, and 500. mu.L/tube are divided into 42 centrifuge tubes, and divided into an experimental group E1 and a control group C1, each group contains 9 bacterial suspensions; and experimental group E2 and control group C2, each of which contains 12 bacterial suspensions;

(3) treatment of the control group: heat-treating the bacterial suspension in groups C1 and C2 in a constant temperature mixer at 90 deg.C for 20 min;

(4) nucleic acid dye treatment: adding 2mmol/L PMA solution to the bacterial suspension in E1 group and C1 group to obtain bacterial suspension with PMA final concentration of 0, 19.80, 27.61, 31.50, 39.22, 58.25, 76.92, 113.21 and 148.15 μmol/L; respectively adding 2mmol/L of PMAXX solution into the bacterial suspensions in the E2 group and the C2 group to obtain bacterial suspensions with the final concentration of PMAXX of 0, 2.00, 2.80, 3.99, 23.72, 27.61, 31.50, 39.22, 58.25, 76.92, 113.21 and 148.15 mu mol/L, uniformly mixing the bacterial suspensions, carrying out dark treatment for 10-30 min, transferring the four groups of centrifugal tubes to ice, irradiating for 10min multiplied by 2 times by using a 650w halogen lamp, and centrifuging to collect precipitates;

(5) and (3) extracting DNA: extracting DNA of the precipitated thallus obtained in the step (4) by using a genome DNA extraction kit, detecting the concentration and quality of the extracted DNA, and adjusting the concentration of the DNA template to be consistent;

(6) fluorescent quantitative PCR amplification, wherein the total volume of an amplification system is 20 mu L, the amplification system contains 10 mu L of real-time fluorescent PCR reaction premixed solution, 0.2 mu mol/L of forward primer and reverse primer respectively, 0.1 mu mol/L of probe and 1-50ng of DNA template, and the amplification conditions are as follows: pre-denaturing at 95 ℃ for 10 mm, then performing 40 cycles of denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 60 seconds, and repeating each sample for 2 times to obtain a Ct value;

(7) selection and determination of optimal concentration of nucleic acid dye: calculating ddCt values according to Ct values of groups E1 and C1 and groups E2 and C2, wherein the calculation formula of the ddCt is as follows: ddCt ═ dCt (dead cell) × dCt (live cell), dCt (dead cell) ═ Ct (dead cell treated with PMAXX) -Ct (dead cell not treated with PMAXX), dCt (live cell) ═ Ct (live cell treated with PMAXX) -Ct (live cell not treated with PMAXX);

by comparing Ct and ddCt values for E1, C1 and E2, C2, the appropriate nucleic acid dye was selected and the optimized PMAXX addition concentration was determined.

The invention has the beneficial effects that:

1. the invention can provide a rapid detection method for live zygosaccharomyces rouxii in honey, and can further improve the detection and/or identification sensitivity of Z.rouxii strains, so that the invention can accurately detect the live cells of the zygosaccharomyces rouxii in honey products, and the detection result is more accurate, and compared with the traditional detection method, the invention greatly shortens the identification period and has good application prospect;

2. the invention applies the reinforced azido propylpyridine bromide (PMAXX) and a real-time fluorescent PCR method (PMAXX-qPCR) to the detection of live Z.rouxii cells in honey and products thereof for the first time, compares the effect of the azido propylpyridine bromide (PMA) and the PMAXX on distinguishing the Z.rouxii dead and live cells, finds that the PMAXX on distinguishing the Z.rouxii dead and live cells is obviously better than the PMA, and selects the PMAXX as a nucleic acid dye used in the research; PMAXX is a charged nucleic acid dye, can selectively enter dead cells with damaged cell wall membranes, and is combined with DNA of the dead cells to inhibit real-time fluorescent PCR amplification of the dead cells; the method has no influence on the living cells with complete cell membranes;

3. the detection process of the invention needs about 6 hours, compared with the detection time of 1-2 weeks of the original culture method, the detection time is greatly saved, and the method can also make up the defects that the original culture detection method can only detect culturable living cells and cannot detect living but non-culturable cells, and is a quick detection method of Z.rouxii living cells with innovation, practicability and application prospect.

Drawings

FIG. 1 is a lower limit of detection of pure cultured Z.rouxii viable cells by PMAXX-qPCR, wherein;

FIG. 2 is the correlation between the Z.rouxii Ct value detected by PMAXX-qPCR method and the plate counting method result;

FIG. 3 is a graph showing the lower limit of detection of viable Z.rouxii cells cultured in 55% and 70% honey solutions according to the present invention;

fig. 4 is a PMAXX-qPCR method detection amplification curve of z.rouxii live cells in 18 honey samples, note: the sample numbers (PC, 10, 12, 5, 2, 7, 3, 6, 9, NC) on the right side of FIG. 4 are the same as those in Table 2; wherein "+" between 9 and NC represents 10 samples with amplification results "n (extracted");

fig. 5 is a PMAXX-qPCR method detection amplification curve of z.rouxii living cells after 8 suspected honey samples are diluted to 50% and cultured, note: the sample numbers (PC, 10, 12, 5, 2, 7, 3, 6, 9, NC) on the right side of FIG. 4 are the same as those in Table 2.

Detailed Description

The present invention will be further described with reference to the following specific examples and drawings, which are not intended to limit the invention in any manner. Reagents, methods and equipment used in the invention are conventional in the art unless otherwise specified; unless otherwise indicated, reagents and materials used in the present invention are commercially available.

The strain information utilized in the present invention is as follows:

zygosaccharomyces rouxii strain (CGMCC 2.1915) purchased from China general microbiological culture collection center and zygosaccharomyces rouxii strains CICC 1417 and CICC 31259 purchased from China industrial microbiological culture collection center are used as standard strains, and other yeast strains used in the invention are separated from honey samples.

Example 1: comparison of the Effect of PMAXX on the discrimination of dead and viable cells of Zygosaccharomyces rouxii from PMA, and optimization of the concentration of PMAXX

In this example, the dimethylsulfoxide was obtained from Tianjin Fuyu Fine chemical Co., Ltd, and azide propyl bromide (PMA) and enhanced azide propyl bromide (PMAXX) were obtained from Biotium, USA.

(1) Preparing a PMA solution with the concentration of 2mmol/L by using dimethyl sulfoxide with the concentration of 20% and PCR-water as solvents, and storing the solution at the temperature of minus 20 ℃ for later use; PMAXX is 20mmol/L water solvent, storing at-20 deg.C in dark place, diluting with PCR-water to 2mmol/L before use, and preparing for use;

(2) inoculating the zygosaccharomyces rouxii standard strain on a malt extract powder agar (MEA) slant culture medium, and culturing for 36 hours in an incubator at 28 ℃;

(3) washing zygosaccharomyces rouxii on MEA slant culture medium with sterile water, and adjusting thallus concentration to 1.0 × 10⁸CFU/mL, split charging in 42 centrifugal tubes, each tube 500 u L; then, they were divided into experimental group E3(9 parts), experimental group E4(12 parts), control group C3(9 parts), control group C4(12 parts);

(4) heat-treating the bacterial suspension in group C3 and C4 at 90 deg.C for 20min in a constant temperature mixing machine;

(5) nucleic acid dye treatment: respectively adding 2mmol/L PMA solution into the bacterial suspensions in the E3 group and the C3 group to obtain bacterial suspensions with the final concentration of PMA of 0, 19.80, 27.61, 31.50, 39.22, 58.25, 76.92, 113.21 and 148.15 mu mol/L respectively, uniformly mixing the bacterial suspensions, carrying out dark treatment for 10-30 min, transferring two groups of centrifuge tubes of E3 and C3 to ice, irradiating for 10min multiplied by 2 times by using a 650w halogen lamp, and centrifugally collecting precipitates;

respectively adding 2mmol/L of PMAXX solution into the bacterial suspensions in the E4 group and the C4 group to obtain bacterial suspensions with the final concentration of PMAXX being 0, 2.00, 2.80, 3.99, 23.72, 27.61, 31.50, 39.22, 58.25, 76.92, 113.21 and 148.15 mu mol/L, uniformly mixing the bacterial suspensions, carrying out dark treatment for 10-30 min, transferring the E4 group and the C4 group onto ice, irradiating the E4 group and the C4 group by using a 650w halogen lamp for 10min multiplied by 2 times, centrifuging and collecting precipitates;

(6) extracting DNA of the precipitated thallus obtained in the step (5) by using a genome DNA extraction kit, detecting the concentration and the quality of the extracted DNA by using NANODRROP (NanoDrop ND-1000, USA), adjusting the concentration of a DNA template to be consistent, and performing real-time fluorescence PCR amplification on Quantstrudio 7 Flex (Applied Biosystems of Life Technologies, USA);

(7) calculating ddCt according to the fluorescence amplification result in the step (6), wherein the calculation formula of the ddCt is as follows: ddCt ═ dCt (dead cell) × dCt (live cell), dCt (dead cell) ═ Ct (dead cell treated with PMAXX) -Ct (dead cell not treated with PMAXX), dCt (live cell) ═ Ct (live cell treated with PMAXX) -Ct (live cell not treated with PMAXX), and the results are shown in table 1.

As can be seen from Table 1, when the concentration of PMA or PMAXX is in the range of 0.00 to 113.21. mu. mol/L, the Ct value of Z.rouxii living cells is not greatly affected by either the treatment with PMA or PMAXX. Indicating that in this concentration range, the viable z. rouxii cell membrane is intact and not substantially affected by PMA or PMAXX.

For heat treated dead z. rouxii, the Ct value after dye addition was significantly higher than for the control without dye addition. When the concentration of PMA is less than or equal to 58.25 mu mol/L, PMAXX and less than or equal to 76.92 mu mol/L, the Ct value is increased along with the increase of the dye concentration, the Ct value of dead cells reaches the maximum value of 27.839 when the concentration of PMA reaches 58.25 mu mol/L, and then the Ct value basically does not change significantly along with the increase of the concentration of PMA, which indicates that a saturation value exists between the concentration of PMA and DNA of dead cells, and the Ct value of dead cells is not obviously changed when the concentration of PMA is increased after the saturation value is reached.

TABLE 1 comparison of the Effect of PMA and PMAXX

In the table:^act and^bct is the mean value of Ct values. + -. Standard Deviation (SD);

^dn: not detected (Undetermined).

For PMAXX, the Ct value of dead cells was at a maximum of 33.171 at a PMAXX concentration of 76.92. mu. mol/L. Thereafter, as the concentration of PMAXX increases, the Ct value instead gradually decreases. Furthermore, according to the value of ddCt, the effect of PMA on distinguishing Z.rouxii dead and live cells is better than that of PMAXX at a concentration of less than or equal to 3.99. mu. mol/L only at a concentration of 39.22. mu. mol/L. And in the range of all test concentrations of PMAXX higher than 23.72 mu mol/L (ddCt between 29.25 and 96.09), the distinguishing effect is better than that of the range of all test concentrations of PMA (ddCt between 1.29 and 19.23). It can be seen that PMAXX has better effect on distinguishing z. rouxii dead and live cells than PMA. As can be seen from Table 1, the concentration of PMAXX is 76.92. mu. mol/L, which has a significant inhibitory effect on dead cells and no significant effect on live cells, so the optimized concentration of PMAXX is 76.92. mu. mol/L.

Example 2: determining the lower limit of the concentration of the living cells of the zygosaccharomyces rouxii detected by PMAXX-qPCR under pure culture conditions

Washing zygosaccharomyces rouxii on MEA slant culture medium with sterile water, adjusting thallus concentration to 1.0 × 10⁸CFU/mL,500 μ L/tube split charging into 4 centrifuge tubes, dividing into experimental group E6 and control group C6 (2 tubes per group), each adding PMAXX to the final concentration of 76.92 μmol/L, standing in dark for 10 min; the two sets of tubes were then placed on ice and irradiated with a 650W halogen lamp for 10min × 2 times. Then, the precipitate was collected by centrifugation, DNA was extracted, and the DNA was diluted 10-fold to 10-fold^-8Multiplying, and carrying out real-time fluorescence PCR amplification to obtain a Ct value; the bacterial suspension was subjected to plate counting simultaneously, and the experimental results are shown in FIGS. 1 and 2.

It can be seen from FIG. 1 that the Ct values of real-time fluorescence PCR amplification of Z.rouxii cells, both live and dead, were almost consistent when not treated with PMAXX, and the cell concentration was 10²---10⁷Ct values between CFU/mL increased as the concentration of the bacterial suspension decreased. Indicating that pure cultured, dead or live z.rouxii cells cannot be distinguished when amplified using only real-time fluorescent PCR without PMAXX treatment. When the bacterial suspension is treated by PMAXX (the final concentration of PMAXX in the bacterial suspension is 76.92uM), when the concentration of Z.rouxii dead cells is 10⁴Below CFU/mL, real-time fluorescent PCR amplification of dead cells will be completely inhibited. The lower limit of detection of PMAXX-qPCR in live Z.rouxii cells is 10³CFU/mL, 10 above the qPCR limit of dead or viable Z.rouxii cells without PMAXX treatment²CFU/mL。

As can be seen from FIG. 2, there is a negative correlation between the logarithm of the Z.rouxii cell concentration (lg CFU/mL) and the Ct value of PMAXX-qPCR. Rouxii cell concentration of 10³-10⁷Ct value and lg CFU/m in the CFU/mL rangeL is well linear with the linear equation y-3.548 x +52.64 and R2 0.999. From this standard curve, it can be seen that the viable Z.rouxii cell concentration can be derived from the Ct value of PMAXX-qPCR.

Example 3: determining the lower limit of the concentration of zygosaccharomyces rouxii living cells in the honey solution detected by PMAXX-qPCR

Diluting sterilized (121 deg.C, 20min) Mel sample to 55% and 70%, inoculating 3mL of activated Z.rouxii standard strain (CGMCC 2.1915) bacterial suspension into sterilized 100mL of 55% and 70% Mel solution (W/W), culturing at 28 deg.C for 5 days, and obtaining the Z.rouxii viable cell concentration of 1.0 × 10 in 55% Mel solution⁸CFU/mL; the concentration of Z.rouxii viable cells in 70% honey solution is 1.0X 10⁶CFU/mL; subpackaging the bacterial suspension into centrifuge tubes at 500 μ L/tube, dividing into experimental group E7 and control group C7, centrifuging to collect thallus, adding 500 μ L sterile water, and performing heat treatment at 90 deg.C for 20 min; then, the DNA was extracted by treating the DNA with an additive PMAXX solution at a concentration of 76.92. mu. mol/L (the conditions and steps of PMAXX treatment and DNA extraction were the same as those in example 1), and the extracted DNA was serially diluted 10-fold with DNA-FREE WATER 10 to 10-fold^-8After the amplification, real-time fluorescence PCR amplification (2 times for each dilution) was performed, and the concentration of the cells in the bacterial suspension was measured by plate counting. The lower limit of detection of Z.rouxii in 55% and 70% honey solution is shown in figure 3.

As can be seen from fig. 3, in the 55% honey solution, the concentration range of culturable viable z.rouxii cells was 10¹-10⁸Within CFU/mL, the Ct value of PMAXX-qPCR and lgCFU/mL have good negative linear relation (R²0.998). In a 70% honey solution, in a culturable viable Z.rouxii cell concentration range of 10¹---10⁶Within the CFU/mL range, the Ct value of PMAXX-qPCR and log CFU/mL have good negative linear relation (R)²0.996). Namely, the Ct value of PMAXX-qPCR and log CFU/mL in 55% and 70% honey solutions both have good negative linear relation, and the linear relation with the negative slope is consistent with the trend of pure Z.rouxii culture by MEA. At the same inoculation concentration, Z.rouxii is dissolved in 55% honeyCulturing at 28 deg.C for 5 days to obtain viable cell concentration of 1.0 × 10⁸CFU/mL, but only 1.0X 10 in 70% honey solution⁶CFU/mL. It can be seen that 55% honey solution is more favorable for growth and reproduction of z.rouxii than 70% honey solution, which further indicates that z.rouxii is a yeast with high osmotic pressure resistance.

Example 4: detection of living cells of zygosaccharomyces rouxii in honey sample

(1) Treating a sample to be tested: selecting 18 different honey samples, wherein each sample is divided into 4 parts, each part is 500 mu L, the honey samples are respectively put into 2mL centrifuge tubes and divided into two groups, each group of samples are divided into two tubes, 1mL sterile water is added into each tube for uniform mixing, then centrifugation is carried out (12000 r/min multiplied by 3 min), precipitates are collected, 500 mu L sterile water is added into the mixture for uniform mixing, one group is an experimental group E8, and the other group is a control group C8;

(2) nucleic acid dye PMAXX treatment: adding PMAXX solution with concentration of 2mmol/L into E8 group bacterial suspension to obtain bacterial suspension with final concentration of 76.92 μmol/L, mixing, dark treating for 10-30 min,

(3) pretreatment: transferring two groups of centrifuge tubes E8 and C8 onto ice, irradiating for 10min × 2 times by using a 650w halogen lamp, centrifuging and collecting precipitates;

(4) and (3) extracting DNA: extracting DNA of the precipitated thallus obtained in step (3) with a genome DNA extraction kit, detecting the concentration and quality of the extracted DNA, adjusting the concentration of the DNA template to be consistent, diluting the extracted DNA to 10 times by using DNA-FREE WATER according to a 10-fold ratio^-8Doubling;

(5) fluorescent quantitative PCR amplification, wherein the total volume of an amplification system is 20 mu L, the amplification system contains 10 mu L of real-time fluorescent PCR reaction premixed solution, 0.2 mu mol/L of forward primer and reverse primer respectively, 0.1 mu mol/L of probe and 1-50ng of DNA template, and the amplification conditions are as follows: pre-denaturation at 95 ℃ for 10 mm, followed by 40 cycles of denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 60 seconds. Each sample is repeated for 2 times to obtain a Ct value;

(6) taking 18 honey samples in the step (1), determining the concentration of the osmophilic yeast by a plate counting method according to GB 14963-2011, and summarizing the concentration and the Ct value in the step (5) to obtain the results shown in the table 2, wherein the amplification curve is shown in the figure 4.

Table 2 results of PMAXX-qPCR, and plate count of z.rouxii live cells in honey samples

Note:^ect, PMAXX-qPCR, representing the Ct value of real-time fluorescent PCR amplification of samples (group E) treated with PMAXX;^fct, qPCR, Ct values for normal qPCR without PMAXX treatment (panel C);^dn represents the Ct value "unexcited", i.e., the Ct value is greater than 40.

As can be seen from table 2 and fig. 4, although the plate count results of culturable z.rouxii in 18 actual honey samples all showed that culturable z.rouxii live cells were not contained therein, the results of qPCR (real-time fluorescence PCR without PMAXX treatment) experiments showed that the qPCR Ct values for all 18 samples read between 24.208 and 38.001. False positives were observed in the qPCR (real-time fluorescent PCR without PMAXX treatment) assay. The detection result of PMAXX-qPCR shows that Ct values of 10 samples are N and are consistent with the result of plate counting; the Ct values for the other 8 samples (44% of the samples tested) ranged from 34.134 to 38.611.

Example 5: validation of suspect samples

8 suspicious samples (namely 8 honey samples with a Ct value between 34.134 and 38.611 after being amplified by PMAXX-qPCR and negative in result by a traditional plate counting method) in example 4 are taken, original honey samples are contained in sterile sample bottles with covers, sterile water is added to dilute the samples until the honey concentration is 50 percent, the samples are cultured for 48 hours at 28 ℃, then a DG18 plate is coated for counting according to a relative osmophilic yeast counting method specified in GB 14963-2011, and the coated DG18 plate is cultured for 7 days at 25 ℃.

Of the 8 samples, 6 developed yeast colonies on DG18 plates. And smearing the single colony of the grown yeast with a methylene blue staining solution, observing under a microscope, comparing with a standard strain, and finding that the yeast with high osmotic pressure resistance grows on DG18 corresponding to 6 samples.

1mL of 50% diluted culture solution of 8 suspicious samples is taken, and after PMAXX is used for treatment, DNA is extracted by using a kit, and real-time fluorescence PCR detection is carried out, namely PMAXX-qPCR detection, and 8 suspicious samples still have amplification curves, which are shown in figure 5.

In summary, of the 8 suspected samples, 6 samples did contain osmotolerant yeast that was not culturable without dilution. DNA contamination was possible with the other 2 suspect samples.

Claims (4)

1. A detection method of zygosaccharomyces rouxii living cells in honey is characterized by comprising the following steps: dividing a sample to be detected into a plurality of parts, centrifuging, collecting precipitates, adding sterile water into the precipitates to obtain a bacterial suspension, treating the bacterial suspension by using a nucleic acid dye, performing exposure treatment, extracting DNA, performing fluorescent quantitative PCR amplification to obtain a Ct value, and obtaining the concentration of the living cells of the zygosaccharomyces rouxii in the sample to be detected from an amplification curve.

2. The method for detecting the living cells of zygosaccharomyces rouxii in honey as claimed in claim 1, wherein the nucleic acid dye is enhanced azido propylidene bromide (PMAXX), and the addition concentration of the nucleic acid dye is in the range of 20.00-120.00 μmol/L; when the cell concentration is 1.0X 10⁸CFU/mL, the concentration of the additive was 76.92. mu. mol/L.

3. The method for detecting the living cells of the zygosaccharomyces rouxii in the honey as claimed in claim 2, wherein the method specifically comprises the following steps:

(1) treating a sample to be tested: dividing a sample to be detected into 4 parts, each 500 mu L of the sample to be detected, respectively filling the 4 parts into 2mL centrifuge tubes, adding 1mL of sterile water, fully mixing, washing, centrifuging (12000 r/min multiplied by 3 min), collecting precipitates, adding 500 mu L of sterile water, mixing uniformly, and dividing the mixture into an experimental group E and a control group C, wherein each group comprises two tubes;

(2) treatment of the control group: heat-treating the bacterial suspension in group C in a constant temperature mixing instrument at 90 deg.C for 20 min;

(3) nucleic acid dye PMAXX treatment: adding PMAXX solution with concentration of 2mmol/L into the bacterial suspension of group E and group C until the final concentration range of PMAXX is 20.00-120.00 mu mol/L, mixing, performing dark treatment for 10-30 min, transferring E, C two groups of centrifuge tubes onto ice, irradiating for 10min × 2 times by using 650w halogen lamp, centrifuging, and collecting precipitate;

(4) and (3) extracting DNA: extracting DNA of the precipitated thallus obtained in the step (3) by using a genome DNA extraction kit, detecting the concentration and quality of the extracted DNA, and adjusting the concentration of the DNA template to be consistent;

(5) fluorescent quantitative PCR amplification, wherein the total volume of an amplification system is 20 mu L, the amplification system contains 10 mu L of real-time fluorescent PCR reaction premixed solution, 0.2 mu mol/L of forward primer and reverse primer respectively, 0.1 mu mol/L of probe and 1-50ng of DNA template, and the amplification conditions are as follows: pre-denaturing at 95 ℃ for 10 mm, then performing 40 cycles of denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 60 seconds, repeating each sample for 2 times to obtain a Ct value, and finding out the concentration of the zygosaccharomyces rouxii living cells in the sample from a standard curve according to the Ct value.

4. A method as claimed in claim 1, wherein the nucleic acid dye is selected and the optimum concentration is determined by the following method:

(1) culturing the strain: inoculating the zygosaccharomyces rouxii standard strain on a malt extract powder agar (MEA) slant culture medium, and culturing for 36 hours in an incubator at 28 ℃;

(2) preparing a bacterial suspension: washing zygosaccharomyces rouxii on MEA slant culture medium with sterile water, adjusting thallus concentration to 1.0 × 10⁸CFU/mL, and 500. mu.L/tube are divided into 42 centrifuge tubes, and divided into an experimental group E1 and a control group C1, each group contains 9 bacterial suspensions; and experimental group E2 and control group C2, each of which contains 12 bacterial suspensions;

(3) treatment of the control group: heat-treating the bacterial suspension in groups C1 and C2 in a constant temperature mixer at 90 deg.C for 20 min;

(4) nucleic acid dye treatment: adding 2mmol/L PMA solution to the bacterial suspension in E1 group and C1 group to obtain bacterial suspension with PMA final concentration of 0, 19.80, 27.61, 31.50, 39.22, 58.25, 76.92, 113.21 and 148.15 μmol/L; respectively adding 2mmol/L of PMAXX solution into the bacterial suspensions in the E2 group and the C2 group to obtain bacterial suspensions with the final concentration of PMAXX of 0, 2.00, 2.80, 3.99, 23.72, 27.61, 31.50, 39.22, 58.25, 76.92, 113.21 and 148.15 mu mol/L, uniformly mixing the bacterial suspensions, carrying out dark treatment for 10-30 min, transferring the four groups of centrifugal tubes to ice, irradiating for 10min multiplied by 2 times by using a 650w halogen lamp, and centrifuging to collect precipitates;

(5) and (3) extracting DNA: extracting DNA of the precipitated thallus obtained in the step (4) by using a genome DNA extraction kit, detecting the concentration and quality of the extracted DNA, and adjusting the concentration of the DNA template to be consistent;

(6) fluorescent quantitative PCR amplification, wherein the total volume of an amplification system is 20 mu L, the amplification system contains 10 mu L of real-time fluorescent PCR reaction premixed solution, 0.2 mu mol/L of forward primer and reverse primer respectively, 0.1 mu mol/L of probe and 1-50ng of DNA template, and the amplification conditions are as follows: pre-denaturing at 95 ℃ for 10 mm, then performing 40 cycles of denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 60 seconds, and repeating each sample for 2 times to obtain a Ct value;

(7) selection and determination of optimal concentration of nucleic acid dye: calculating ddCt values according to Ct values of groups E1 and C1 and groups E2 and C2, wherein the calculation formula of the ddCt is as follows: ddCt ═ dCt (dead cell) × dCt (live cell), dCt (dead cell) ═ Ct (dead cell treated with PMAXX) -Ct (dead cell not treated with PMAXX), dCt (live cell) ═ Ct (live cell treated with PMAXX) -Ct (live cell not treated with PMAXX);

by comparing Ct and ddCt values for E1, C1 and E2, C2, the appropriate nucleic acid dye was selected and the optimized PMAXX addition concentration was determined.

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