TWI692528B - Methods for detecting E. coli and molecular markers used - Google Patents

Abstract

The invention provides a method for simultaneously detecting Escherichia coli ( Escherichia coli ) and specific pathogenic E. coli, and a molecular marker for detecting E. coli. The detection method has high sensitivity and can detect a variety of E. coli. The molecular marker can be used to detect various pathogenic E. coli.

Description

Methods for detecting E. coli and molecular markers used

The invention relates to a method for detecting E. coli and a molecular marker for detecting E. coli. In particular, it relates to a method for detecting E. coli and specific pathogenic E. coli using random amplified polymorphism (RAPD) and single nucleotide polymorphism (SNP).

Food hygiene and food-borne diseases are topics of great concern in recent years, usually because pathogenic bacteria enter the human body through the contamination of food or drinking water, resulting in the occurrence of food-borne diseases. Among them, Escherichia coli ( Escherichia coli ) is the main infectious pathogen causing diarrhea symptoms, and it will further lead to the occurrence of intestinal or extraintestinal diseases with high lethality. Since these diseases are mainly transmitted through inadequately processed livestock products, manure, or contaminated raw food, water sources, human feces, etc., in the food and other industries, in order to prevent the transmission of E. coli through humans and other carriers, it is necessary to Technical means for effective identification and testing of E. coli.

The traditional identification of food-borne microorganisms is to confirm the bacterial species through morphology and staining after cultivation. In recent years, in order to further improve the efficiency of strain identification, the application of enzyme-linked immunosorbent assay (ELISA), flow cytometry, enzyme-linked fluorescence analysis (ELFA), and in situ fluorescence hybridization methods have been developed in the identification , Pulse gel electrophoresis, polymerase chain reaction (PCR), real-time polymerase chain reaction technology. However, as an inspection for the food industry The technology of measuring E. coli, these technologies still have the problem of high time and labor cost.

For the detection technology of Escherichia coli that requires relatively low time and experimental requirements, RAPD-PCR to establish the genetic map of each bacterial species through random amplified polymorphism (RAPD) can be cited. For example, Packey et al. (refer to Non-Patent Document 1) disclosed a 3H primer used for RAPD-PCR. This 3H primer can distinguish E. coli NC101 in a sterile mouse specimen based on the difference in the fragment size of the amplicon (amplicon) , Escherichia coli K12 and other strains. However, for how to further detect various pathogenic E. coli strains and improve their efficiency and sensitivity, based on the limited experimental results of Packey et al., the technology using only the aforementioned 3H primers is not a technical means that can fully solve these problems. .

【Prior Technical Literature】

[Non-Patent Document 1] Packey CD, Shanahan MT, Manick S, Bower MA, Ellermann M, Tonkonogy SL, Carroll IM, Sartor RB (2013) Molecular detection of bacterial contamination in gnotobiotic rodent units. Gut Microbes, 4(5), 361-370. doi:10.4161/gmic.25824

In view of the above problems, the present invention aims to provide a technical means capable of detecting E. coli, which is a technical means capable of simultaneously detecting E. coli and various specific pathogenic E. coli, and is ideal from the viewpoint of time and labor cost Technical means.

The inventors conducted in-depth research and found that by using the nucleic acid sequence of c-terminal part of glutamate-ammonia-ligase of c-terminal region of E. coli as a molecular marker, For detection of various E. coli, the nucleic acid sequence is 687 base pairs. The sequence is shown as the 7th to 693th bases in SEQ ID No: 9.

Based on the nucleic acid sequence of the molecular marker, the inventor also designed a first primer set (having the nucleic acid sequences of SEQ ID No: 1 and SEQ ID No: 2 respectively) that can detect the molecular marker through polymerase chain reaction, and the second A primer set (having nucleic acid sequences of SEQ ID No: 3 and SEQ ID No: 4 respectively) and a third primer set (having nucleic acid sequences of SEQ ID No: 5 and SEQ ID No: 6 respectively).

At the same time, the inventor found that by using the above primer set, combined with the technique of high-resolution melting curve (HRM) and real-time polymerase chain reaction, the large intestine in the specimen can be detected in real time by analyzing the melting temperature of the high-resolution melting curve Bacillus

At the same time, the inventors found that by using 3H primers (with nucleic acid sequence of SEQ ID No: 17) for high-resolution melting curve (HRM) analysis, semi-quantitative analysis of E. coli can be performed.

The inventor further conceived a method for detecting E. coli, which uses the first primer set of the present invention and the aforementioned 3H primer at the same time. The first step of the first step is to perform RAPD-HRM PCR with the 3H primer, and the second step of the method The first primer set of the invention (having the nucleic acid sequences of SEQ ID No: 1 and SEQ ID No: 2 respectively) was subjected to HRM-PCR. By analyzing the melting curve of HRM-PCR, the presence of E. coli can be detected.

After further research, the inventor also found that by analyzing the single nucleotide polymorphism of each E. coli strain on the molecular marker, it can be used to identify different E. coli strains.

Therefore, the present invention provides the following technical means.

(1) A molecular marker for detecting Escherichia coli , which is characterized in that it contains the nucleic acid sequence of the glutamic acid-amino-ligase adenylyltransferase gene of the c-terminal region of E. coli, and the sequence It is 687 base pairs.

(2) A primer set for detecting E. coli, characterized in that the primer set is selected from the group consisting of a first primer set, a second primer set, and a third primer set, and the first primer set has SEQ ID No: 1 and SEQ ID No: 2, the second primer set has the sequences of SEQ ID No: 3 and SEQ ID No: 4, respectively, and the third primer set has the sequences of SEQ ID No: 5 and SEQ ID No: Nucleic acid sequence of 6.

(3) A probe for detecting pathogenic E. coli, characterized in that its sequence is selected from the group consisting of SEQ ID No: 10 and SEQ for detecting extraintestinal infection of E. coli (ExPEC) ID No: 11; SEQ ID No: 12 for detecting invasive E. coli (AIEC); SEQ ID No: 13, SEQ ID No: 14, SEQ ID No for detecting enterohemorrhagic E. coli (EHEC) : 15; SEQ ID No: 16 for detecting Toxigenic Escherichia coli (ETEC).

(4) A method for detecting E. coli, characterized in that the steps include: (a) For a test sample, the first primer set is used to perform a polymerase chain reaction to detect whether the reaction product contains E. coli c- The nucleic acid sequence of the glutamic acid-ammonia-ligase adenylyl transferase gene in the terminal region, the reaction product contains glutamine in the c-terminal region of E. coli The nucleic acid sequence of the acid-ammonia-ligase adenylyl transferase gene, that is, the presence of E. coli is detected in the specimen; wherein the first primer set has the sequences of SEQ ID No: 1 and SEQ ID No: 2, respectively.

(5) The method according to item 4, wherein the steps of the foregoing method further include: (b) performing a polymerase chain reaction on the specimen using a 3H primer to detect whether the reaction product contains an expansion of 702 base pairs in size Increaser, the reaction product contains an amplicon with a size of 702 base pairs, that is, the presence of E. coli is detected in the sample; and, the 3H primer has the sequence of SEQ ID No:17.

(6) The method according to item 4 or 5, wherein the method for detecting the nucleic acid sequence and the amplicon is at least one selected from nucleic acid staining and high-resolution melting curve analysis (HRM).

(7) A kit for detecting E. coli, which is characterized in that the kit includes a test sample and more than one set of primers, and the aforementioned set of primers is selected from a first primer set, a second primer set, and a third A group of primer sets and 3H primers, wherein the first primer set has the sequences of SEQ ID No: 1 and SEQ ID No: 2, respectively; the second primer set has SEQ ID No: 3 and SEQ ID No: The sequence of 4; the aforementioned third primer set has the nucleic acid sequences of SEQ ID No: 5 and SEQ ID No: 6, respectively; the aforementioned 3H primer has the sequence of SEQ ID No: 17.

(8) The kit according to item 7, wherein the kit further includes more than one probe, and the probe can detect the glutamate-ammonia-ligase in the c-terminal region of pathogenic E. coli A sequence of single nucleotide polymorphism on the adenylyl transferase gene.

(9) A screening method for Escherichia coli, which is characterized by comprising: detecting the expression of glutamate-amino-ligase adenylyltransferase gene in the c-terminal region of E. coli for a test sample Whether the protein is present, and if the protein is present, the presence of E. coli is detected in the specimen.

(10) The method according to item 9, wherein the detection step is performed using an antibody that recognizes the protein.

The invention provides an applicable method and molecular marker for detecting E. coli, which is a technical method that can simultaneously detect E. coli and a variety of specific pathogenic E. coli, and has high sensitivity, up to 1.26×10 ² / ml colony forming units. From the viewpoint of time and labor cost, the method used in the present invention can quickly detect E. coli directly from the specimen, and the experimental operation is relatively difficult, which is an ideal technical means.

[Figure 1] A single nucleotide polymorphism site showing the nucleic acid sequence of glutamate-ammonia-ligase adenylyltransferase in the c-terminal region of different pathogenic E. coli.

[Figure 2] shows the RAPD-PCR fingerprint of E. coli in Example 4, M in the figure represents the DNA molecular weight control standard, 1-10 represents the E. coli DNA derived from human feces, NTC table A control group without a DNA template is shown, and P.a. indicates a P. aeruginosa exotoxin A (ETA) gene expression control group.

[Figure 3A] shows the high-resolution melting curve obtained by using the 3H primer in the purified 702bp amplicon and human fecal E. coli DNA samples 1-10 in Example 5, NTC represents the control group without DNA template, green The melting curve of exotoxin A (ETA) gene expression of Pseudomonas was another control group.

[FIG. 3B] It shows the average melting temperature of E. coli DNA samples 1-10 from human feces in the results of high-resolution melting curve analysis using 3H primers in Example 5.

[Figure 3C] shows a semi-quantitative analysis chart based on the analysis results of E. coli DNA at different concentrations in the high-resolution melting curve analysis using 3H primers. The Y-axis value in the figure is the amount of fluorescent signal (dF/dt)

[FIG. 4A] This shows a high-resolution melting curve obtained from a human fecal sample and the purified 702bp amplicon in Example 5 using 3H primers after RAPD-HRM PCR.

[FIG. 4B] This is a high-resolution melting curve obtained from a human fecal sample and the purified 702bp amplicon in Example 5 after HRM PCR using the first set of primers of the present invention.

[Figure 4C] Agarose gel electrophoresis results of human fecal samples and the purified 702bp amplicon in Example 5 using 3H primers for RAPD-HRM PCR

[Figure 4D] The results of agarose gel electrophoresis of PCR products obtained from human fecal samples and the purified 702bp amplicon in Example 5 after performing HRM PCR using the first set of primers of the present invention

[Figure 5] Line graph showing the sensitivity of RAPD-HRM to E. coli detection, X axis Denotes the degree of dilution. The Y-axis is the number of colonies. The number on each point is the number of colonies measured at this dilution level, so as to know the colony concentration of the E. coli stock solution sample.

One of the embodiments of the present invention is to provide a probe for detecting a variety of specific types of pathogenic E. coli. Specifically, the aforementioned probe is used to identify various pathogenic E. coli in the c-terminal region valley A single nucleotide polymorphism sequence on the alanine-amino-ligase adenylyl transferase gene confirms whether the specimen detected by the probe contains the pathogenic Escherichia coli.

For example, the probe can be designed to identify more than one nucleotide variation point on glutamate-ammonia-ligase adenylyltransferase located in the c-terminal region shown in Table 1 or FIG. 1.

One embodiment of the present invention is to provide a screening method for E. coli, which detects glutamic acid-amino-ligase adenylyltransferase in the c-terminal region of E. coli for a test sample Whether the protein expressed by the gene is present, and if the protein is present, the presence of E. coli is detected in the specimen.

The step of detecting a protein in the aforementioned method can be accomplished using an antibody that recognizes the protein.

For the preparation of the aforementioned antibody, for example, the glutamic acid-amino-ligase adenylyltransferase gene of the c-terminal region of Escherichia coli can be expressed as a protein by a fungal system, and this protein can be used as an antigen to generally produce a single plant Or multiple antibodies to produce antibodies.

In another embodiment of the present invention, it can be further combined with a general method of preparing reagents or test papers to detect glutamate-amino-ligase adenylyltransferase base in the c-terminal region of E. coli Due to the antibodies of the protein expressed, reagents or test papers can be quickly screened for E. coli.

In this specification, the term "pathogenic E. coli" refers to E. coli that may cause clinical illness in humans, which includes adherent invasive E. coli (AIEC), toxigenic E. coli (ETEC), and extraintestinal infection E. coli ( ExPEC), enterohemorrhagic Escherichia coli (EHEC), etc. In the text or diagrams of the manual, for conciseness of expression, the abbreviations in parentheses may also be used.

The following describes the embodiments of the present invention with examples, but the scope of the present invention is not limited to these.

【Example】

Example 1: Collection of specimens from human feces

Stool samples from 10 healthy human donors (4 males and 6 females) with recorded age and eating habits were taken. Place 0.5-1g of the stool sample in a 1.5ml microcentrifuge tube, add 1ml of sterilized water to the sample to homogenize, centrifuge and collect the supernatant, as a stool sample for subsequent analysis.

Example 2: Morphological identification of E. coli in the specimen

E. coli from feces was pre-screened using eosin methylene blue agar (EMB Agar, Difco™, Heidelberg, Germany). An appropriate amount of supernatant was taken in a disposable inoculation loop, inoculated on the aforementioned eosin methylene blue agar by a four-zone drawing method, and cultured at 37°C. After culturing at 37°C for 12 hours, single colonies with green metallic luster were selected from the aforementioned eosin methylene blue agar medium, and the TSI test (BD Difco, Heidelberg, Germany) and Compact Dry were performed according to the manufacturer's instructions for the kit used in the following tests EC plate test (R- Biopharm AG, Darmstadt, Germany), Gram stain (BASO®, Taipei, Taiwan). In each test, E. coli strain ATCC 23815, Pseudomonas aeruginosa strain ATCC 31156, and E. coli DH5α were used as control groups.

The test results are shown in Table 1. Compared with the E. coli strain ATCC 23815 obtained from the American Type Culture Collection, all E. coli isolated from human fecal samples are Gram-negative bacteria, belonging to the E. coli group, Oxyglucose fermenting bacteria are gas-producing bacteria that do not produce hydrogen sulfide. In addition, in the Compact Dry EC plate test, all colonies appeared blue, indicating that they were E. coli with β-glucuronidase activity against X-Gluc.

Example 3: Purification of E. coli DNA

Genomic DNA from E. coli from feces was purified by QIAamp DNA Mini kit (QIAGEN, Hilden, Germany). According to the kit manufacturer's instructions, 1 mL of the bacterial suspension was centrifuged and the supernatant was removed. Add the buffer in the QIAamp DNA Mini kit to the pellet and centrifuge. Then use the QIAamp Mini filter column provided by the QIAamp DNA Mini kit for DNA separation. The isolated DNA was quantified by Nanodrop ultra-micro spectrophotometer (Maestrogen, Hsinchu City, Taiwan) Stool DNA concentration, and stored at -20 ℃.

Example 4: RAPD-PCR analysis

RAPD-PCR reaction was carried out using the E. coli genome of the foregoing Example 3 as a DNA template. The reaction was carried out in a final volume of 20 μL. The reaction solution contained 90 ng DNA template, 2U Taq polymerase (JMR, London, UK), 3 μL MgCl ₂ (25 mM), 2.5 μl 3H primer (20 μM), 2 μl dNTP (10 mM) (Promega, WI, USA). Among them, the primer is a 3H primer, a primer published by Packey et al. (Packey et al. 2013), and the primer has the nucleic acid sequence shown in SEQ ID No: 17. In addition, a primer set for detecting ETA exotoxin of Pseudomonas aeruginosa was used as a control group. In this primer set, the forward primer and the reverse primer had the nucleic acids shown in SEQ ID No: 7 and SEQ ID No: 8 respectively sequence.

The amplification of RAPD-PCR was carried out in the Thermalcycler polymerase chain reaction reactor (Biometra GmbH, Gottingen, Germany) under the following reaction conditions: 95°C for 5 minutes; 95°C for 1 minute, 36°C for 1 minute, and 72°C for 2 minutes. This repeated 36 cycles; the DNA extension was completed in 7 minutes at 72°C. PCR products were stored at 4°C.

The PCR product was taken on a 1.7% agar gel and subjected to 80V electrophoresis for 35 minutes to confirm whether an 702 bp amplicon was obtained. The results are shown in Figure 2. After the E. coli DNA isolated from the feces of healthy human donors was amplified by RAPD-PCR with 3H primers, the size of the amplicons was the same, 702bp.

Example 5: RAPD-HRM analysis using 3H primers

The 702bp amplicon of the foregoing Example 4 was further condensed with Qiaex II The gel extraction kit (QIAGEN, Hilden, Germany) is extracted and purified. The purification steps are according to the manufacturer's instructions. Next, the human fecal E. coli genomic DNA of the aforementioned Example 3 and the purified amplicon DNA were used as DNA templates to perform RAPD-HRM analysis using the aforementioned 3H primer.

According to the study of Tulsiani et al. (Tulsiani et al., 2010), the conditions were modified so that each tube of 25 μL of reaction solution contained: 12.5 μl HRM mix (QIAGEN, Hilden, Germany), 0.5 μL 25 mM MgCl ₂ , 1 μL 20 μM primer set, 9 μL DNA template (90ng) and 2μL molecular grade water. Among them, the primer set is the same as in the foregoing Example 3, and a 3H primer is used (the primer has the nucleic acid sequence shown in SEQ ID No: 17).

The reaction was RAPD-HRM with green fluorescence, which was performed in the Rotor-Gene Q real-time polymerase chain reaction instrument (QIAGEN, Hilden, Germany) under the following reaction conditions: 95°C for 10 minutes; 94°C for 1 minute, 40°C for 1 minute, Repeat 72 cycles at 72°C for 2 minutes; 7 minutes at 72°C. During the RAPD-HRM, fluorescence data is obtained at the end of the PCR's adhesion/extension cycle. In HRM analysis, the temperature is gradually increased between 75°C and 95°C at a temperature increase rate of 0.1°C/2 seconds. Use Microsoft Excel 2010 to statistically analyze the data of the melting curve from the experimental data of the three replicates, and express the data as the mean ± SD.

The analysis results are shown in Figure 3A. The melting temperature of the purified 702bp amplicon in the melting curve is 88.08°C. Relatively, a dominant peak appears in the melting curve obtained from the reaction of human fecal E. coli genomic DNA and the average temperature As shown in FIG. 3B, it is 88.1±0.22°C.

Example 6: RAPD-HRM detection of large intestine using 3H primer and primer set of the present invention Bacillus

Using the human stool sample from the foregoing Example 1, five fresh stool samples obtained directly from the subject were divided into two steps to detect Escherichia coli. In the first step, RAPD-HRM PCR was performed with 3H primers. In the second step, the first primer set of the present invention is used for HRM-PCR.

(1) RAPD-HRM analysis using 3H primers

According to the study of Tulsiani et al. (Tulsiani et al., 2010), the conditions were modified so that each tube of 25 μL of reaction solution contained: 12.5 μl HRM mix (QIAGEN, Hilden, Germany), 0.5 μL 25 mM MgCl ₂ , 1 μL 20 μM primer set, 9 μL DNA template (90ng) and 2μL molecular grade water. Among them, the primer set is the 3H primer published in the aforementioned previous document (the primer has the nucleic acid sequence shown in SEQ ID No: 17).

The reaction was carried out in the RAPD-HRM with green fluorescent emission in a Rotor-Gene Q real-time polymerase chain reaction apparatus (QIAGEN, Hilden, Germany) under the following reaction conditions: 95°C for 10 minutes; 94°C for 1 minute, 40°C for 1 minute, Repeat 72 cycles at 72°C for 2 minutes; 7 minutes at 72°C. During the RAPD-HRM, fluorescence data is obtained at the end of the PCR's adhesion/extension cycle. In HRM analysis, the temperature is gradually increased between 75°C and 95°C at a temperature increase rate of 0.1°C/2 seconds.

(2) HRM-PCR analysis using the first primer set of the present invention

Each tube of 25 μL of reaction solution contains: 12.5 μL HRM mixture (QIAGEN, Hilden, Germany), 1.75 μL primer set (10 μM), 2 μL DNA template and 8.75 μL molecular-grade water. Among them, the primer set is the first primer set of the present invention (the forward primer and the reverse primer have the nucleic acid sequences shown in SEQ ID No: 1 and SEQ ID No: 2 respectively) Column).

According to the manufacturer's instructions (Type-it HRM PCR kit, QIAGEN, Hilden, Germany), real-time PCR amplification was performed in Rotor-Gene Q (QIAGEN, Hilden, Germany). The reaction conditions were: hot start step at 95°C 5 minutes; denaturation step 95 ℃ 10 seconds, bonding step 58 ℃ 30 seconds, extension step 72 ℃ 25 seconds, and thus repeat 35 cycles; the final HRM melting step, the temperature is increased from 65 ℃ to 95 ℃, the speed is 0.1 °C/step, hold each step for 2 seconds. The fluorescence threshold of all samples is set manually.

The results showed that, as shown in FIG. 4A, if only 3H primers were used to screen E. coli in fresh untreated human feces by RAPD-HRM, the 702bp amplicon as the control group at the melting temperature of 88.45℃ Compared with the peaks, although the melting curve obtained from the human stool samples also has a peak at 88.48±0.13℃, it is not obvious.

However, when the first primer set of the present invention is further used for the second stage of HRM-PCR, the results are shown in FIG. 4B. The melting curves obtained from the 702bp amplicon and 5 human fecal samples are all at the melting temperature of 88.35±0.11℃ There are obvious peaks and the average peak is high.

The RAPD-HRM and HRM-PCR products were confirmed by agarose gel electrophoresis. As shown in FIGS. 4C and 4D, amplicons of 702 bp and 687 bp in size were confirmed, which were consistent with the target DNA molecular weight. Although the control group without template also has a band of about 600bp in Figure 4D, the corresponding peak of the PCR product on the HRM melting curve differs from the melting temperature of the target peak used for screening by about 3°C, so it can be obvious The difference between this product and the non-specific PCR product does not affect the judgment during actual detection.

Example 7: Semi-quantitative analysis

The human fecal E. coli genomic DNA of the aforementioned Example 3 was collected at different concentrations, and subjected to an HRM reaction under the same conditions as in Example 5, and then subjected to semi-quantitative analysis using Graph Pad Prism version 7 (GraphPad Software, CA). Taking the DNA concentration as the X axis and the fluorescent signal (dF/dt) as the Y axis, the analysis data is expressed as the average ±SD in the XY graph, and the Spearman correlation coefficient (r) and one-tailed probability test (one-tailed probability test) are calculated test) calculated value P. If the P value is <0.05, it is considered statistically significant.

The results of RAPD-HRM quantitative analysis of different concentrations of E. coli DNA (90ng, 180ng, 270ng and 360ng) are shown in Figure 3C. The fluorescent signal increases with the increase of E. coli DNA concentration, and the two are positively correlated, Spearman correlation The calculated values of the coefficient (r) and the one-tailed probability test (p) are r=0.9, P<0.05, and N=5. This result shows that RAPD-HRM analysis using 3H primers can semi-quantitate E. coli in the test specimen.

Example 8: Sensitivity of RAPD-HRM to E. coli detection

First, according to the research by Haase et al. (Haase et al., 2017), after modifying the conditions, calculate how many original colony type units (CFU) the DNA extracted in Example 3 originated from. After screening and purifying E. coli with eosin methylene blue agar, the purified E. coli was selected using an inoculation loop, inoculated into LB Broth (Neogen, Lansing, MI, USA), cultured at 37°C for 12 hours, and a group of 10 times was prepared after cultivation For serial dilutions, 10 μL of each dilution was inoculated on LB Agar (MDBio, Inc, Xinbei, Taiwan) plates and incubated at 37° C. for 12 hours. By counting the colonies cultured in serial dilutions, differentiated colonies were observed and used to count the number of viable bacteria in the original solution. 5, the calculation results show that the original 1ml broth containing 1.48 × 10 ⁵ colony forming units, and containing the E. coli DNA 105,652ng. Since the amount of DNA required for each reaction in the PCR reaction in the foregoing Example 6 was 90 ng, further backstepping can be used to determine the detection sensitivity of 1.26×10 per ml of E. coli when using 3H primers for RAPD-HRM detection. ² colony forming units. Compared to the prior art known to the highest sensitivity of detection of E. coli in fecal samples, i.e., ¹⁰² to ¹⁰³ colony forming units (Dutta et al.2001; Persson et al.2007 ) the PCR using a conventional, favorably.

Example 9: Detection of 687bp fragment of pathogenic E. coli strain

In the same way as in Example 3, genomic DNA was extracted from five known pathogenic E. coli strains UMN026, O83:H1, O104:H4, O157:H7 and O169:H41. Obtained by the deposit center. Using the second set of primers of the present invention (having the nucleic acid sequences shown in SEQ ID No: 3 and SEQ ID No: 4 respectively) or the third set of primers (having the nucleic acid sequences of SEQ ID No: 5 and SEQ ID No: 6 respectively), Probes with fluorescent labels different from those shown in Table 3 (designed according to the nucleic acid sequences of SEQ ID No: 10 to SEQ ID No: 16, respectively) were subjected to real-time PCR through SNP genotype detection. Each 25 μL PCR reaction solution contains: 12.5 μl PCR master mix (Type-it Fast SNP Probe PCR Kit, Qiagen), 0.9 μM primer, and 0.2 μM probe. The PCR reaction was performed in the Rotor-Gene Q real-time polymerase chain reaction apparatus (QIAGEN, Hilden, Germany) under the following reaction conditions: 95°C for 5 minutes; 95°C for 15 seconds, and 60°C for 30 seconds, thereby repeating 36 cycles. After the reaction, confirm whether the fluorescent label of each probe of each strain is a positive signal.

【Possibility of industrial utilization】

The invention provides an applicable method and molecular marker for detecting E. coli, which is a technical method that can simultaneously detect E. coli and a variety of specific pathogenic E. coli, and has high sensitivity, up to 1.26×10 ² Colony forming unit. From the viewpoint of time and labor cost, the method used in the present invention can quickly detect E. coli directly from the specimen, and the experimental operation is relatively difficult, which is an ideal technical means.

Further, the molecular marker provided by the present invention, that is, the nucleic acid sequence of the glutamic acid-amino-ligase adenylyl transferase in the c-terminal region of E. coli, is suitable for developing reagents or kits for testing microorganisms, especially Reagents or kits for testing for pathogenic E. coli.

<110> National Pingtung University of Science and Technology

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Claims (8)

A molecular marker for detecting Escherichia coli , which is characterized in that it contains the nucleic acid sequence of the glutamic acid-amino-ligase adenylyl transferase gene of the c-terminal region of E. coli, and the sequence is as SEQ ID No: 7 to 693 bases in 9. A primer set for detecting E. coli, characterized in that the primer set is selected from the group consisting of a first primer set, a second primer set, and a third primer set, and the first primer set has SEQ ID No: 1 and SEQ ID No: 2 sequences; the aforementioned second primer set has the sequences of SEQ ID No: 3 and SEQ ID No: 4, respectively; the aforementioned third primer set has the sequences of SEQ ID No: 5 and SEQ ID No: 6 respectively Nucleic acid sequence. A probe for detecting pathogenic Escherichia coli, characterized in that its sequence is selected from the group consisting of SEQ ID No: 10 and SEQ ID No for detecting extraintestinal infection of E. coli (ExPEC): 11; SEQ ID No: 12 for detecting invasive E. coli (AIEC); SEQ ID No: 13, SEQ ID No: 14, SEQ ID No: 15 for detecting enterohemorrhagic E. coli (EHEC): SEQ ID No: 16 for the detection of Toxigenic E. coli (ETEC). A method for detecting Escherichia coli , characterized in that the steps include: (a) For a test sample, the first primer set is used to perform a polymerase chain reaction to detect whether the reaction product contains E. coli c -The nucleic acid sequence of the glutamic acid-amino-ligase adenylyl transferase gene in the terminal region, and the reaction product contains the nucleic acid sequence of the glutamic acid-amino-ligase adenylyl transferase gene in the c-terminal region of E. coli, namely The presence of E. coli was detected in the specimen; where the first primer set had the sequences of SEQ ID No: 1 and SEQ ID No: 2, respectively. The method as described in item 4 of the patent application scope, wherein the steps of the aforementioned method further include: (b) performing a polymerase chain reaction on the aforementioned sample using a 3H primer to detect whether the reaction product contains an expansion of 702 base pairs in size Increaser, the reaction product contains an amplicon with a size of 702 base pairs, that is, the presence of E. coli is detected in the sample; and, the 3H primer has the sequence of SEQ ID No:17. The method as described in item 4 or 5 of the patent application scope, wherein the method for detecting the aforementioned nucleic acid sequence and amplicon is at least one selected from nucleic acid staining and high-resolution melting curve analysis (HRM). A kit for detecting Escherichia coli, characterized in that the kit includes a test specimen and more than one primer set, the primer set is selected from a first primer set, a second primer set, a third primer set and A group of 3H primers, wherein the first primer set has the sequences of SEQ ID No: 1 and SEQ ID No: 2, respectively; the second primer set has the sequences of SEQ ID No: 3 and SEQ ID No: 4 respectively The aforementioned third primer set has the sequences of SEQ ID No: 5 and SEQ ID No: 6, respectively; the aforementioned 3H primer has the sequence of SEQ ID No: 17. The kit as described in item 7 of the patent application scope, wherein the kit further includes more than one probe, and the aforementioned probe can detect pathogenic E. coli in the c-terminal region glutamate-ammonia-ligase A single nucleotide polymorphism in the adenylyltransferase gene.

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