CN111235144A - Method for extracting intestinal tract aggregated Escherichia coli DNA and application of standard substance thereof - Google Patents

Abstract

The invention belongs to the field of nucleic acid standard substance preparation and molecular biology detection, and particularly relates to an extraction method of intestinal tract aggregated escherichia coli genome DNA, and application of the intestinal tract aggregated escherichia coli genome DNA method in preparation of an intestinal tract aggregated escherichia coli nucleic acid detection standard substance. The invention provides a method for extracting intestinal tract aggregative Escherichia coli genome DNA, which is mainly characterized in that before cracking thalli, lysozyme is used for treating cultured bacteria liquid, before cracking, lysozyme is used for treating thalli, and a large amount of protease K is used for treating in the cracking process. The invention lays a foundation for the extraction work of preparing a large number of nucleic acid qualitative standard samples, and provides a reference for the extraction method of bacterial genome DNA with difficult extraction of the genome DNA.

Description

Method for extracting intestinal tract aggregated Escherichia coli DNA and application of standard substance thereof

Technical Field

The invention belongs to the field of nucleic acid standard substance preparation and molecular biology detection, and particularly relates to an extraction method of intestinal tract aggregated escherichia coli genome DNA, and application of the intestinal tract aggregated escherichia coli genome DNA method in preparation of an intestinal tract aggregated escherichia coli nucleic acid detection standard substance.

Background

Enterobacter diarrheagenic bacteria are the main pathogenic bacteria of bacterial food poisoning and also the main cause of infectious diarrhea, and can be classified into 5 groups, namely Enterotoxigenic escherichia coli (ETEC), Enteroinvasive escherichia coli (Enteroinvasive e.coli, EIEC), enteropathogenic escherichia coli (EPEC), enterohemorrhagic escherichia coli (enterohemorrhagic escherichia coli, EHEC) and Enteroaggregative escherichia coli (EAEC), the EAEC was first reported in 1987, and was first isolated in children with intractable diarrhea, and is a novel class of diarrheagenic escherichia coli, the EAEC does not invade intestinal epithelial cells, but can cause intestinal fluid accumulation, does not produce heat-stable enterotoxin, does not produce heat enterotoxin, does not produce shiga toxin, and is only characterized in that it can form adhesion to Hep-2 cells, also called escherichia coli-2, the EAEC has no difference with common Escherichia coli in morphology, is gram-negative short bacillus, has the optimum growth temperature of 37 ℃, and has no special nutritional requirement.

EAEC takes food or water as main transmission medium, is transmitted through the fecal oral route, is generally susceptible to human beings, and has the symptoms of poisoning of adults as moderate diarrhea and persistent diarrhea of more than 2 weeks after infection of infants. In addition to enterotoxigenic E.coli infections, EAEC is also a major cause of diarrhea in travelers, and it is currently turning worldwide from sporadic to fulminant epidemics. Food-borne pathogenic bacteria cause serious food safety problems, so that public health safety is seriously threatened, scientific, rapid and accurate detection of food-borne pathogenic bacteria is an important means for preventing and controlling food safety problems caused by food-borne diseases, compared with the traditional microbiological culture method, Polymerase Chain Reaction (PCR) with high sensitivity, strong specificity, short time consumption and wide application range is widely applied, however, food microorganism reference substances used as positive control or quantitative standard of PCR detection are deficient in the whole world, and the reference substances suitable for food microorganism detection on a reference substance resource sharing platform are few, and basically stay in the stage of pure species provided by a strain preservation organization. Therefore, establishing the nucleic acid reference substance for detecting the food-borne pathogenic bacteria is a key method for changing the deficiency status of the reference substance. In the extraction process of EAEC genome DNA, different from general escherichia coli, the thalli of EAEC is not easy to crack, the viscosity of thalli cracking liquid is far higher than that of other escherichia coli, so that the extraction of EAEC genome DNA is difficult, and the preparation of nucleic acid reference substances cannot be directly extracted by using the bacterial genome extraction kit provided on the market at present. At present, no special research is available for EAEC genomic DNA extraction and nucleic acid reference substance preparation.

Disclosure of Invention

In order to solve the technical problems, the invention mainly optimizes the TaKaRa and the whole type golden bacteria genome DNA extraction kit by comparing the extraction effects of various kits on EAEC genome DNA, thereby improving the extraction amount of EAEC genome DNA and providing an efficient extraction method for the preparation of early samples in the research and development of food microorganism nucleic acid reference substances.

On one hand, the invention provides an extraction method of intestinal tract aggregative escherichia coli genome DNA, which is mainly characterized in that before the thalli is cracked, lysozyme is used for treating cultured bacteria liquid, and before the thalli is cracked, lysozyme is used for treating the thalli and a large amount of proteinase K is used for treating in the cracking process.

Further, when lysozyme treatment is adopted, 3-5 mg of lysozyme is used for treating bacteria liquid per ml, and the bacteria liquid is treated for 35-45 min at the temperature of 35-40 ℃; preferably, the lysozyme treatment is carried out at 37 ℃ for 40min by using 4mg of lysozyme per ml of bacterial liquid.

Further, when the thalli is cracked, 0.7-0.9 mg of proteinase K is used for each ml of bacterial liquid, and the bacterial liquid is treated for 30min at the temperature of 55 ℃;

preferably, 0.8mg of proteinase K is used per ml of bacterial liquid when the bacteria are cracked, and the bacterial liquid is treated for 25-35 min at 50-60 ℃.

Further, the whole type gold bacterium genome DNA extraction kit EE161-01 or TaKaRa bacterium genome DNA extraction kit 9763 was used for extraction.

Further, the extraction uses a whole type aureobacterium genome DNA extraction kit.

Further, the method for extracting the intestinal tract aggregating Escherichia coli genome DNA comprises the following steps:

(1) 1mL of overnight-cultured intestinal tract aggregated Escherichia coli suspension is taken, centrifuged, supernatant is discarded, and 200 mu L of resuspension buffer (RB11) containing 4mg of lysozyme is added; incubating at 35-40 ℃ for 35-45 min, centrifuging, and removing supernatant;

(2) adding 100. mu.L of lysis buffer (LB11) and 40. mu.L of 20mg/mL proteinase K solution to the product after supernatant removal in (1); incubating for 25-35 min at 50-60 ℃; after the incubation is finished, adding 20 mu L of RNase A, uniformly mixing and standing for 10 min;

(3) adding 400 μ L of Binding Buffer (BBII) to the product left standing in (2), and vortexing for 30 s; repeating the previous steps, and collecting 3 parts of lysate prepared from 1mL of thalli to the same centrifugal column through centrifugation; and rinsing and eluting to obtain the genome DNA.

The application of the method in preparing the intestinal tract aggregated escherichia coli nucleic acid detection standard substance; and the intestinal tract aggregated Escherichia coli genome DNA product prepared by the method and the application of the intestinal tract aggregated Escherichia coli genome DNA product as an intestinal tract aggregated Escherichia coli nucleic acid detection standard substance are all within the protection scope of the invention.

In the method of the present invention, the whole gold bacterium genomic DNA extraction kit EE161-01 or TaKaRa bacterium genomic DNA extraction kit 9763 is a preferable kit, and the actually usable kit/extraction method is not limited to these two, and other kit/extraction method may use the steps of lysozyme treatment and a large amount of proteinase K treatment in the present application.

Drawings

FIG. 1 is a diagram showing the results of agarose gel electrophoresis analysis of EAEC genomic DNA extracted from various kits; wherein M: full gold D15000+2000DNA Marker (production); 1: EAEC genome extracted by the full-scale gold kit; 2: EAEC genome extracted by OMEGA kit; 3: EAEC genome extracted by the Tiangen kit; 4: EAEC genome extracted by TaKaRa kit;

FIG. 2 is a diagram showing the result of extracting EAEC genomic DNA by the optimized TaKaRa kit for agarose gel electrophoresis analysis; wherein M: tiangen D15000+2000DNA Marker; 1-4: EAEC genome extracted by TaKaRa reagent box after optimization;

FIG. 3 is a diagram showing the result of extracting EAEC genomic DNA by the whole gold kit after lysozyme lysis by agarose gel electrophoresis analysis; wherein M: tiangen D15000+2000DNA Marker; 1-3: EAEC genome DNA extracted after lysozyme cracking;

FIG. 4 is a diagram showing the result of extracting EAEC genome DNA by a full-scale gold kit for analyzing lysozyme lysis by agarose gel electrophoresis and increasing the dosage of proteinase K; wherein M: tiangen D15000+2000DNA Marker; 1-4: EAEC genomic DNA;

FIG. 5 is a diagram showing the agarose gel electrophoresis analysis of the amplification products of characteristic genes astA, aggR, pic, and uidA amplified by PCR using the extracted EAEC genomic DNA as a template.

Detailed Description

Example 1

Experimental materials, instruments and materials

The EAEC strain has a strain preservation number of CICC 24186, the characteristic gene primer sequence is shown in Table 1, and the characteristic virulence gene primer refers to the national standard GB 4789.6-2016.

TABLE 1 EAEC characteristic genes and primer sequences thereof

The culture method comprises weighing 24.5g brain heart leachate broth culture medium dry powder, dissolving in 1L distilled water, subpackaging, autoclaving at 121 deg.C for 20min, cooling, inoculating 1% of EAEC, and culturing on 37 deg.C shaker at 180rpm overnight for about 12 h.

Brain heart infusion broth medium (BHI) was purchased from beijing luoqiao technologies, inc; tris-boric acid buffer powder was purchased from Biotechnology engineering (Shanghai) GmbH; agarose was purchased from Beijing Quanzijin Biotechnology Ltd; absolute ethanol was purchased from the national pharmaceutical group; d15000+2000Marker was purchased from Tiangen Biotechnology, Inc.; GelStain was purchased from Beijing Quanjin Biotechnology Ltd; OMEGA bacterial genome DNA extraction kit (D3350-01); a rhizobacterium genome DNA extraction kit (DP 302); a complete type gold bacterium genome DNA extraction kit (EE 161-01); TaKaRa bacterial genome DNA extraction kit (9763).

The vertical high-pressure steam sterilizer is purchased from Shanghai Shenan medical instrument factory; the constant temperature culture shaker was purchased from shanghai noon cheng analytical instruments manufacturing ltd; double purification benches were purchased from suzhou purification equipment ltd; electronic balances were purchased from Sidoolis (Beijing) Inc.; eppendorf centrifuges were purchased from Ed. Co, Germany; the water bath was purchased from Beijing eastern Jingrui science and technology development, Inc.; ice-making machines are available from north kyo dantian she technologies; thermo Nanodrop 2000/2000c was obtained from GeneLimited, and gel imaging systems were obtained from Beijing Junyi Oriental electrophoresis Equipment, Inc.; the electrophoresis apparatus was purchased from Beijing Junyi Oriental electrophoresis Equipment Co.

Example 2

Extraction of EAEC DNA by multiple kits

Extraction kit and method

(1) OMEGA bacterial genomic DNA extraction kit (D3350-01): the bacterial genome extraction procedure was referenced to kit instructions and the genomic DNA samples were stored at-20 ℃.

(2) Rhizobacteria genomic DNA extraction kit (DP 302): the bacterial genome extraction procedure was referenced to kit instructions and the genomic DNA samples were stored at-20 ℃.

(3) Whole formula gold bacterium genome DNA extraction kit (EE 161-01): the bacterial genome extraction procedure was referenced to kit instructions and the genomic DNA samples were stored at-20 ℃.

(4) TaKaRa bacterial genome DNA extraction kit (9763): the bacterial genome extraction procedure was referenced to kit instructions and the genomic DNA samples were stored at-20 ℃.

Detection of DNA concentration, purity and integrity

1 mu L of each of the genomic DNA samples extracted by the four kits was used to test the purity and concentration of the extracted EAEC genomic DNA using Thermo Nanodrop 2000/2000 c.

Respectively taking 2 mu L of genome DNA samples, uniformly mixing with a proper amount of loading buffer solution (10X), loading the samples into 0.8% agarose gel containing one ten thousandth of GelStain, carrying out electrophoresis at constant pressure of 150V for 35min, adopting 0.5 xTris-boric acid (TBE) buffer solution as a buffer system, and analyzing the integrity and purity of the genome samples in a gel imager after the electrophoresis is finished.

Detection of EAEC characteristic virulence genes

Primers (example 1, table 1) were synthesized according to the primer sequence of the characteristic virulence gene of EAEC provided by the national standard for food safety food microbiology test of national standard GB 4789.6-2016 escherichia coli test (shanghai) and the characteristic virulence genes astA, aggR, pic, uidA were cloned using EAEC genomic DNA as a template, the PCR amplification reaction system is shown in table 2 and the PCR amplification reaction program is shown in table 3.

TABLE 2 EAEC characteristic gene PCR reaction System

TABLE 3 EAEC characteristic virulence gene PCR reaction program

After the PCR is finished, 5 mu L of PCR product is sampled and spotted in 1% agarose gel, electrophoresis is carried out for 25min at a constant voltage of 175V, a buffer system adopts 0.5 XTris-boric acid buffer solution, after the electrophoresis is finished, GelStain is used for developing, and the PCR result is observed in a gel imager.

Results

1mL of each overnight-cultured EAEC bacterial suspension was aspirated, and EAEC genomic DNA was extracted according to the standard method provided by the four kits, with the results shown in Table 4 and the results of electrophoretic analysis shown in FIG. 1.

TABLE 4 comparison of purity and concentration of EAEC genomic DNA extracted from four kits

	Full type gold	OMEGA	Tiangen (root of heaven)	TaKaRa
					Concentration (ng/. mu.L)	14.6	18.6	10.2	28.4
A260/A280	1.39	1.56	1.41	1.64
					A260/A230	0.89	1.12	0.76	1.09

The concentration of the extracted genome DNA of the four kits is 10-30 ng/. mu.L, the value of A260/A280 is about 1.60, the extracted genome sample is presumed to be polluted by protein, the value of A260/A230 is about 1.00, and the genome DNA sample may contain pollutants such as organic reagents or salt ions and the like. As shown in the bands 1-4 of FIG. 1, the genome bands extracted by the four kits have good integrity and no RNA pollution, but the concentration of the genome extracted by the four methods is low, the sample extraction work in the early stage of nucleic acid standard sample preparation consumes a large amount of resources, and the extraction steps need to be optimized for improving the yield. In view of the time-consuming extraction work of the EAEC genomic DNA from the four kits: TaKaRa < Tiangen < holotype gold < OMEGA, the extraction process of the OMEGA kit takes the longest time, and the extraction concentration of the Tiangen kit is too low, so the standard extraction methods of the TaKaRa kit and the holotype gold kit are optimized.

Example 3

Optimization method for extracting EAEC genome DNA by TaKaRa kit

The method is characterized in that a gram-positive bacteria extraction method is adopted in a standard extraction method of a TaKaRa kit, and the method is additionally provided with the following steps of treating a bacterial suspension on the basis of the gram-negative bacteria extraction method: taking 1mL of EAEC bacterial suspension which is cultured overnight, centrifuging, taking supernatant, adding 500 mu L of Buffer BS, adding lysozyme to the final concentration of 2mg/mL, optimizing on the basis of the extraction method, and prolonging the incubation time of 56 ℃ to 40min from the original 30min after adding protease K in the first step of material treatment. The extraction results are shown in Table 5, and the results of electrophoretic analysis are shown in FIG. 2.

TABLE 5 purity and concentration of EAEC genome extracted from TaKaRa kit after optimization

	1	2	3	4
					Concentration (ng/. mu.L)	34.7	32.1	36.5	33.2
A260/A280	1.56	1.54	1.57	1.56
					A260/A230	0.69	0.67	0.69	0.68

As shown in Table 5, the extraction concentration of the genome DNA of EAEC is about 36.5 ng/muL after optimization by adopting a TaKaRa kit gram-positive bacterium genome extraction method, the time consumption of the extraction process is increased by 1.5h, the values of the genomes A260/A280 and A260/A230 are lower than those before optimization, as shown in figure 2, the main band of the extracted genome DNA after optimization is clear, and the RNA pollution is avoided. The optimized extraction effect cannot achieve the purpose of quickly extracting a large amount of high-quality genome DNA.

Example 4

Method for extracting EAEC genome based on full-scale gold kit

The DNA extraction method referred to in the full gold kit (EE161-01) in example 1 was as follows: 1mL of EAEC bacterial suspension cultured overnight is taken, centrifuged, supernatant is discarded, and 100. mu.L of lysis buffer and 20. mu.L of protease K (20mg/mL, total 0.4mg) are added; mixing, and incubating at 55 deg.C for 15 min; after the incubation is finished, adding 20 mu L of RNase A, uniformly mixing and standing for 10 min; add 400. mu.L binding buffer and vortex for 30 s; adding all the solution into a centrifugal column, centrifuging and then discarding effluent; and rinsing and eluting to obtain EAEC genomic DNA.

The improvement process is as follows:

after reading the relevant literature, the inventors speculate that the peptidoglycan layer of the EAEC cell wall may be thick and therefore attempted to use lysozyme for treatment.

1mL of EAEC bacterial suspension cultured overnight is taken out, centrifuged and supernatant is discarded, and 200 mu L of resuspension buffer (containing 4mg of lysozyme) is added; incubating at 37 ℃ for 40 min; centrifuging and removing supernatant; add 100. mu.L of reagent LB11 and 20. mu.L of protease K (20mg/mL, total 0.4 mg); incubating at 55 deg.C for 30 min; after the incubation is finished, adding 20 mu L of RNase A, uniformly mixing and standing for 10 min; add 400. mu.L BBII vortex 30 s; adding all the solution into a centrifugal column, centrifuging and then discarding effluent; and rinsing and eluting to obtain EAEC genomic DNA. The results are shown in Table 6, and the results of the electrophoretic analysis are shown in FIG. 3.

TABLE 6 purity and concentration of EAEC genome extraction after Lysozyme cleavage treatment

	1	2	3
				Concentration (ng/. mu.L)	37.5	36.9	42.0
A260/A280	1.65	1.68	1.70
				A260/A230	1.19	1.24	1.36

As can be seen from Table 6, the extraction amount of EAEC genomic DNA after lysozyme incubation treatment increased nearly three times, as shown by bands 1-3 in FIG. 3, the extracted genomic bands were uniform and free from RNA contamination. In the extraction process, after the reagent LB11 and proteinase K are added and incubated at 55 ℃ for 30min, the bacterial liquid is still very viscous, so that the components in the EAEC bacterial cells are presumed to be complex, possibly the protein content is high, and the added proteinase K cannot completely digest the bacterial protein, so that the lysate is very viscous, and the subsequent extraction of genome DNA is not facilitated.

1mL of EAEC bacterial suspension cultured overnight is taken out, centrifuged and supernatant is discarded, and 200 μ L of RB11 (containing lysozyme 4mg) is added; incubating at 37 ℃ for 40 min; centrifuging and removing supernatant; add 100. mu.L of reagent LB11 and 40. mu.L of protease K (20mg/mL, total 0.8 mg); incubating at 55 deg.C for 30 min; after the incubation is finished, adding 20 mu L of RNase A, uniformly mixing and standing for 10 min; add 400. mu.L BBII vortex 30 s; collecting thalli lysate with three times of volume into the same centrifugal column by centrifugation; and rinsing and eluting to obtain EAEC genomic DNA.

After the dosage of the protease K is increased, the viscosity of the thallus lysate is obviously reduced, and after the thallus lysate is transferred to an adsorption centrifugal column for centrifugation, liquid can easily pass through an adsorption film, so that 3 times of volume of the thallus lysate can be used for extracting DNA (when the viscosity is high, the adsorption film is easy to block, and even 1 time of volume of extract is difficult to pass through), the genome extraction effect is shown in table 7 after the dosage of the protease K is increased and the column loading times is increased, and the electrophoresis analysis result is shown in fig. 4.

TABLE 7 purity and concentration of EAEC genomes extracted after increasing proteinase K dosage

	1	2	3	4
					Concentration (ng/. mu.L)	198.7	195.6	188.3	208.2
A260/A280	1.63	1.63	1.62	1.64
					A260/A230	0.32	0.32	0.35	0.40

As can be seen from Table 7, the concentration of EAEC genomic DNA is increased by about 14 times after the gram-positive bacterium genomic DNA extraction method is adopted and the dosage of proteinase K is increased by one time, which indicates that the optimization is reasonable and has obvious effect on improving the concentration of genomic DNA. As shown in FIG. 4, the main band of the extracted EAEC genome is obvious, no RNA pollution exists, and the result shows that the extracted genomic DNA can be used for preparing qualitative nucleic acid standard samples.

Example 5

EAEC virulence gene PCR amplification detection

The extracted EAEC genomic DNA was tested according to the test standard of national standard 4789.6-2016. The extracted EAEC genomic DNA was used as a template, characteristic genes astA, aggR, pic, and uidA thereof were amplified by PCR, and the amplified products were analyzed by agarose gel electrophoresis, and the results are shown in FIG. 5. The astA gene, the aggR gene and the pic gene are characteristic virulence genes of the EAEC, the uidA gene can be detected in 97% of escherichia coli, the detection results of the four genes are positive, the size of a PCR product strip is consistent with the size of a target fragment, the extracted genome is determined to be EAEC genome DNA, and although the purity indexes of the extracted genome DNA, namely A260/A280 and A260/A230, do not reach 1.8, the prepared genome DNA sample has no influence on the detection result of a qualitative nucleic acid standard sample according to the virulence gene amplification result and can meet the requirement.

According to the invention, the extraction effects of various bacterial genome extraction kits on EAEC genomic DNA are compared, and the EAEC cell lysate is very viscous and not beneficial to subsequent genome extraction, so that the concentration and purity of the EAEC genomic DNA extracted by the used kit are very low. The steps for extracting the genome by the full-scale gold kit are simple and convenient, the consumed time is less, so that the extraction process of the full-scale gold bacteria genome DNA extraction kit is optimized, the extraction concentration of the EAEC genome DNA is improved by 14 times by increasing 30min of lysozyme incubation, one time of the using amount of proteinase K and 2 times of the volume of thallus lysate, the extracted genome DNA is used as a template for PCR detection of the virulence genes of the EAEC, and the result shows that 3 virulence genes are positive, thereby meeting the preparation requirements of the early-stage sample of the nucleic acid qualitative reference substance for food detection. The invention lays a foundation for the extraction work of preparing a large number of nucleic acid qualitative standard samples, and provides a reference for the extraction method of bacterial genome DNA with difficult extraction of the genome DNA.

Claims (9)

1. A method for extracting intestinal tract accumulating escherichia coli genome DNA is characterized in that a cultured bacterium solution is treated by lysozyme before thalli are cracked.

2. The method for extracting the intestinal tract aggregated Escherichia coli genome DNA as claimed in claim 1, wherein the lysozyme treatment is carried out at 35-40 deg.C for 35-45 min by using 3-5 mg lysozyme per ml of bacterial solution; preferably, the lysozyme treatment is carried out at 37 ℃ for 40min by using 4mg of lysozyme per ml of bacterial liquid.

3. The method for extracting the genome DNA of the enteroaggregative Escherichia coli according to claim 1, wherein 0.7-0.9 mg of proteinase K is used per ml of bacterial liquid when the bacteria are lysed, and the treatment is carried out at 55 ℃ for 30 min;

preferably, 0.8mg of proteinase K is used per ml of bacterial liquid when the bacteria are cracked, and the bacterial liquid is treated for 25-35 min at 50-60 ℃.

4. The method for extracting genomic DNA from enteroaggregative Escherichia coli according to claim 3, wherein the whole gold bacterium genomic DNA extraction kit EE161-01 or TaKaRa bacterium genomic DNA extraction kit 9763 is used for the extraction.

5. The method for extracting genomic DNA from enteroaggregative Escherichia coli according to claim 4, wherein the whole gold bacterium genomic DNA extraction kit EE161-01 is used for the extraction.

6. The method for extracting genomic DNA of enteroaggregative Escherichia coli according to claim 5, comprising the steps of:

(1) taking 1mL of overnight cultured intestinal tract aggregated Escherichia coli suspension, centrifuging, discarding supernatant, and adding 200 μ L of resuspension buffer containing 4mg of lysozyme; incubating at 35-40 ℃ for 35-45 min, centrifuging, and removing supernatant;

(2) adding 100 mu L of lysis buffer solution and 40 mu L of 20mg/mL protease K solution into the product from which the supernatant is removed in the step (1); incubating for 25-35 min at 50-60 ℃; after the incubation is finished, adding 20 mu L of RNase A, uniformly mixing and standing for 10 min; (3) adding 400 mu L of binding buffer solution into the product standing in the step (2), and performing vortex operation for 30 s; repeating the previous steps, and collecting 3 parts of lysate prepared from 1mL of thalli to the same centrifugal column through centrifugation; and rinsing and eluting to obtain the genome DNA.

7. The use of the method for extracting genomic DNA of enteroaggregative Escherichia coli according to any one of claims 1 to 6 for preparing a standard substance for detecting nucleic acid of enteroaggregative Escherichia coli.

8. An enteroaggregative E.coli genomic DNA product prepared by the method for extracting enteroaggregative E.coli genomic DNA according to any one of claims 1 to 6.

9. Use of the enteroaggregative E.coli genomic DNA product according to claim 8 as a standard for detection of enteroaggregative E.coli nucleic acid.

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