WO2009091110A1

WO2009091110A1 - Method of concentrating and detecting various miocroorganisms in food by using hollow-fiber ultrafilter

Info

Publication number: WO2009091110A1
Application number: PCT/KR2008/005818
Authority: WO
Inventors: Gwang-Pyo Ko; Hee-Yeon Kim; Jae-Chang Cho; Kyu-Ho Lee; Kun-Soo Kim; You-Hee Cho; Soon-Jung Park
Original assignee: Snu R&Db Foundation; Hankuk University Of Foreign Studies Research And Industry-University Cooperation Foundation
Priority date: 2008-01-14
Filing date: 2008-10-02
Publication date: 2009-07-23
Also published as: KR100951903B1; KR20090078136A

Abstract

The present invention relates to a isolating, concentrating and detecting method of at least a microorganism in food by using a hollow fiber. According to the present invention, the method can make it possible to detect various microorganisms in food such as very small sized virus, and bacteriophage rapidly and accurately, thereby contributing food hygiene, and automation and industrialization of detecting method.

Description

METHOD OF CONCENTRATING AND DETECTING VARIOUS MIOCROORGANISMS IN FOOD BY USING HOLLOW-FIBER

ULTRAFILTER

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a process of isolating, concentrating and detecting at least a microorganism in food with a hollow fiber. The present invention provides a simple, rapid and accurate method of detecting various microorganisms such as virus, bacteriophage and the like.

(b) Description of the Related Art

Even though the developed technology, it is very difficult to prevent the food poisoning completely. The mixing and propagation of the microorganism is not capable of being detected with the naked eye for the food poisoning caused by microorganisms, and is required to technical skill. Thus, microorganism-borne food poisonings believed to be mostly difficult to prevent.

Food-borne diseases in human are about 250 caused by about 25 kinds of pathogens, and mostly related with stock farm products. Important pathogens propagating via stock farm products include Salmonella species, E.coli:O157, Listeria monocytogenes, Camphylobacter jejunii, Staphylococcus aureus, Clostridium perfringens, etc. and cause food poisoning in human.

The number of subjects with food poisoning and viral food-mediated gastroenteritis increases continuously each year. Although the social and economical cost increase sharply each year, the virus and food causing the diseases cannot be found out clearly, thereby making it difficult to control food-borne infectious diseases. In case of the epidermal diseases, it is necessary to diagnose and treat rapidly and accurately, and to prevent the spread of disease. Bacterial food poisoning is divided to toxin type and infection type according to onset type. The infection type food poisoning occurs when bacteria contained in contaminated food proliferates in intestine. The bacteria causing the infection type food poisoning include Salmonella spp, Vibrio par aheamolyticus, Escherichia coli O157:H7 and the like. The bacteria causing toxin type food poisoning include Staphylococcus aureus, Clostridium botulinum and the like.

In a method of detecting microorganisms, it is very important to develop the control system where a microorganism causing mass outbreak of the food-borne bacterial diseases in a short time is detected early and the mass outbreak or the spread of the disease are prevented efficiently.

In general, to identify a microorganism causing a food poisoning accurately, microorganisms in specimens or contaminated food are cultured and then analyzed with biochemical tests, but it takes at least three days to several weeks. The culturing and biochemical analysis method is widely used as an official test, but is too late for preventing the mass spread of disease. For example, the detecting method of Salmonella spp. includes the steps of pre-culturing in Buffered Peptone Water (BPW), detecting preliminarily by culturing in selective media, and performing biochemical test and serological analysis. It takes about 5 to 6 days to detect.

While it is somewhat easy to culture and analyze food-poisoning bacteria, it is impossible to diagnose rapidly viral food poisoning due to a difficulty in culturing and observing a virus, and to detect various viral mutants by using common seroimmunologic method. It is reported that an infection and enteritis are caused by

Astro virus and Rotavirus. Recently, a Noro virus infection increases in all seasons.

Norovirus is a member of human Caliciviridae family and has a genomic RNA sized about 7,000 bp. Norovirus is also called as SRSV. Main source of Norovirus infection is food and is detected in children's acute gastroenteritis, which reveals the transmissible disease between humans. Because the detection of Norovirus is very important for public health and food quality control, there is a need for detecting most or all subtype by using PCR with high accuracy, sensitivity and speed. Rotavirus belongs to Reoviridae and causes acute diarrhea and gastroenteritis in infant and child which occur continuously in developed and undeveloped countries. The rotavirus-mediated gastroenteritis can be treated by supplementing water and electrolyte which are removed by vomiting and diarrhea. However, an effective vaccine for rotavirus has not been developed until now.ddbal

Particularly, when two or more types of viruses infect simultaneously, the virus types must be detected accurately. The accurate and rapid detection method is required on the basis of molecular biology. Recently, pathogenic virus is detected by a test method using polymerase chain reaction (PCR) and Reverse Transcription polymerase chain reaction (RT-PCR).

There are a still need for early detecting and preventing the pathogenic microorganism. A bacteria causing bacterial food poisoning can be detected by a method of pre-culturing the sample in selective media and observing colony formed on the media with a naked eye, and other indentifying method, for example PCR. However, conventional PCR method requires sufficient amount of sample to be detected and thus the microorganism in the sample must be cultured, thereby taking one day or more for detecting the microorganism. In addition, the method further includes a additional step for identifying the amplified gene product, such as electrophoresis and staining.

General detection method for microorganism takes four to seven days, because of a culturing step of microorganism. In addition, any microorganism cannot be cultured on various culturing conditions in laboratory and be isolated from contaminated food and patient. It is difficult to classify the microorganism clearly due to the unclear morphologic characteristics. The detection method, which is developed to resolve the problem, includes commercially used small biochemical kit, DNA/RNA or ELISA test, and improved automotive detection method, etc. However, the methods incline too heavily toward quantitative analysis, and thus have a difficulty in identifying the microorganism, because of many kinds of microorganisms in food, and determination of biochemical and immunological reaction (W. E. Hill et al., Bacteriological Analytical Manual 8th ed., 1998; S. Brunelle, IVD Technology published on June: 55-61, 2001; M. A. Pfaller, Emerging Infectious Diseases 7: 312-318, 2001). Furthermore, the methods still require the pre-culture of sample before analysis (W. E. Hill et al., Bacteriological Analytical Manual 8th ed., 1998). Additional detection methods developed to resolve the low accuracy, high cost and long time are also disadvantageous in big sized apparatus and high cost, and thus a new detection method is still required for detecting and diagnosing the microorganism causing food poisoning.

Until now, the concentration and purification method are selected depending on the kinds of food-poisoning microorganism, which makes it difficult to detect the microorganism rapidly and easily. Thus, there is still need for rapid and accurate method of detecting microorganism is still without pre-culturing step before analysis.

SUMMARY OF THE INVENTION

The present invention relate to the detection of pathogenic microorganisms such as Norovirus contained in a low concentration in a food. It is very important to develop a method of rapid and accurate detecting the infection of food poisoning microorganisms. The method is essential for the automation and/or industrialization of detecting method.

An object of the present invention is to provide a method of isolating and concentrating a small amount of microorganisms in food efficiently. Another object of the present invention is to provide a method of detecting food-poisoning microorganisms such as Norovirus rapidly and accurately.

Further object of the present invention is to provide a method of isolating and concentrating food-poisoning microorganisms contained in a small amount in food, thereby largely decreasing the cost and time for analyzing the food-poisoning microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a schematic flow chart a process of isolating, concentrating and purifying the microorganisms from food contaminated with food-poisoning microorganisms by using hollow-fiber filtration according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENTS The present invention is partially funded by Korean Food and Drug

Administration (08212 food 050) and Gyeonggi GRRC program (PR07011).

To achieve the object of present invention, the present invention provides a method of isolating and concentrating microorganisms from food comprising a step of performing ultra-filtration of the microorganism solution with a hollow fiber where the microorganism solution is obtained by treating a food with an isolating solution.

The microorganisms are at least one selected from the group consisting of bacteriophage, virus, bacteria, prokaryote and spore. The virus is at least one selected from the group consisting of Rotavirus and Norovirus. The microorganisms is contained at a various concentration in food, for examples small amount, for example, 1 to IxIO² CFU, or large amount, for example IxIO² to IxIO⁶ Or more. In particular, the present method is more useful, when the microorganism is at small amount in food.

In addition, the present invention provides a method of isolating and concentrating comprising a step of detecting isolated microorganisms with PCR.

The present invention is described in more detail. The present invention relates to a method of isolating and concentrating various microorganisms in food at a small amount.

The isolating solution for the microorganism are at least one solution selected from the group consisting of distilled water, beef extract, a mixed solution of glycine and NaCl, leucine solution, and Tris-HCl solution. Various isolating solutions can be used for isolating microorganisms in food at a small amount. Examples of the isolating solutions are indicated in Table 1, more preferably a solution of 0.05 to 0.5M glycine and NaCl, or 1 wt% to 5 wt% Beef extract solution.

Table 1

Isolating solution (5L) | pH

The microorganisms to be detected in the present invention include virus, bacteria, bacteriophage, spore, prokaryote and the like. The method of the present invention can isolate various viruses, for examples Norovius and Rotavirus, but does not limited thereto. For examples, the food-poisoning bacteria includes Salmonella spp, Vibrio paraheamolyticus, Escherichia coli O157:H7, Staphylococcus aureus, Clostridium botulinum, Bacillus cereus, Vibrio parahaemolyticus, Listeria monocytogenes, Yersinia enter icolitica, Clostridium perfringens, Salmonella spp., Shigella spp., Camphylobacter jejunii and the like.. In an embodiment, the hollow fiber used for ultra-filtration is made of various materials such as polysulfone . In general, the hollow-fiber ultra-filtration was used for haemodialysis in kidney dialysis subject and water sampling of prokaryote such as Cryptosporidium.

The hollow fiber used for ultrafiltration is a circular membrane having 0.2-1.0mm of inner diameter and 0.3-1.5mm of outer diameter, and is between microfilter and reverse osmosis membrane based on the pore size. The hollow fiber is a separating membrane operated by the difference in pressure. Unlike the microfilter having symmetric pore structure, the ultrafilter has a asymmetric structure including a active layer having lμm or smaller of pore size controlling the separating capability and filtering volume, and a support layer where solvent pass freely. The hollow fiber is made in a hollow fiber module to increase membrane area and packing density per unit volume.

The materials of ultrafiltration membrane include polysulfone, Cellulose Acetate (CA), Regenerated Cellulose, polyimide, Polyvinylidenefluoride (PVDF), Polyacrylonitrile (PAN) and the like. In particular, polysulfone-based ultrafilter is widely used in medical use and home use, because it is resistant to acid, base, and hydrolysis, chemicals.

An embodiment of ultrafϊltering apparatus according to the present invention is shown in Fig. 1. All tube connected to the apparatus is sterilized, and the pressure gauge is washed with 10% bleach solution. The examples of hollow fiber ultrafiltering apparatus are listed in Table 2 but not limited thereto.

Table 2: Examples of hollow fiber

In an embodiment, before performing the ultrafiltering, the filter can be pre- treated with at least a blocking solution selected from the group consisting of bovine calf serum, NaPP, Fetal bovine serum (FBS), glycine-added FBS, Beef extract, bovine serum albumin (BSA) and nutrient broth. More specifically, before filtering the microorganism solution, the filter is pre-treated with the blocking solution for 1 hour and then unbound blocking material is washed with distilled water. The examples of the blocking solutions are listed in Table 3.

Table 3

No blocking

5% calf serum No blocking

0.1% NaPP

0.01% Napp

No blocking

5% FBS

5% FBS added with 0.05M glycine

No blocking

1% Beef Extract

3% beef extract

5% beef extract

No blocking

5% BSA

10% BSA

No blocking

3% nutrient broth

5% nutrient broth

To test an effect of counter-washing of ultrafilter on the isolating rate, the different surfactants are used. The surfactant has a hydrophobic region and hydrophilic region, and minimizes the interaction between microorganism and surface of ultrafilter. It is reported that Tween 80, nonionic surfactant is effective for minimizing the adhesion of microorganism on the filter. The examples of surfactant include Tween 20 having a concentration of 0.01%, 0.5% or 1% and Tween 80 having a concentration of 0.01%, 0.5%, or 1%.

The present invention comprises a step of detecting a microorganism with PCR by using specific primers. In addition, the present invention provides a detection kit for food-poisoning microorganism including PCR primers, reaction buffer and Taq polymerase.

According to a method of isolating the microorganism, the microorganism is isolated from food, and then detected by amplifying specific region of the microorganism with PCR and identifying the specific PCR product according to an identifying method, for example, electrophoresis.

The PCR is any PCR method, preferably conventional PCR or real time PCR, and more preferably real time PCR. The conventional PCR is performed by amplifying specific DNA and then identifying the amplified product, with another identifying method, such as electrophoresis.

The template DNA is one extracted from the isolated microorganism. The template DNA is extracted according to any method of conventional DNA extraction, preferably alkaline extraction method, hot water extraction method, Column extraction method or Phenol/Chloroform extraction method, and more preferably alkaline extraction method. Optionally, before extraction of DNA template, the sample containing the microorganism is crushed with addition of sterilized water or saline, and then the supernatant is taken.

In an embodiment, the sample DNA extraction is performed, after the microorganism is isolated and concentrated according to the present invention, and suspended in sterilized water, or 0.85% saline, treated with Proteinase K, and then the pellet is collected. RT-PCR includes the steps of synthesizing a specific region of RNA as a template to produce cDNA(complementary DNA), and then amplifying, and specifically, 1) preparing cDNA from RNA with reverse transcriptase and 2) amplifying specific region by using cDNA. 2) step is the same as the method of amplifying specific region of gene from genomic DNA. The detection kit for microorganism can be a detecting kit for pathogenic RNA virus including the pairs of primers used for RT-PCR include a pair of primers specific for Rotavirus, a pair of primers specific for Astrovirus and pairs of primers specific for

Norovirus genogroup I and II . In addition, the detection kit uses multiplex RT-PCR.

The reaction condition of PCR can be determined by considering the type of PCR, PCR apparatus, and kinds of primers. The PCR includes real time PCR, conventional PCR, nested PCR, multiplex PCR, single PCR and micro PCR.

The detection method for pathogenic microorganism with PCR includes the steps of amplifying a microorganism with specific primers and identifying the PCR product according to electrophoresis. PCR is advantageous in efficiently detect a small amount of sample. The conventional detection method by using PCR are disclosed in KR 1999-65107Al where four genes of E.coli O157:H7 is detected with multiplex-PCR, and KR 1999-88425 Al where a method for detecting a vero (phonetic transcription) toxin gene and an 0157 antigen-synthesizing region gene as pathogenic factors in enteric hemorrhagic E. coli(EHEC) (or verotoxin producing E. coli(VTEC)) is provided to perform gene amplification by using oligonucleotides functioning as primers in the reaction of synthesizing nucleic acids, thereby providing a simple, rapid and highly sensitive clinical examination particularly in the examination of food poisoning and diarrhea, particularly EHEC symptoms causing severe disorders. The pairs of primers useful for the present invention can be any one amplifying the microorganism to be detected specifically. The detection kit is PCR primers specific for the microorganism to be detected and common components for PCR (reaction buffer solution, Taq DNA polymerase, etc.). An example of PCR detection kit includes:

(1) PCR Primers for specific microorganism to be detected; (2) Reaction Buffer; and

(3) Taq DNA polymerase.

In an embodiment of the present invention, the microorganism can be detected according to the following method.

Bacteriophage MS2 can be assayed with single agar assay (SAL, USEPA. 2001). MS2 was purified from infected cell lysate in SAL plaque assay plate. The purified sample was added with the same amount of chloroform as that of confluent lysis, centrifuged at 400Og rpm for 30 minutes and supernatant was taken and preserved at refrigerator. The concentration of MS2 was assayed with SAL.

The concentration of microorganisms is determined by taking 2.5 μJL sample from 500ml concentrate, performing PCR with primers and calculating RT-PCRU. The isolating rate is determined by dividing RT-PCRU with initial concentration inoculated on 5L buffer and converting to 100%. The examples of primers specific for each microorganism are as follows: Table 4

In Table 4, Letter "N" is A, T, G or C.

The concentration of Salmonella enterica is determined by taking 2.5μJi sample from 500ml concentrate, performing PCR with primers (SEQ ID NOs: 9 and 10), and calculating RT-PCRU. Salmonella enterica showed 329bp band in electrophoresis.

Forward primer (SEQ ID NO: 9)

5'-GCGCGAATTCAGCTTTTAATTACCGGTGGATGTGGCTTCC-S'

Backward primer (SEQ ID NO: 10) 5'-GCGCGAATTCGCCGTACTGCCGCAAGTAAATTTAAAGTTC-S'

The concentration of Vibrio cholera is determined by taking 2.5jΛ sample from 500ml concentrate, performing PCR with primers (SEQ ID NOs: 11 and 12), and calculating RT-PCRU. The isolating rate is determined by dividing RT-PCRU with the initial concentration inoculated in 5L buffer and converting to 100%. Vibrio cholera showed 419bp band in electrophoresis.

Forward primer (SEQ ID NO: 11)

5'-GCGCGAATTCTATATTGATCGCTTCGAAACTGAGTTTGCG-S '

Backward primer (SEQ ID NO: 12) 5'-GCGCGAATTCATCGCCAAATGTACCTACCTACTTTTTTGCATT-S'

The concentration of Cryptosporidium parvum oocyst (human and mouse strain

AZ-I) is determined with direct immunofluorescence assay according to U.S.

Environmental Protection Agency method 1623. The isolating rate is determined by dividing RT-PCRU with initial concentration inoculated in 5L buffer and converting to

100%. A pair of primers specific for Cryptosporidium parvum are as follows:

Forward primer (SEQ ID NO: 13)

5'-ACCGCTTCTCAACAACCATCTTGTCCTC-S'

Backward primer (SEQ ID NO: 14) 5'-CGCACCTGTTCCCACTCAATGTAAACCC-S'

As described above, the hollow-fiber ultrafiltration of present invention can detect even small amount of various microorganisms effectively, and food-poisoning microorganisms such as Norovirus rapidly and accurately. In addition, various microorganisms are detected by isolating, concentrating and purification simultaneously and has advantages in cost and time

The following examples are provided to demonstrate preferred embodiments of the present invention and the invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Example 1: Preparation of microorganism sample and Analysis of isolating rate

To detect various microorganisms in food, various samples were prepared. 1.1. Bacteriophage MS2 TATCC 15597-B1)

MS2 (ATCC 15597-B1) can be assayed with single agar assay(SAL, USEPA. 2001). MS2 was purified from infected cell lysate in SAL plaque assay plate. The purified sample was added with the same amount of chloroform as that of confluent lysis, centrifuged at 400Og rpm for 30 minutes and supernatant was taken and preserved at refrigerator. The concentration of MS2 was assayed with SAL.

The concentration was calculated as PFU(plaque forming unit) with using Single agar Layer assay(U.S. Environmental Protection Agency method 1602) adopting Escherichia CoIi(ATCC 11775) as a host.

1.2. Ampicillin resistant E.coli DH5α

E.coli (ATCC 11775) was transformed with a plasmid having Ampicillin resistant gene to produce Ampicillin resistant E.coli DH5α. E.coli was cultured in LB broth containing Ampicillin at 50/zg/ml at 37 ^°C, 200rpm for 16 hours. The transformant was spread on LB agar medium containing Ampicillin and measured as colony forming unit (CFU). Then, the isolating rate was calculated by dividing CFU with initial CFU of DH5α-amp+ inoculated in 5L buffer. Phosphate buffer saline (PBS) was used for diluting all experimental samples. Letter "M" means A or C, letter "W" is A or T and letter "Y" is T or C in the following sequence listings. Forward primer (SEQ ID NO: 15)

5'-GAG AGT TTG ATY MTG GCT CAG-3'

Backward primer (SEQ ID NO: 16)

5'- GAA GGA GGT GWT CCA RCC GCA-3'

1.3. Noro virus

Norovirus was received from Korea Center for Disease Control and prevention (KCDC).

Forward primer (GISKF) (SEQ ID NO: 1)

5 ' -CTGCCCGAATTYGTAAATGA-S ' Backward primer (GISKR) (SEQ ID NO: 2)

5¹-CCAACCCARCCATTRTACA-3^l

Forward primer (G II KF) (SEQ ID NO: 3)

5 ' -CNTGGGAGGGCGATCGCAA-S '

Backward primer (G II KR) (SEQ ID NO: 4) 5' CCRCCNGC ATRHCCRTTRTACATS'

Norovirus stool sample was amplified with RT-PCR and the concentration was calculated with RT-PCRU.

The used primers were GISKF, GISKR, GIISKF, and GIISKR. Depending on the type of Norovirus type, a pair of GISKF and GISKR, and another pair of GIISKF and GIISKR were used for RT-PCR and the concentration of Norovirus was calculated with RT-PCRU. The isolating rate was obtained by dividing RT-PCRU with initial concentration of Norovirus inoculated on 5L buffer, and converting 100 percentages.

Example 2: isolating rate depending on the composition of isolating solution

The sample was prepared by using 5L eluting buffer solution where bacteriophage MS2 of Example 1 was inoculated at a concentration of 1010MS2 /\00βi. As shown in Table 1, the isolating solutions were distilled water(isolating solution 1), 3% Beef extract solution (isolating solution 2), 0.05M Glycine-0.14M NaCl solution (pH 7.5) (isolating solution 3), and 10OmM Tris-HCl solution (pH 9.5) (isolating solution 4).

5L isolating solution inoculated with microorganisms was filtered with

Fresenius Hemoflow F8HPS filtering apparatus to final volume of 500 mL. The filtering apparatus was operated by Cole-Parmer model 7524-45 peristaltic pump at cross-flow of 1700ml/min. All tests were at three times and then standard error was calculated. The nucleic acid was extracted from 50OmL of concentrated solution to obtain the isolating rate. The isolating rate was The concentration was calculated as PFU with using Single agar Layer assay(U.S. Environmental Protection Agency method 1602) adopting Escherichia coli{ ATCC 11775) as a host.

Isolating rate = an amount of microorganisms remaining in 500 mL solution/(an amount of microorganisms inoculated on 5L) X 100 %

The experimental results were summarized in Table 5. 0.05M Glycine-0.14M NaCl(pH7.5) showed highest isolating rate among 4 kinds of isolating solutions. Table 5: Isolating rate of MS2 depending on isolating solution type

Example 3: Isolating rate of E.coli depending on use of blocking solution and type of isolating solution

The sample of this example was about 108ml of Ampicillin resistant DH5α- amp+ E.coli obtained from Example 1 which was cultured in LB broth containing Ampicillin and preserved in refrigerator.

1 mL of DH5α-amp+ E.coli was added to 3 different isolating solutions and diluted to obtain each 5L isolating solutions.

The hollow-fiber was blocked by using 5% beef extract 50OmL as a blocking solution, before filtering the isolating solution inoculated by microorganisms. The unbound blocking material was removed by distilled water. Then, the ultra-filtration was performed with three types of isolating solution inoculated by microorganisms. According to the method of Example 2 using Fresenius Hemoflow F8HPS ultrafiltering apparatus, 5 L eluting buffer solution inoculated by microorganisms was filtered to 500ml by using Cole-Parmer model 7524-45 peristaltic pump at cross-flow of 1700ml/min. All tests were at three times and then standard error was calculated. The nucleic acid was extracted from 50OmL of concentrated solution to obtain the isolating rate. The isolating rate was The concentration was calculated as PFU with using Single agar Layer assay(U.S. Environmental Protection Agency method 1602) adopting Escherichia coli(ATCC 11775) as a host. The test results were summarized in Table 6.

In case that the hollow fiber was not blocked as a pretreatment, the experiment was performed by the same method of the above ultrafiltration, except that the filter was blocked with blocking solution. The isolated E.coli was spread on LB agar medium containing Ampicillin and measured as CFU. Then, the isolating rate was calculated by dividing CFU with initial CFU of DH5α-amp+ inoculated in 5L buffer. The test results were summarized in Table 6.

Table 6: Isolating rate of DH5α-amp+ E.coli (%)

As shown in Table 6, the result of filter blocking by using 5% beef extract was compared with that of no blocking treatment. The blocking showed higher isolating rate than no blocking treatment. Among all blocking solutions, 0.05M Glycine-0.14M NaCl (pH7.5) was highest isolating rate.

Example 4: Simultaneous isolating of Norovirus and E.coli

1 mL of DH5α-amp+E.coli (108/ml) and Norovirus G I -4 at a concentration of 4xlO⁸/ml obtained in Example 1 was diluted in 0.05M Glycine-0.14M NaCl (pH7.5) to obtain 5L isolating solution.

Except for the isolating solution, the test was performed as the same method of Example 2. The isolated E.coli was spread on LB agar medium containing Ampicillin and measured as CFU. Then, the isolating rate was calculated by dividing CFU with initial CFU of DH5α-amp+ inoculated in 5 L buffer. Norovirus stool sample was amplified with RT-PCR and the concentration was calculated with RT-PCRU. The used primers were GISKF, GISKR, GIISKF, and GIISKR. Depending on the type of Norovirus type, a pair of GISKF and GISKR, and another pair of GIISKF and GIISKR were used for RT-PCR and the concentration of Norovirus were calculated with RT- PCRU. The isolating rate was obtained by dividing RT-PCRU with initial concentration of Norovirus inoculated on 5L buffer, and converting 100 percentages. The test result was shown in Table 7. Table 7: Isolating rate (%) of Norovirus G-4 and DH5α-amp+ E.coh

Mean isolating rate (%)

Norovius GI-4 DH5α-amp+ E.coli

50 34.8

The concentration of Noro virus GI-4 was 4xlO⁸/ml, and the isolating rate was 50% by 1 ml of inoculating volume. The isolating rate of DH5α-amp+ E.coli was 34.8%, which was higher than inoculation of DH5α-amp+ E.coli alone (14.64% of Table 6).

Example 5: Detection of microorganism in Food

Fruit was treated with 100/^(4x10 ) of Norovirus G II -4 at a concentration of 4xlO⁴/ml, dried completely, and isolated in 100ml of 3% Beef extract for 4 hours at

20Og rpm and room temperature. To remove contaminant, the isolating solution was added by chloroform at an amount of half volume of the isolating solution and centrifuged at 4^°C, 2,00Og for 10 minutes. Then, the virus in supernatant was concentrated and dissolved completely with the addition of 8% PEG 10,000 to final concentration of 0.3M NaCl. The solution was precipitated at 4^°C for 4 hours and then centrifuged at 4^°C, 12,00Og rpm for 30 minutes. The supernatant was discarded, and virus-containing pellet was suspended again in 2 mL of phosphate buffered saline. The

RNA was extracted from the solution, amplified with RT-PCR, and the isolating rate was calculated.

Table 8: Isolating rate with 3% Beef extract

The fruits was inoculated by 100M(10¹⁰) of each MNV-I and HPSl, dried completely and isolated in 1 L of 0.05M Glycine-0.14M NaCl (pH7.5) for 30 minutes (200rpm, room temperature). Like the method of Example 2, the ultra-filtration was performed with Fresenius Hemoflow F8HPS ultrafiltering apparatus, 5L isolating buffer solution inoculated by microorganisms was filtered to 500ml by using Cole-Parmer model 7524- 45 peristaltic pump at cross-flow of 1700ml/min. All tests were at three times and then standard error was calculated. The isolated HPSl was spread on LB agar medium containing Ampicillin and CFU was measured. Then, the isolating rate was calculated by dividing CFU with initial CFU of HPSl-amp+ inoculated in 1 L buffer. The isolating rate of MNV-I was obtained by performing real-time RT-PCR. The test results were summarized in Table 9.

Table 9: Isolating rate (%) of microorganism with 0.05M Glycine-0.14M

NaCl(pH7.5)

Example 6: Detection of various microorganisms Like the method of Example 2, the ultra-filtration was performed with

Fresenius Hemoflow F8HPS ultra-filtering apparatus, 5L isolating buffer solution inoculated by microorganisms was filtered to 500ml by using Cole-Parmer model 7524- 45 peristaltic pump at cross-flow of 1700ml/min. All tests were at three times and then standard error was calculated. Table 10 is isolating rate (%) of various microorganisms by isolating each microorganisms with Distilled Water and 0.05M Glycine-0.14M NaCl(pH7.5) as isolating solutions

Table 10

Table 11 is isolating rate (%) of various microorganisms by simultaneous-isolating with Distilled Water and 0.05M Glycine-0.14M NaCl (pH7.5) as isolating solutions.

Table 11

As shown in Tables 10 and 11, when isolating a microorganism along and isolating various microorganisms simultaneously, the simultaneous-isolating method showed higher isolating rate. When the filter was pretreated with blocking solution, the isolating rate increased as three times as simultaneous-isolating method. When simultaneous-isolating method was performed with blocking treatment, the isolating rate for MNV showed ten times or higher.

Claims

WHAT IS CLAIMED IS;

1. A method of isolating and concentrating at least a microorganism comprising a step of performing ultrafiltration of the microorganism solution with a hollow fiber where the microorganism solution is obtained by treating a food with an isolating solution.

2. The method of Claim 1, wherein the isolating solution is at least one selected from the group consisting of distilled water, beef extract solution, a solution including glycine and NaCl, leucine-containing solution, Tris-HCl solution, and a mixture thereof.

3. The method of Claim 2, wherein the isolating solution is 0.05 to 0.5M of a solution including glycin and NaCl, or 1 wt% to 5 wt% of beef extract.

4. The method of Claim 1, wherein, before performing the ultra-filtration, the hollow fiber is treated with at least a blocking solution selected from the group consisting of bovine calf serum, NaPP, glycine-added FBS, beef extract, bovine serum albumin (BSA), and a nutrient broth including peptone and Beef extract.

5. The method of Claim 1, wherein the microorganism is at least one selected from the group consisting of bacteriophage, virus, bacteria, and spore.

6. The method of Claim 5, wherein the virus is selected from the group consisting of Rotavirus and Norovirus.

7. The method of Claim 1 , wherein the microorganism is contained in the food in an amount of 1 to 1x10² CFU.

8. A method of detecting a microorganism in food, comprising a step of identifying the microorganism isolated and concentrated by a method of any one of Claims 1 to 7.

9. The method of detecting a microorganism in food of Claim 8, wherein the identifying step is preformed by identifying a product amplified from a nucleic acid extracted from the isolated and concentrated microorganism, or by identifying with biochemical property of microorganism after culturing the isolated and concentrated microorganism.