KR101451874B1 - Methods for the fastest detection of microorganism using real-time PCR - Google Patents

Abstract

The present invention relates to a method for detecting the shortest time of a microorganism using real-time PCR. More specifically, a detection limit of real-time PCR of a specific microorganism is first determined, and then a WGA is performed to generate a graph of DNA concentration over time. X) is expressed as a function of a specific DNA concentration (Y), then the detection limit value is substituted into the function Y to determine the shortest time of WGA, the specific microorganism is performed for the shortest time of WGA, And a method for detecting the shortest time of a microorganism using PCR.

Description

[0001] The present invention relates to a method for detecting the shortest time of a microorganism using real-time PCR,

The present invention relates to a method for detecting the shortest time of a microorganism using real-time PCR. More specifically, a detection limit of real-time PCR of a specific microorganism is first determined, and then a WGA is performed to generate a graph of DNA concentration over time. X) is expressed by a function of a specific DNA concentration (Y), then the detection limit value is substituted into the function Y to determine the shortest time of WGA, the specific microorganism is performed for the shortest time of WGA, And a method for detecting the shortest time of a microorganism using PCR.

Clostridium difficile is a bacterium that produces endogenous spores and toxins that are highly resistant to gram-positive, anaerobic, heat, radiation, and chemical disinfectants. It is a bacterium that produces diarrhea in antibiotic-associated colitis patients , Pseudomembranous colitis, diarrhea caused by C. difficile (Clostridium difficile associated diarrhea, CDAD) (Boer, E. et al., Int . J. Food Microbiol . 144: 561564 (2011)). It is present in the intestinal flora and is about 2% in the healthy adults but it is present in the high rate of 10-20% in the neonates within 1 month of the elderly and acts as opportunistic infectious disease when the host immunity is weak. C. difficile produces two types of enterotoxin, toxin A, and toxin B, a cytotoxin, which is pathogenic. Toxin A mainly damages epithelial cells in the intestinal tract and causes bleeding. Toxin B exhibits strong cytotoxicity more than 100 times stronger than toxin A (Lee, JY et al., J. Exp. of Bacteriol . Virol . 35: 217-226 (2005)).

Pathogenic C. difficile is recognized as a potential cause of food poisoning as it is separated from ground meat, but food-related studies are reported only as a result of monitoring in confined meat, which is a limited sample. Rodriquez-Palacios et al. Was isolated a 60 12 (20%) from the ground meat of C. difficile purchased at retail (Rodriguez-Palacios, et al, Emerg Infect Dis 13:.... 485-487 (2007)) in the study, such as C. difficile Bouttier pork sausage, have not been detected in the sample, and separating the second (1.9%) from the pulverized beef dog 105 (Bouttier, S. et al, Emerg Infect Dis 16....: 733-735 (2010)). Also Songer and the like to remove the 37 (42%) dogs from 88 to sample showed a high detection rate (Songer, JG et al, Emerg Infect Dis 5:.... 819821 (2007)). It has also been reported that pigs, cows, and chickens are separated from meat that they eat.

In the Food and Drug Administration (FDA) (2012), the conventional method for detecting microorganisms, which is a standard method, is listed. However, since the culture method consists of enrichment culture, isolation culture, confirmation test and serum test, . In addition, the selective medium method is a golden standard for isolating microorganisms, but it is known to be an inefficient detection method because it requires a lot of time and labor and has low sensitivity (Hyeon, JY et al., Korean J. Food Sci . Ani . Resour . 29: 506-512 (2009)). Recently, in order to minimize such disadvantages, various methods such as an immunological method such as a lateral flow system, an electric conductivity method, and a PCR method using DNA have been developed and used (Shearer , AE et al., J. Food Protect . 64: 788-795 (2001)).

The microorganism detection method requires the minimum bacterial concentration required for detection according to each method and is called the detection limit. Since food poisoning bacteria are present in very small amounts in food, most of them are lower than the detection limit, and therefore, a bacterial process that increases the number of microorganisms is absolutely necessary.

Therefore, a method with a low detection limit is advantageous for quickly detecting food poisoning bacteria. This is because it can shorten the time for increasing the number of microorganisms to the detection limit. It is the real-time PCR method that has the lowest detection limit of the microorganism measurement method to date and generally has a detection limit of 10 ¹ to 10 ³ cfu / mL level in the medium (Arvind, A. et al., J. Food Microbiol . 84: 217224 (2003)). In addition to low detection limit, there is no need to confirm the amplification product by electrophoresis. Because it is not only easy and quick to obtain the result but also the risk of contamination is low, recently, (Lee, HM et al., Koeran J. Apiculture 20: 109-116 (2005)). Since clostridium difficile is known to exist in a small amount in crushed meat, it can not be detected immediately by using any kit, and therefore, a bacterial process is required. In this way, rapid detection of microorganisms in trace amounts in foods requires a process of culturing above the detection limit and generally takes about 8 hours or more (Catarame, TMG et al., J. Food Safety 26: 1-15 (2006)).

Whole genome amplification (WGA) is a mechanism by which a random primer anneals a lot of template DNA to amplify a large number of DNA simultaneously and can amplify a large amount of DNA at 30 ° C using Phi29 DNA polymerase (Handyside, AH et al., Mol . Hum . Reprod . 10: 767772 (2004)). Since amplification using Phi29 DNA polymerase is carried out under the same temperature conditions, it is a great advantage that no equipment such as a thermal cycler is required. It is also possible to amplify nano gram DNA of various samples (eg, single cell, blood card, buccal swab, soil, plant, formalin-fixed paraffin-embedded tissue etc.) to obtain total genomic DNA of more than 10 μg (Geigl, JB et al., Nucleic Acids Res . 37: 105 (2009)).

The fastest detection method for microorganisms is the incubation time, which is the most time-consuming and fastest detection method. Therefore, in order to detect food poisoning bacteria present in food in real time by real-time PCR, it is possible to isolate and detect DNA by increasing the number of bacteria to the limit of detection through a microorganism enrichment process. Or by increasing the genomic DNA to the limit of detection without microbial growth, real - time PCR will be possible. Therefore, in the present invention, in place of the microbial enrichment process, a small amount of DNA was rapidly amplified using WGA to examine the possibility of detection by real-time PCR. Finally, the microbial enrichment process was replaced with a rapid DNA amplification process, And to develop a reduced speed detection technology.

Accordingly, a main object of the present invention is to provide a method for detecting the shortest time of a microorganism using a real-time PCR capable of detecting the pathogenic bacteria from a sample in which pathogenic microorganisms are present in a minimum amount of time.

According to one aspect of the present invention, there is provided a method for detecting the shortest time of a microorganism using real-time PCR comprising the steps of:

a) extracting DNA from a 10-diluted continuous microorganism concentration and performing real-time PCR to determine a detection limit of a real-time PCR of a specific microorganism;

b) performing a whole genome amplification (WGA) using DNA of a microorganism concentration not detected in the real-time PCR to generate a graph of DNA concentration by WGA time;

c) representing the graph as a function of DNA concentration (Y) for each WGA time (X), and substituting the detection limit into the function Y to obtain the shortest time of WGA; And

d) performing WGA on the microorganism for a shortest time of the WGA and then performing real-time PCR.

In the method of the present invention, the step (a) is a step of determining whether the DNA concentration of the microorganism contained in the sample is lower than the real-time PCR detection limit for the specific microorganism, and the sample containing the DNA concentration not detected by real- It can be an object of analysis of the present invention.

As one example according to step a), in the examples, the detection probability was determined by using real-time PCR on the concentration of decoded diluted microorganisms in Clostridium difficile, which is one of food poisoning bacteria, as 1.16 × 10 ¹ to cfu / ml level of the target gene was possible to show the detection is amplified in only 37.24 cycle (Ct, threshold value), as shown in Table 5, the detection limit is 1.16 × 10 ¹ cfu / ml. In real-time PCR, when the Ct value is arranged on the Y-axis, the slope is -3.3665 and the amplification efficiency is 98.17%. Therefore, clostridium difficile can be an object of the present invention when it is less than 11.6 cells per ml in the analytical sample.

In the method of the present invention, the step b) is a step of amplifying microbial DNA below the real-time PCR detection limit value using WGA, and can be detected by real-time PCR when the DNA concentration is increased using WGA.

As one example according to the step b), DNA isolated from a microorganism concentration of 1.16 x 10 < ⁰ > cfu / ml, which was not detected in the examples, was amplified with WGA to detect microorganisms at a concentration of 1.16 x 10 ^And the time required to increase the DNA concentration to 308 μg / ml from ¹ cfu / ml was calculated. (Y) as a function of amplified DNA concentration while performing WGA.

In the method of the present invention, the step (c) is a step of obtaining the WGA shortest time from the DNA concentration change function according to the WGA execution time to the real time PCR detection limit value. For example, supposing one clostridium difficile is present in a 25 g sample, one cell will be present at 250 g, including the diluent, And the concentration is calculated to be 3 × 10 ^-8 μg / ml. Since this concentration is below the detection limit, DNA amplification must be performed until detection limit of 3 × 10 ² μg / ml is detected using WGA (see FIG. 7). This is inserted into the WGA amplification curve of FIG. 6 to calculate the time required for amplification, which is 32 seconds or more.

In the method of the present invention, the microorganism can be detected by all the microorganisms which can be detected by real-time PCR after enrichment, so that there is no particular limitation on the microorganism to be detected. However, in the conventional food microorganism test method As shown, a food poisonous bacterium that exists in a low concentration in the food (ie, a concentration of 0.04 cfu / g if there is one cell at 25 g) and needs rapid detection so that it should not be detected in the 25 g sample . And is preferably Clostridium difficile among the food poisoning bacteria present in the food at a concentration of 10 < ^-3 > to 10 < ¹ > cfu / g.

Conventionally known microorganism detection has a disadvantage in that a considerable time is required in the process of raising the microorganism to a level exceeding the 'detection limit' which is the minimum bacterial concentration required for the detection according to the method. Especially, food poisoning bacteria are present in a very small amount in food, so most of them show bacterial concentration lower than the detection limit. Therefore, it takes a lot of time to enrich the microorganisms. Even if real-time PCR, which has the lowest detection limit among the microorganism detection methods, is currently used, the detection of a microorganism existing in a trace amount in a food generally takes about 8 hours or more (Catarame, TMG et al., J. Food Safety 26: 1-15 (2006)).

The method for detecting the shortest time of a microorganism according to the present invention is a method for detecting the shortest time of microorganisms by replacing the microorganism culturing process which has taken a long time in the conventional detection methods described above with WGA and finding the shortest time of WGA for a specific microorganism Can be dramatically reduced.

In the present invention, the shortest time of the WGA can be obtained through a function expressed by the following equation (1).

[Formula 1]

Here, X is WGA time and Y is DNA concentration.

In the present invention, Formula 1 is calculated from a graph in which DNA concentration is amplified by real-time PCR using WGA shown in FIG. That is, to compare the speed between WGA and microbial growth, WGA was also modeled by Equation 1 using a modified Pearson's model. By doing this, the induction unit and the growth rate can be obtained.

As described above, studies for separating Clostridium difficile from a patient's sample using a molecular biological method such as real-time PCR have been reported, but no studies on the method of isolating from food have been reported yet. In addition, the detection method using the conventional medium is known to be difficult to detect a trace amount of food poisoning bacteria existing in meat and vegetables when a high level of uptake bacteria naturally existing in food (Shin, BM et al., Korean J. Lab Med . 26: 27-31 (2006)). Therefore, real-time PCR, a molecular biologic detection method using DNA specificity, and the WGA method, a molecular amplification method, have inherent selectivity for food poisoning bacteria present in foods, so that a very small amount of Clostridium difficile can be detected accurately and quickly can do.

As described above, according to the present invention, the shortest time of the WGA can be calculated by substituting the real-time PCR detection limit of a specific microorganism into the graph represented by the DNA concentration per WGA time or its corresponding equation, and the WGA Followed by real-time PCR to detect specific microorganisms in the shortest time. Therefore, the method of the present invention is expected to be very useful in the field of food production, distribution, and management since it can detect a specific microorganism in a food in a trace amount of food poisoning bacteria in the shortest time.

Figure 1 is a summary diagram of a real-time PCR method.
2 is a summary diagram of the whole genome amplification (WGA) method.
Figure 3 shows the growth curve of microorganisms in BHI medium and the time required for real-time PCR detection.
Figure 4 shows the detection limit of microorganisms measured using real-time PCR in BHI.
5 shows the real-time PCR amplification efficiency of the microorganism target gene.
Figure 6 shows the concentration of DNA in real time PCR using WGA.
Figure 7 shows an amplification plot for real time PCR using WGA (30 sec, 1 min, 5 min and 10 min).

Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

Example 1 Strain Culture and Isolation

Clostridium difficile (KCTC 5009) was anaerobically incubated at 37 ° C in a chamber (Whitley A35 anaerobic chamber; Don Whitley Scientific Ltd., Shipley, UK) using a brain heart infusion broth (Oxoid, Hampshire, England) For 48 hours. Then, a single colony showing green / yellow fluorescence in a long-wave ultraviolet light was applied to a selective agar (BD BBL, Sparks, USA) and used for the experiment.

Example 2. Growth curve model in BHI

Clostridium Dipisilyl isolated in Example 1 was added to BHI containing 0.5% yeast extract, DL-cysteine (Sigma-Aldrich) and 0.1% sodium taurocholate (Sigma-Aldrich) The cells were cultured at 37 ° C for 24 h at 37 ° C. Cells were cultured at 37 ° C for 1 h at 10 ° C / 10 ° C. Cells were then decanted and cefoxitin-cycloserine fructose agar (Oxoid, Hampshire, England) And then the number of bacteria was measured.

Example 3. Application of Gum Pots Model

The data obtained from Example 2 were analyzed using a program (GraphPad Prism 4.0 Software; Graphad Software, San Diego, Calif., USA) applied to the modified Gompertz, time (LT) and growth rate log (GR) were obtained. The DNA amplification by WGA was applied to the deformed beater pads and the equations were obtained.

Equation: Gompertz *

Y = N ₀ + C? Exp [-exp {(2.718.mue / C) (lag -X) + 1}]

Here, N _{0 is the} log initial number of cells, C is the difference between the initial and final cell numbers, lag is the delay before growth, the same units are as X, m is the maximum specific growth rate, X is time,

Example 4. Extraction of genomic DNA

Genomic DNA was extracted from BHI-cultured Clostridium difficilium culture using the AccuPrep genomic DNA extraction kit (Bioneer Co., Daejeon, Korea) according to the manufacturer's instructions. To measure the amount of separated DNA, the concentration was measured at 260 nm and 280 nm using a spectrophotometer (Gehealtcare, Ultrospec 2100 pro, San Francisco, USA).

Example  5. Real time PCR  Condition

PCR products of each sample were obtained using the PCR primer set described in Table 1 below. For the real-time PCR, the reaction mixture consisted of 10 μL of TaqMan Universal Master Mix (Applied Biosystems, Foster City, USA), 3 μL of DNA solution, 2 μL of 1000 nM forward and reverse primers, and 1 μL of probe (250 nM). Real-time PCR was performed in a 96-well microplate using a StepOne Plus real-time PCR system (Applied Biosystems). The reaction was carried out at 50 ° C. for 2 minutes and at 95 ° C. for 10 minutes, followed by reaction at 95 ° C. for 15 seconds and at 60 ° C. for 1 minute for a total of 40 cycles (see Table 2). Ct (threshold cycle) values were analyzed with StepOne ™ software (Applied Biosystems).

Target
gene Primer
/ probe sequence (5 '- > 3') tcdB Forward GAAAGTCCAAGTTTACGCTCAAT Reverse GCTGCACCTAAACTTACACCA Probe FAM ^{1 )} -ACAGATGCAGCCAAAGTTGTTGAATT-TAMRA ^{2 )}

¹⁾ FAM, 6-carboxyfluorescein (the reporter dye)

²⁾ TAMRA, 6-carboxytetramethylrhodamine (the quencher dye)

System UNG
incubation Polymerase
activation PCR Hold Hold cycle (40 cycles) Denature Anneal / extend  Temperature (° C) 50 95 95 60  Time (sec) 120 600 15 60  Reaction volume (μL) 20

Example  6. WGA  Condition

(SEQ: TempliGen ™ WGA kit; SolGent co., Daejeon, Korea). The total reaction solution was prepared by adding 1 μL of sample DNA and 1 μL of denaturation buffer per reaction tube, Minute. The final volume was adjusted to 20 μL by adding 2 μL of neutralization buffer, 6 μL of sample buffer, 9 μL of reaction buffer, and 1 μL of enzyme mixture. (C1000 ™ Thermal Cycler, Bio-Rad Laboratories Inc, CA, USA) at 70 ° C. for 5 minutes.

Experimental results 1. Growth curves in medium

Since clostridium difficile is present in a low concentration in food, proliferation should first be carried out using a culture medium for rapid detection. BHI was artificially inoculated with clostridium difficile and cultured for 20-30 hours. The culture was taken over time, and the number of bacteria was measured to prepare a growth curve. It was also calculated using the modified Gompertz equation as a growth prediction model (see FIG. 3).

Figure 3 shows the growth curve of Clostridium difficile on BHI medium and the time required for real-time PCR detection. It takes time to increase the cell number to 1.16 × 10 ¹ CFU / mL from 1 cell per 25 g sample to the detection time, and it is estimated to be 6 hours and 30 minutes inferred from the growth curve.

formula:

¹⁾ X: time

²⁾ Y: log cell

In order to predict the growth of bacteria, the induction unit (LT) and the growth rate (GR), which are growth indicators representing the growth of bacteria, were determined as shown in Table 3 below. The Gompertz model is one of the most widely used forms of expression of bacterial growth. However, the Gompertz model has been reported most frequently and the pathogen modeling program program, PMP) and the Food and micro models (food micromodel, FMM) has been used in the model bears Perth (Moon, SY et al, Korean J. Food Sci Techno 36:... 349-354 (2004)). Clostridium difficile showed 3.775 in LT and 0.863 in BHI. The R ² value, which indicates the statistical fit of the predicted growth index, was 0.9894, which was close to 1.

Parameters of modified Gompertz equation ^{1 )} GR ²⁾ LT ³⁾ R ^{2 4)} Clostridium difficile 0.863 3.775 0.9894

^{1) The} modified Gompertx equation was

_{Y = N 0 + C · exp} {-exp [2.718 · μ / C) · (lag - X) + 1]}.

²⁾ GR: maximum growth rate (per hour).

³⁾ LT: the lag time before growth (hours).

⁴⁾ R ² : coefficient of determination.

Experimental results 2. Real time PCR  Detection limit

In general, clostridium difficile detection in food is carried out in a non-selective medium such as BHI, cooked meat media, and cultured for 48 to 72 hours using selective medium, CCFA. The conventional method uses a selective medium using biochemical properties, so it has a disadvantage in that it does not know whether toxins are produced in the isolates. In order to detect clostridium difficile rapidly, we have investigated the possibility of rapid detection using real - time PCR which is reported to have low detection limit among various detection techniques. First, the most important detection limits were measured in rapid detection technology. As a result of detection experiments using real-time PCR after extracting DNA from 10 consecutive diluted bacterial concentrations, the detection limit value was measured to be 1.16 × 10 ¹ cfu / ml and the detection limit of 10 ¹ level (See Fig. 4). In addition, as shown in Table 5 below, Ct, which is the threshold value of the fluorescence indicated by the fluorescence dye, that is, the threshold value at which the target DNA was detected, was estimated to be 38 cells per cell and 35 cycles to 10 Cells, and 32 cycles per 100 cells. The PCR amplification efficiency was calculated from the amplification curve slope for Clostridium difficile. In the experiment, the slope was measured at -3.3665 and the amplification efficiency was measured at 98.17% (see FIG. 5). The PCR amplification efficiency was calculated as (10 ^{-1 / slope} ) -1, which is known to be in the range of 80-120%.

Target Threshold cycle (Ct) 1.16 × 10 ⁸¹⁾ 1.16 x 10 ⁷ 1.16 × 10 ⁶ 1.16 x 10 ⁵ 1.16 x 10 ⁴ 1.16 x 10 ³ 1.16 x 10 ² 1.16 × 10 ¹ 1.16 × 10 ⁰ tcdB 16.37 19.14 22.98 26.72 30.62 34.20 37.36 37.24 -

¹⁾ CFU / mL

Detection limit R ² Slope Efficiency (%) ¹⁾ 1.16 × 10 ¹ 0.9821 -3.3665 98.17

¹⁾ Efficiency = (10 ^{-1 / slope} ) -1

Experimental results 3. Estimation of the shortest detection time using the growth curve

따라서 시료 25 g에 1 세포가 존재할 경우 tcdB 유전자(톡신 B의 유전자)를 target으로 하여 BHI 배지를 이용하여 증균 배양할 경우, DNA 추출시간 30분과 실시간 PCR 수행시간 1시간 반을 모두 합하여 약 8시간 30분 이내에 검출이 가능할 것으로 판단하였다.
The shortest detection time for detecting clostridium difficile by real - time PCR method was calculated. After adding 9 volumes of enrichment medium at the initial concentration (0.04 CFU / mL) assuming the presence of 1 cell of Clostridium Diphysyl in 25 g of sample, the real-time PCR detection limit at 0.004 CFU / mL, the microbial concentration It was considered that microbial growth of up to 11.6 CFU / mL was required before detection. If we convert this to log (log), we need to increase from -2.398 to 1.064, which is about 6 hours and 30 minutes when we substitute it into the following equation.
[formula]

Therefore, when 1 cell is present in 25 g of sample, when tcdB gene (toxin B gene) is used as a target and enrichment culture is carried out using BHI medium, the DNA extraction time is 30 minutes and the real time PCR execution time is 1 hour and 30 minutes. It was judged that detection could be made within 30 minutes.

Experimental Results WGA  Detection limit

Generally, according to the method provided by the manufacturer, WGA time is recommended within 2 hours. Therefore, to shorten the time for rapid detection of microorganisms, the detection limit was measured to find the point at which amplification of the concentration that can be detected by real-time PCR during the WGA process was amplified. WGA was performed using 1.16 × 10 ⁰ CFU / ml of DNA that was not detected in real-time PCR. Figure 6 shows the concentration of DNA in real time PCR using WGA. In the presence of 1 cell in 25 g of sample, time required to increase the detectable DNA amount to 308 μg / ml in the isolated DNA concentration was calculated to be 32 seconds inferred from the model equation (1) by WGA.

formula:

¹⁾ X: time

²⁾ Y: Concentration of DNA

As a result of the detection experiment, the threshold cycle (Ct) of 36.32 was measured in DNA subjected to WGA for 30 seconds as shown in Table 6 below. It was determined that the number of microorganisms had a detection limit value similar to 1.16 × 10 ¹ to 10 ² CFU / ml.

Reaction time (min) 0 0.5 One 5 10 30 120 Threshold cycle (Ct) ND ^{1 )} 36.32 37.83 36.80 36.93 35.73 36.67

¹⁾ ND: not detection

Experimental results 5. WGA Estimate the shortest detection time using

After the amplification of a small amount of DNA by WGA method, we tried to calculate the shortest detection time for detection of Clostridium difficile using real - time PCR. As shown in the following Table 7, assuming that 1 cell of Clostridium Diphysyl is present in 25 g of the sample, the initial DNA concentration is 3 × 10 ^-7 μg / mL, 9 times the amount of the enrichment medium, DNA amplification from 10 ^-8 μg / mL to real-time PCR detection limit of 3 × 10 ² μg / mL was considered to be detectable. FIG. 7 shows that when WGA was performed at a DNA concentration of 3 × 10 ^-8 μg / mL for 0.5 (30 seconds), 1 minute, 5 minutes, 10 minutes, 30 minutes and 120 minutes, the target Clostridium difficile Of the genes were detected. That is, clostridium difficil was detected. WGA time is short, it is found that about 32 seconds is required to substitute the equation shown in FIG. 6 for more accurate understanding. Therefore, if one cell is present in 25 g of sample, WGA can be detected for more than 32 seconds without microbial enrichment. Therefore, DNA amplification time, DNA extraction time of 30 minutes, It was judged that detection was possible in about 1 hour and 30 minutes.

WGA time 0 ¹⁾ 0.5 One 5 10 30 120 DNA conc. (ug / ml) 3 × 10 ^-7,2) 308 393 411 442 547 583

¹⁾ Time (minutes)

^{2) The} DNA concentration at 1.16 x 100 cfu / mL was 3 x ^10-7 ug / ml

In summary, the present invention has devised a method for detecting clostridium difficile present in food and recognized as a potential cause of food poisoning in the shortest time using real time PCR and WGA method in the presence of trace amounts. LT was 3.775 and GR was 0.863 when calculated using the modified Gompertz equation, which was transformed into a growth prediction model for the enrichment process. Real-time PCR was performed using the target gene, and the detection limit was measured to be 1.16 × 10 ¹ cfu / ml and to have a detection limit of 10 ¹ level. Assuming that 1 cell is present in 25 g of sample, detection by real-time PCR was possible by incubation in BHI medium for about 6 hours and 30 minutes. However, WGA was used to replace the microbe microarray by DNA amplification , It was amplified with a detectable amount of DNA after about 32 seconds. Therefore, the use of WGA significantly reduces the time required for the enrichment process, so that rapid detection of food poisoning bacteria is possible.

Claims (3)

A method for detecting the shortest time of a microorganism using real-time PCR comprising the steps of:
a) extracting DNA from a 10-diluted continuous microorganism concentration and performing real-time PCR to determine a detection limit of a real-time PCR of a specific microorganism;
b) performing a whole genome amplification (WGA) using a DNA having a microorganism concentration below the detection limit of the real-time PCR to generate a graph of DNA concentration by WGA time;
c) representing the graph as a function of DNA concentration (Y) for each WGA time (X), and substituting the detection limit into the function Y to obtain the shortest time of WGA; And
d) performing WGA on the microorganism for a shortest time of the WGA and then performing real-time PCR.
The method according to claim 1, wherein the microorganism is Clostridium difficile present in the food at a concentration of 10 ^-3 to 10 ¹ cfu / g.
2. The method according to claim 1, wherein the function of step (c) is the following formula (1).
[Formula 1]

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