KR20090095751A - Method for rapid detection infectious microorganisms of unknown sample - Google Patents

Abstract

A method for detecting infectious microorganism in unknown sample is provided to quickly and selectively detect the microorganism and used for food test of agricultural, cattle, and marine products. A method for detecting infectious microorganism in unknown sample comprises: a step of inoculating unknown sample in animal cells and culturing; a step of collecting cells; a step of analyzing through real-time PCR; and a step of comparing the amount of nucleic acid and detecting microorganism.

Description

Method for rapid detection infectious microorganisms of unknown sample

The present invention relates to a method for rapidly detecting an infectious microorganism present in an unknown sample, and more specifically, to detect only an infectious microorganism present in an unknown sample in a short time by using a real-time polymerase chain reaction method and a cell culture method. It is about a method.

Pathogenic microorganisms around the world cause many diseases by contaminating water and human drinking water and food. Conventionally, methods for detecting them have been developed and used, but each detection method has a disadvantage in that it takes a long time or does not accurately identify a harmful sample.

There are two main methods for identifying viruses, harmful microorganisms in water systems: Information Collection Rule (ICR), Virus Monitoring Protocol for the Information Collection Rule: Laboratory Manual, 1995, EPA Number: 814B95002, and gene detection. Yoe-jin Choo and Sang-Jong Kim, Detection of human adenoviruses and enteroviruses in korean oysters using cell culture, integrated cell culture-PCR, and direct PCR, J of Micro., 2006)

Cell culture is a method for separating and identifying pathogenic viruses and non-pathogenic viruses, and is determined by whether a cytopathic effect (CPE) occurs. In general, it takes a long time of 1 to 4 weeks until the cell lesion effect is seen. In addition, not all cell culture methods have been developed. One virus can infect several cell types, and one cell can infect several virus types. Norwalk virus, for example, has not yet been developed as a cell culture technology and cannot be observed as a cytopathic effect. Adenovirus can infect Hela cells, A549 cells, etc. BGMK) cells can be infected with a variety of enteroviruses, including polioviruses, echoviruses, and coxsackieviruses. Poliovirus can also infect Vero cells, coxsackievirus A9 can infect HFF cells, and coxsackievirus B5 can infect Hela cells. Therefore, according to the cell culture method, it can be seen that the virus exists, but there are disadvantages of not knowing what kind of virus and what kind. Since the cell culture method takes a long time to detect, the utility value is insufficient to determine the presence of harmful microorganisms in the water and to prepare for the countermeasure.

The polymerase chain reaction method, which is one of gene diagnosis methods, is a method of amplifying a small amount of DNA or RNA, and thus, sensitivity, specificity, and rapidity are much superior to cell culture methods, thereby overcoming the disadvantages of cell culture methods. The polymerase chain reaction method is a method of extracting a nucleic acid of a microorganism directly from an aqueous sample, amplifying the nucleic acid, and then determining the presence of the microorganism by gel electrophoresis. However, if a small amount of harmful microorganisms in the water or a large amount is lost in the nucleic acid extraction process, there is a disadvantage that can be judged negative when detected by the polymerase chain reaction method. That is, even if not detected by the polymerase chain reaction method, microorganisms may exist. In addition, as a fundamental problem of the polymerase chain reaction method, it is difficult to quantitatively analyze and has a high risk of false positives due to contamination.

In order to solve this problem, the 'Integrated cell culture-Polymerase chain reaction (ICC-PCR) method' has been developed.This method infects water samples and incubates for 72 hours. After increasing the amount, the nucleic acid of the microorganism is extracted and detected by the polymerase chain reaction method. This method takes more time than the general polymerase chain reaction method, but it is shorter than the conventional cell culture method and has an advantage of higher detection probability than the general polymerase chain reaction method by increasing the amount of microorganisms.

In addition to the ICC-PCR method, there is also a PCR-nested PCR method. This method amplifies the gene once and then confirms it by gel electrophoresis, although it is not detected, the polymerase chain reaction product is used as a template. As a judgment method, detection sensitivity is higher than that of a general polymerase chain reaction method. However, in this case, there is a problem in that inactivated microorganisms that are not infected with a cell are detected only if the gene is present.

The above PCR method, ICC-PCR method, PCR-nested PCR method (SH Lee et. Al., The simultaneous detection of both enteroviruses and adenoviruses in environmental water samples including tap water with an integrated cell culture-multiplex-nested PCR procedure.J App. Mic. 2005) has a shorter detection time compared to cell culture, but it is not detected or still takes 2 to 3 days, or it is cumbersome to double the polymerase chain reaction.

In order to overcome the limitations of existing polymerase chain reaction method, real-time PCR was developed and the detection method of microorganisms in water using this was developed (Nlham T. et. All., Quantification of in vitro and in vivo cryptosporidium parvum infection by using real-time PCR, APP.ENV.Mic., 2006; Real-Time PCR for quantification of giardia and cryptosporidium in environmental water samples and sewage, App.Env.Mic. 2003; Z Gu, et. All., Multiplexed, Real-Time PCR for Quantitative detection of human adenovirus, J. Cli. Mic., 2003). The real-time polymerase chain reaction method quantitatively determines the initial concentration of a specific gene in a sample by measuring the fluorescence value in each cycle and identifying the cycle in which a certain amount of fluorescence value is generated in the polymerase chain reaction without the gel electrophoresis procedure. This method can be used for quantitative analysis compared to general polymerase chain reaction method and is less likely to be polluted since it is detected in a closed container. However, this method also amplifies the nucleic acid, if there is only a nucleic acid of the microorganism in the sample has the disadvantage that it is detected regardless of the infectivity of the microorganism. To pass drinking water standards, you need to know exactly how many harmful microorganisms are actually infectious. However, the sample may contain infectious harmful microorganisms and may also contain non-infectious microorganisms, such as those that are naturally dead or inactivated by disinfection. Therefore, the real-time polymerase chain reaction method also has a disadvantage that it is not possible to selectively quantitate live infectious microorganisms.

The present invention is to overcome the disadvantages of the conventional real-time polymerase chain reaction (Real-Time PCR) method and cell culture method, by applying the cell culture method to the real-time polymerase chain reaction method The present invention was completed by presenting a method capable of analyzing only infectious microorganisms in a short time.

Accordingly, an object of the present invention is to provide a method for rapidly detecting infectious microorganisms in an unknown sample using real-time polymerase chain reaction and cell culture.

The present invention is to provide a method for quickly and accurately identifying and detecting the presence and type of microorganisms having infectious properties that can cause fatal damage to an unknown sample. The unknown sample may infect microorganisms including drinking water, drinking water including tap water, rivers, water purification plants, foods such as agricultural, livestock and aquatic products, and secretions such as saliva and runny nose, and biological samples such as blood or feces. Refer to all samples present. Also, Infectious microorganism means an infectious microorganism which can affect the human body or an animal, such as viruses and protozoa. The detection method of the present invention can rapidly process quantitative analysis of infectious microorganisms as well as whether or not microorganisms are infected in the sample.

The present invention will be described in more detail.

The present invention comprises the steps of inoculating the unknown sample in animal cells; Recovering the cells to obtain nucleic acids; And detecting the nucleic acids by a Real-Time polymerase chain reaction (Real-Time PCR) method. The unknown sample refers to all samples that can be infected by microorganisms, including water sources such as drinking water, drinking water, foods such as agricultural, livestock, and marine products, and secretions such as saliva, and biological samples such as animal blood or feces.

Through the infectious microorganism detection method according to the invention it is possible to quantitatively analyze the amount of infection of microorganisms. The present invention is inoculated with animal cells to incubate the unknown sample to confirm the presence of infectious microorganisms in the unknown sample, and then, by detecting the nucleic acid from the cultured animal cells by time and comparing the amount of nucleic acid, and detecting the time Intervals shall be incubated for 2, 4, 6, 8, 24, 48 or 72 hours. At this time, the time to incubate to know the first amount of infectious microorganisms in the unknown sample infected with the animal cells is 2 hours to 8 hours. This is to determine whether the sample is infectious or quantitative, and that 2 to 8 hours after inoculation can detect infectious microorganisms in the sample. In addition, the amount of virus was increased by detecting and comparing the amount of nucleic acid obtained from the cultured animal cells according to the time interval. Therefore, even if a very small amount of virus is present, increasing the incubation time to 72 hours is sufficient unknown sample. Infectious microorganisms present in can be quantitatively detected.

Infectious microorganisms in the unknown sample detectable through the analysis method of the present invention include viruses and protozoa. Wherein the virus is an adenovirus (Adenovirus); Enteroviruses , including poliovirus , Coxsackievirus , Echovirus ; Astrovirus ; Hepatitis A virus ; Hepatitis E virus ; Reovirus; Rotavirus ( Rotavirus ) can be selected from the group consisting of, but not limited thereto.

In addition, the protozoa can be selected from Giardia (Giardia), the group consisting of Cryptosporidium, and can be selected from (Cryptosporidium) the group consisting of, and more particularly, Giardia person Calabria (Giardia lamblia), Cryptosporidium Parr boom (Cryptosporidium parvum) It is not limited thereto.

The detection method of the present invention can be applied to anything that is intended to detect a virus or protozoan in which the cell infection method has been developed. For example, even viruses such as Norwalkvirus , which have not yet been developed, can be detected by the method of the present invention if a cell infection method is developed.

The present invention uses animal cells capable of culturing the virus and protozoa to detect infectious microorganisms in unknown samples, wherein the animal cells are HeLa cells, A549 cells, Vero cells, HFF cells, BGMK cells, FRhK-4 cells and HCT-8 cells are selectable from, but are not limited to.

Hereinafter, the present invention will be described in detail.

At this time, if there is no other definition in the technical terms and scientific terms used, it has a meaning commonly understood by those of ordinary skill in the art.

In addition, repeated description of the same technical configuration and operation as in the prior art will be omitted.

In the present invention, the term 'polymerase chain reaction' or 'PCR' encompasses general (non-quantitative) PCR and quantitative PCR, and includes, for example, both general PCR and real-time PCR. Can be used as a concept to refer to 'generic PCR'.

In order to detect infectious viruses and protozoa separately from non-infectious viruses and protozoa according to the present invention, the following methods are used.

First, the animal cells are infected with viruses and protozoa. The method of infecting cells is cultured in a CO ₂ incubator at 37 ° C. until the cell occupies 80-90% of the cell culture area, and then the virus or protozoa. Animals were infected. At this time, the type of cells to be infected varies depending on the virus. Adenoviruses infected HeLa cells, and enteroviruses polioviruses, echoviruses, and coxsackieviruses infected BGMK cells. In addition, hepatitis A virus infected FRhK-4 cells and protozoa Giardia lambria infected HCT-8 cells. After infection, cells were recovered by time such as 2 hours, 4 hours, 6 hours, 8 hours, 24 hours, 48 hours, and 72 hours to extract DNA or RNA. After that, the Ct value of each amplification was measured by real-time polymerase chain reaction, and the difference was observed with time. As a result, after 2 hours, 4 hours, 6 hours, and 8 hours after infection, the cells showed almost similar results, but after 48 hours or 72 hours, the results were increased by 10 to 100 compared to 2 hours. In other words, when culturing microorganisms to know the first amount of infectious microorganisms, it is not necessary to cultivate a long time, and 2 hours to 8 hours of culturing is sufficient to know the first amount of microorganisms. In addition, up to 72 hours of incubation is required to detect very small amounts of microorganisms, but it can be seen that the initial amount is increased by about 100.

Through the above results, infectious virus or protozoa can infect a cell and can separate infectious virus or protozoa from non-infectious virus or protozoa in a few hours. Since the more sensitive real-time polymerase chain reaction method is used, it does not need to be cultured for 3 days like the cell culture-based polymerase chain reaction method. In addition, since cell culture-based polymerase chain reaction cannot detect a small amount of virus by the polymerase chain reaction method, it is difficult to know the first amount because its purpose is to increase the amount by cell culture method.

However, the method aims to determine the amount of infectious virus present in the original sample. According to this method, even if a small amount is detected by the high-sensitivity real-time polymerase chain reaction method can detect all.

In addition to the virus or protozoa mentioned in the present invention, any other virus or protozoa capable of being infected by culturing animal cells can detect harmful microorganisms by the method according to the present invention. For example, astroviruses, leoviruses, etc. can be cultured in Caco2 cells {R.M. Plnco, et. all., Detection of infectious astroviruses in water, App. Env. Mic., 1996} and the protozoan Cryptosporidium parboom is capable of infecting HCT-8 cells {Alexandra R. Keegan, et. all., Cell culture-Taqman PCR Assay for evaluation of Cryptosporidium parvum Disnfection, App. Env. Mic., 2003} can be detected in the same manner as above.

As described above, the present invention is a method that can be detected quickly and accurately in the detection of infectious microorganisms in addition to the dining table and the cooking room even when trying to determine whether the pathogen is contaminated hygienically in the school or company's lunch restaurants. Where it is possible to collect pathogens in or by using a sampling tool such as cotton swab to easily collect by culturing in the culture medium can be quantitative detection and detection of bacteria and identification of the target bacteria. In addition, it can be used to test for infectious microorganisms on animal blood, feces or secretions, and foods, including agricultural, livestock, and aquatic products, and microorganisms can be detected even with a small amount of samples.

As described above, according to the present invention, the cell culture time for separating the microorganisms in the sample into infectious and non-infectious takes about 2 to 4 hours, and the nucleic acid is extracted to perform real-time polymerase chain reaction. It takes about 3 hours to determine the presence of infectious microorganisms in 4-7 hours, which significantly shortens the time compared to the existing cell culture method, polymerase chain reaction method and cell culture-based polymerase chain reaction method. It is also judged whether a practical effect is expected to identify infectious microorganisms in unknown samples. This detection method can determine the presence and type of infectious microorganisms present in the sample in a wide range of targets, such as waterborne microorganisms and food test samples, as well as pathogenicity, so that drinking water test, restaurant hygiene test, agricultural, livestock and marine products It can be effectively used because it can be used for food test and microbial infection test of biological samples such as blood, feces and secretions.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited by the following examples, and various modifications or changes can be made within the spirit and scope of the present invention to those skilled in the art.

Example 1 Animal Cell Culture and Intracellular Virus and Protozoan Infection

BGMK cells (Korea Cell Line Bank), HeLa cells (Korea Cell Line Bank), FRhK-4 cells (Korea Cell Line Bank) and HCT-8 cells (Korea Cell Line Bank) were put in a 6-well culture vessel at 37 ° C and CO ₂ incubator. (Heal Force, Shanghailishen Scientific Equipment.Co, Ltd, China).

The cells were infected when the cultured cells occupied 90% of the cell culture area under a microscope. Viruses to infect cells include adenovirus 41 (VR-930, ATCC, USA), enteroviruses poliovirus 1 (VR-1464, ATCC, USA), ecovirus 6 (VR-1044, ATCC, USA), and cock Sakivirus B5 (VR-185, ATCC, USA) and hepatitis A virus (VR-1357, ATCC, USA) were used, and protozoa were Giardia lambria (ATCC50803, ATCC, USA). Adenovirus was infected to HeLa cells, poliovirus 1, Echovirus 6 and coxsackievirus B5, respectively, to BGMK cells. Hepatitis A virus infected FRhK-4 cells and protozoa Giardia lambria infected HCT-8 cells.

 As a negative control of each virus and protozoa, each virus and protozoa inactivated by heat treatment were used. The heat treatment is to destroy the virus's outer membrane, and the virus is inactivated by treatment at 100 ° C. for 10 minutes.

The specific method of infecting each cell with virus is to remove all of the medium in the cell culture container, leaving about 0.5 ml of medium, and then dissolving 100 μl of the virus solution dissolved in a 37 ° C water bath (Jeio Tech, Korea) in 7 cell culture containers. Add and shake well. The same virus and protozoa inactivated with negative control were added to 7 cell culture vessels at 100ul each and shaken well. Then 37 ° C, CO ₂ It was placed in the incubator and shaken every 15 minutes to allow the virus to fully infect the cells. After culturing for one hour, remove the virus-infected cell container to remove the virus that is not infected with the cells, remove all the medium, wash it thoroughly with Phosphate Buffered Saline (PBS, Gibco, invtrogen), and then clean the new medium. 3 ml of the cells were incubated for one more hour to recover cells infected with the virus. In addition, after 4 hours, 6 hours, 8 hours, 24 hours, 48 hours, 72 hours after infection, the virus-infected cells were collected and stored at -20 ° C for use in experiments. Inactivated virus and protozoa were treated the same.

Example 2 DNA and RNA Extraction

Cells collected by time were centrifuged at 3000 rpm for 10 minutes and the supernatant was used for adenovirus and Giardia lambria for DNA extraction kit (Accuprep® Genomic DNA Extraction kit, Bioneer, Korea), and for other viruses for RNA extraction kit (Accuprep). DNA and RNA were extracted using the Viral RNA Extraction kit, Bioneer, Korea).

Example 3 Real Time Polymerase Chain Reaction

Real-time quantitative PCR was performed using Exicycler ^™ Quantitative Thermal Block (Bionia, Korea) as a template with the DNA and RNA extracted in Example 2. Primer and probe sequences used in real time polymerase chain reaction are shown in Table 1 below.

Adenovirus 41 and Giardia lambria were subjected to real time polymerase chain reaction at 95 ° C for 5 minutes 1 cycle, 95 ° C for 10 seconds, and 57 ° C for 30 seconds using 45 cycles using the following primers and probes: Dualstar (Bioneer, Korea) It was. Other viruses, RNA viruses, were reacted at 42 ° C for 1 hour using a reverse transcriptase (RTase), Moloney Murine Leukemia Virus (MMLV), to convert RNA to cDNA, followed by real-time polymerase chain reaction. It was.

Adenovirus 41

Forward primer: 5'-AGCAGTGACCCTTAAGTATTCACC- 3 '(SEQ ID NO: 1)

Reverse primer: 5'-AGAGAGGTGCCGATGTTGTTAAG-3 '(SEQ ID NO: 2)

Probe: 5'-CCCCGCTTACAGTAAAAGGCCCGCC-3 '(SEQ ID NO: 3)

Poliovirus 1

Forward primer: 5'-GGTATTTCGAACGCCTATTCACAC-3 '(SEQ ID NO: 4)

Reverse primer: 5'-TTTAGAGATGCTGCACCATAGAGG-3 '(SEQ ID NO: 5)

Probe: 5'-ACCTAGTGCTGCCGACTGGTCCTTCA-3 '(SEQ ID NO: 6)

Ecovirus 6

Forward primer: 5'-GACATGGTGCGAAGAGTCTATTGA- 3 '(SEQ ID NO: 7)

Reverse primer: 5'-CACCTGCTCCGCAGTTAAGATT-3 '(SEQ ID NO: 8)

Probe: 5'-CCTCCGGCCCCTGAATGCG-3 '(SEQ ID NO: 9)

Coxsackievirus B5

Forward primer: 5'-GGTACCAGTCCCATGTTCTCCTA-3 '(SEQ ID NO: 10)

Reverse primer: 5'-AGACCGATGGCACCGTGTT-3 '(SEQ ID NO: 11)

Probe: 5'-TTTCCGAGCCAGGCGACTGTGG-3 '(SEQ ID NO: 12)

Hepatitis A Virus

Forward primer: 5'-CACGCTTTCTGTCTTCTTTCTTCC- 3 '(SEQ ID NO: 13)

Reverse primer: 5'-ATCTGTGTCCCCAATTTAGACTCC-3 '(SEQ ID NO: 14)

Probe: 5'-TGCTAATCATGGAGTTGACCCCGCCG- 3 '(SEQ ID NO: 15)

Giadia Rambria

Forward primer: 5'-AAACGGAGGGTAGAGCATGCT-3 '(SEQ ID NO: 16)

Reverse primer: 5'-GCAACAAGTTGGCCGAGACT-3 '(SEQ ID NO: 17)

Probe: 5'-TGGCGCATCTATGCACCCTCTTTCC- 3 '(SEQ ID NO: 18)

Table 1.

As a result, all virus and protozoa showed a similar tendency for 2 hours, 4 hours, 6 hours, and 8 hours after infection, and increased more than 100 hours after 2 hours after infection. This means that within 2 to 8 hours after infection with a cell, the amount of microorganisms is similar to that of the initial stage of infection. Inactivated viruses and protozoa, negative controls, were not detected by real-time polymerase chain reaction. This is because the inactivated virus was not infected with the cells and was washed away when washed with PBS. Real-time polymerase chain reaction was performed by directly extracting DNA or RNA from the negative control group, which did not infect cells, and showed an amplification curve. This is because the outer membranes of viruses and protozoa have been destroyed but have DNA or RNA.

After inoculating the samples into animal cells to infect the microorganisms through the above experiments, the cells were recovered several hours later and the nucleic acid was extracted to perform real-time polymerase chain reaction to detect only the infectious microorganisms as well as quantitative analysis. It can be seen that this is possible.

Figure 1a is a standard real-time polymerase chain reaction graph of adenovirus 41, each curve 1-7 shows 10 ^7-10 copies.

Figure 1b is a graph showing the real-time polymerase chain reaction of the adenovirus 41 by infecting the HeLa cells after recovery by time, curve 1 shown in the figure is the response of the experimental group infected for 72 hours, curves 2 to 5 Results of the response of the experimental group infected for 2, 4, 6 and 8 hours, respectively, curve 6 represents the negative control group.

Figure 2a is a standard real-time polymerase chain reaction graph of coxsackievirus B5, each curve 1-7 shows 10 ^7-10 copies.

Figure 2b is a graph showing the results of real-time polymerase chain reaction by recovering time after infection with coxsackievirus B5, curve 1 shown in the figure is the reaction result of the experimental group infected for 72 hours, curves 2 to 5 are each 2, 4 , 6, 8 hours, the response of the experimental group was infected, curve 6 represents the negative control.

Figure 3a is a standard real-time polymerase chain reaction graph of hepatitis A virus, each curve 1-7 shows 10 ^7-10 copies.

Figure 3b is a graph showing the real-time polymerase chain reaction of the hepatitis A virus infected with the cells and recovered by time, the curve 1 shown in the figure is the response of the experimental group infected for 72 hours, curves 2 to 5 Results of the response of the experimental group infected for 2, 4, 6 and 8 hours, respectively, curve 6 represents the negative control group.

Figure 4a is a standard real-time polymerase chain reaction graph of Giardia lambria, each curve 1-7 shows 10 ^7-10 copies.

Figure 4b is a graph showing the real-time polymerase chain reaction and recovered by time after infection with HCT-8 cells, curve 1 shown in the figure is the response of the experimental group infected for 72 hours, curves 2 to 5 are each 2 , 4, 6, 8 hours the response of the experimental group was infected, the curve 6 shows a negative control.

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Claims (8)

Inoculating an unknown sample into an animal cell and culturing it; Recovering the cells to obtain nucleic acids; And analyzing the nucleic acids by a real-time polymerase chain reaction (Real-Time PCR) method. The method of claim 1, The method of detecting an infectious microorganism, characterized in that the unknown sample is inoculated into animal cells and cultured to obtain nucleic acid from the cultured animal cells over time to compare nucleic acid amounts. The method of claim 2, The time interval is a detection method for infectious microorganisms, characterized in that the culture for 2, 4, 6, 8, 24, 48 or 72 hours. The method of claim 1, Incubating the unknown sample in animal cells and then culturing to know the initial amount of the microorganisms is 2 hours to 8 hours detection method for infectious microorganisms. The method of claim 1, The infectious microorganism in the unknown sample includes a virus and a protozoa. The method of claim 5, Wherein the virus is an adenovirus (Adenovirus); Enteroviruses , including poliovirus , Coxsackievirus , Echovirus ; Astrovirus ; Hepatitis A virus ; Hepatitis E virus ; Reovirus; Rotavirus ( Rotavirus ) detection method for infecting microorganisms, characterized in that selected from the group consisting of. The method of claim 5, Detection of an infectious microorganism, characterized in that the protozoa are selected from the group consisting of Giardia (Giardia), Cryptosporidium (Cryptosporidium). The method of claim 1, The animal cell is a method for detecting infectious microorganisms, characterized in that selected from the group consisting of HeLa cells, A549 cells, Vero cells, HFF cells, BGMK cells, FRhK-4 cells and HCT-8 cells.

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