CN114606330A - Detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization - Google Patents

Detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization Download PDF

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CN114606330A
CN114606330A CN202210419012.2A CN202210419012A CN114606330A CN 114606330 A CN114606330 A CN 114606330A CN 202210419012 A CN202210419012 A CN 202210419012A CN 114606330 A CN114606330 A CN 114606330A
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徐颖
刘海江
陈晓光
董梅
梁洁
张瑾
滕新栋
徐翮飞
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Qingdao International Travel Health Care Center Qingdao Customs Port Outpatient Department
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Abstract

The invention provides a detection kit for rapidly detecting Escherichia coli O157: H7 by RPA visualization, which comprises: a reference tube with the concentration of EHECO157: H7, a positive control standard with the concentration of EHECO157: H7, a negative control standard with the concentration of EHECO157: H7, a sample processing tube, a detection tube, a buffer solution I, a buffer solution II and a color developing agent; the invention has low requirements on instruments in the nucleic acid extraction process and the nucleic acid amplification process, strong detection specificity and no cross reaction with other diarrhea pathogens, the sensitivity is 10-100 times of that of PCR detection, the result interpretation is simple, the direct interpretation by naked eyes, and the food supervision, rapid detection site and basic level laboratory can meet the conditions and carry out detection, thus being suitable for popularization and use.

Description

Detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization
Technical Field
The invention relates to an extraction and detection kit for diarrhea pathogenic bacteria, in particular to a detection kit for Escherichia coli hemorrhagic O157: H7.
Background
Hemorrhagic Escherichia coli O157: H7 (Enteromorrhagic Escherichia coli, EHEC O157: H7) is a common serotype of Enterohemorrhagic Escherichia coli, with contaminating food and water as the major transmission pathways. There are outbreaks and epidemics of EHEC O157: H7 of different sizes around the world, with 280 million people infected each year, and are therefore of concern worldwide. The infection of EHEC O157: H7 can cause diarrhea, hemorrhagic colitis, hemolytic uremic syndrome and even death of patients, and the death rate can reach 5-10%. In addition, the pathogenic amount of EHEC O157: H7 is very low, and diseases can be caused by eating less than 10 bacteria. EHEC O157: H7 is very viable, even when subjected to various harsh conditions in the human food chain, allowing it to survive for long periods in food. At present, the infection mechanism of EHEC O157: H7 is not completely clear, and an effective treatment method is clinically lacked, so that the establishment of a method for quickly and sensitively detecting EHEC O157: H7 has important significance for preventing and controlling the transmission of EHEC O157: H7 in environment and food.
At present, a plurality of methods are available for detecting EHEC O157: H7, and common methods for detecting EHEC O157: H7 include traditional bacterial detection methods, immunological analysis methods, molecular biological detection methods, microfluidic sensor detection methods and the like. Although the methods are widely used, the analysis time of the traditional bacteria detection method is usually 2-3 days, and the time and the labor are consumed; although the immunological analysis method is shortened, the sample pretreatment time is long, the sample is easy to be polluted, and false positive results are easy to appear; compared with the traditional detection method, the nucleic acid detection method has many advantages, such as good specificity and sensitivity of PCR detection, but the detection speed is still about 120min, and expensive instruments and equipment are required.
The current national standard GB4789.6-2016 is characterized in that bacteria are added to a sample, a flat plate method is used for multiple screening and biochemical test identification, and finally PCR is used for confirmation test, so that the operation is complicated, the period is long, an accurate and expensive PCR instrument and a perfect experimental environment are required, and the application in a basic laboratory and a field is limited.
Disclosure of Invention
In view of the defects of the prior art, the invention designs the RPA detection primer according to the rfbE gene conserved sequence of EHEC O157: H7, uses SYBR Green I as a nucleic acid developer, establishes the visual RPA detection method, and detects the specificity and sensitivity of the visual RPA detection method, and the method established by the research can be used for rapidly detecting EHEC O157: H7 on site, does not need complex instruments and equipment, is simple to operate, has high sensitivity, and can be applied to hospitals, port banks, food monitoring sites, some basic laboratories and the like.
In order to achieve the above objects and other related objects, the present invention provides a detection kit for rapidly detecting Escherichia coli O157: H7 by RPA visualization, comprising: a reference tube with the concentration of EHEC O157: H7, a positive control standard substance with the concentration of EHEC O157: H7, a negative control standard substance with the concentration of EHEC O157: H7, a sample processing tube, a detection tube, a buffer solution I, a buffer solution II and a color developing agent; the EHEC O157H 7 concentration reference tube is a PCR 8 cascade tube filled with 5 tubes of liquid with different colors; the EHEC O157: H7 positive control standard is a solution of EHEC O157: H7 standard strain genome DNA, and the concentration of the solution is 10-2 ng/muL-100 ng/muL, preferably 1 ng/muL; the EHEC O157: H7 negative control standard is sterile deionized water containing no nucleic acid; the sample processing tube is a sterile EP tube containing 100-500 μ L of EHEC O157: H7 nucleic acid extraction reagent; the detection tube is a twist Amp reaction tube containing an RPA universal reaction reagent freeze-dried preparation; the buffer solution I is RPA universal reaction buffer solution containing an EHEC O157: H7 detection upstream primer F2 and an EHEC O157: H7 detection downstream primer R2, and the buffer solution I contains an EHEC O157: H7 detection upstream primer F2 with the concentration of 0.28 mu M-0.85 mu M; the concentration of the downstream primer R2 detected by the EHEC O157: H7 is 0.28-0.85 muM; the buffer solution II is a solution containing 280mM magnesium acetate; the color developing agent is SYBR Green I solution, and the concentration of the color developing agent is between 100 x and 10000 x.
Preferably, the EHEC O157: H7 concentration reference tube acquisition method comprises the following steps: the method for detecting EHEC O157: H7 in the sample to be detected detects that the concentration of EHEC O157: H7 genome DNA is 10-3ng/μL、10-4ng/μL、10-5ng/mu L of three samples, EHEC O157: H7 negative control standard and EHEC O157: H7 positive control standard, 5 colored detection tubes are obtained after detection, 5 parts of staining solution with corresponding consistent colors are prepared by orange and green dyes according to the colors of the detection tubes, the 5 parts of staining solution are sequentially added into 5 holes of the PCR 8-linked comb tube, 8-linked comb tube covers are covered, and the outer side of the cover or the 8-linked comb tube is sequentially marked with 10-3ng/μL、10-4ng/μL、10-5ng/mu L, negative and positive, and taking the whole as a reference tube of EHEC O157: H7 concentration; the liquid capacity in the EHEC O157: H7 concentration reference tube is 50-200 muL, preferably 200 muL; the result colors of the 5 colored detection tubes are green, yellow-green, light yellow-green, orange-red and green in sequence; the method for detecting EHEC O157: H7 in the sample to be detected comprises the following steps: (1) extracting nucleic acid of a sample to be detected; (2) preparing a detection system of a sample to be detected, which is disclosed by the invention: adding 33.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 mu L of buffer solution II into a detection tube, and blowing, beating and uniformly mixing; dropping 2 microliter of color developing agent on the inner side of the cover of the detection tube; (3) covering a cover of the detection tube, and placing the detection tube at 25-45 ℃ for reaction for 10-25 min; (4) and (4) detecting a result: turning the detection tube upside down, and fully and uniformly mixing the liquid in the detection tube; placing the detection tube at room temperature for 1-5 min; synchronously detecting one part of an EHEC O157: H7 positive control standard and one part of an EHEC O157: H7 negative control standard in each detection reaction; (5) and (4) interpretation of results: firstly, detecting a detection tube of an EHEC O157: H7 positive control standard, comparing the concentration of EHEC O157: H7 with that of a reference tube of EHEC O157: H7, wherein the color of the tube marked as 'positive' in the reference tube of the EHEC O157: H7 is green, and detecting the negative color of EHEC O157: H7Comparing the detection tube of the sexual control standard substance with the EHEC O157: H7 concentration reference tube, wherein the color of the tube marked as 'negative' in the EHEC O157: H7 concentration reference tube is orange red, judging that the detection result is credible, otherwise, the result is not credible and needs to be detected again; comparing the detected sample with a reference tube of EHEC O157: H7 concentration, wherein the tube is orange red and has the same color with the tube marked as 'negative', and judging the tube is negative; the color of the detection tube for detecting the sample to be detected is green, is similar to the color of the tube marked as 'positive' in the EHEC O157: H7 concentration reference tube, the detection tube is judged to be positive, and simultaneously, the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be more than 10-3ng/mu L; the color of the detection tube for detecting the sample to be detected is yellow green, and the reference tube of the EHEC O157: H7 concentration is marked as 10-4ng/microliter' tubes are similar in color and are judged to be positive, and meanwhile, the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be close to 10-4ng/mu L; the color of the detection tube for detecting the sample to be detected is light yellow green, and the reference tube for detecting the concentration of EHEC O157: H7 is marked as 10-5The tube with the ng/mu L color is judged to be positive, and the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be close to 10-5ng/mu L; the specific operation of the step (1) of extracting the nucleic acid of the sample to be detected is as follows: 1) when the sample to be detected is liquid, 100-500 mu L of each sample is taken, and when the sample to be detected is colloidal, paste, semisolid or solid, 100-500 mg of each sample to be detected is taken and fully sheared into pieces; 2) adding the sample to be detected into a sample processing tube, and fully and uniformly mixing; 3) placing the sample processing tube at 90-100 ℃ and incubating for 5-25 min; 4) and (3) centrifuging 12000g of the sample processing tube for 1min-5min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid to be detected.
Preferably, in the step 1) of extracting nucleic acid from a test sample according to the present invention, the sample volume of the test sample is preferably the same as the volume of the EHEC O157: H7 nucleic acid extraction reagent in the sample treatment tube; more preferably, the sample processing tube contains 200. mu.L of EHEC O157: H7 nucleic acid extraction reagent; more preferably, 200 μ L of the sample is taken for each sample when the sample to be tested is liquid, and 200mg of the sample to be tested is taken for each sample when the sample to be tested is colloidal, pasty, semisolid or solid.
Preferably, the sample to be tested is incubated for 10min at 100 ℃ in the nucleic acid extraction step 3); in the step 4) of extracting nucleic acid from the sample to be tested, the sample to be tested is more preferably centrifuged at 12000g for 3 min.
Preferably, the temperature in the step (3) of detecting EHEC O157: H7 in the sample to be detected is more preferably 35 ℃; wherein the preferable reaction time is 10min-25 min.
Preferably, the reaction time of the step (3) and the standing time of the detection tube of the step (4) for detecting the EHEC O157: H7 in the sample to be detected are 15min in total.
Preferably, the EHEC O157H 7 positive control standard acquisition method comprises the following steps: extracting genome DNA of EHEC O157: H7 standard strain according to general bacterial genome DNA extraction kit, obtaining DNA from bacterial sample, dissolving in TE solution or sterile deionized water, detecting nucleic acid concentration, diluting EHEC O157: H7 standard strain genome DNA with TE solution or sterile deionized water to 10-2ng/. mu.L-100 ng/. mu.L, and more preferably the concentration of genomic DNA of the diluted EHEC O157: H7 standard strain is 1 ng/. mu.L.
Preferably, the reagent for extracting nucleic acid from EHEC O157: H7 in the sample processing tube is: 0.5mol/LNaOH, 2% Chelex-100 and 0.05mol/L TrisHCl, and the three are prepared according to the volume ratio of 1:1: 1.
Preferably, the sequence of the upstream primer F2 detected by the EHEC O157: H7 is 5'-AACTATTACTACAGGTGAAGGTGGAATGGT-3' (SEQ ID NO3), and the sequence of the downstream primer R2 detected by the EHEC O157: H7 is 5'-AATCATCAGCTTGTTCTAACTGGGCTAATC-3' (SEQ ID NO 4).
Preferably, the detection system of the sample to be detected is preferably a 50 μ L system, which is: 12 mu L of nucleic acid to be detected obtained after the sample to be detected is extracted, 33.5 mu L of buffer solution I, 2.5 mu L of buffer solution II and 2 mu L of color developing agent; 50 mu.L of buffer I, the buffer I is RPA universal reaction buffer containing 0.7 mu M of EHEC O157: H7 detection upstream primer F2 and 0.7 mu M of EHEC O157: H7 detection downstream primer R2, and the color developing agent is SYBR Green I solution containing 1000X.
As described above, the detection kit for rapidly detecting the Escherichia coli O157: H7 by RPA visualization has the following beneficial effects:
(1) the requirements of the nucleic acid extraction process and the nucleic acid amplification process on instruments are low, and the requirements can be met only by one water bath kettle or hot water kettle and one centrifugal machine, and the requirements can be met by food supervision, a rapid detection field and a basic laboratory; (2) the reagent can specifically detect EHEC O157: H7, and has no cross reaction with other diarrhea pathogens; (3) the sensitivity for detecting EHEC O157H 7 is good, and the lower limit of detection can reach 10-5ng/muL is 10-100 times of PCR detection; (4) the nucleic acid extraction efficiency is high, the nucleic acid extraction of EHEC O157: H7 is not influenced by the feces pollution during diarrhea, the nucleic acid detection sensitivity is high, and the detection efficiency is good (5) the nucleic acid extraction method is simple, the detection steps are few, the interpretation method is simple, and the requirement on operators is low; (6) the detection of EHEC O157: H7 in a sample can be completed within 20-60 min from nucleic acid extraction to result interpretation, and the sensitivity and specificity are met and the speed is higher than that of other methods; (7) the result is easy to judge, the result is directly judged by naked eyes, the longer the time is, the better the color development effect is, and the EHEC O157: H7 concentration reference tube is compared, so that the EHEC O157: H7 concentration in the sample can be judged, and the sample cannot be judged to be false negative because the judgment is not accurate due to the too low concentration.
Drawings
FIG. 1 shows the results of the electrophoresis of the method of the present invention for detecting EHEC O157: H7 using different amplification primers, where M is Marker, 1-5: primer amplification products 1-5, 6: blank control.
FIG. 2 shows the results of electrophoresis of the present invention for detecting EHEC O157: H7 with different amplification temperatures, where M: marker, 1-5: 4 deg.C, 15 deg.C, 25 deg.C, 35 deg.C, 45 deg.C.
FIG. 3 shows the results of electrophoresis of EHEC O157: H7 for different amplification times in the methods of the invention, where M: marker, 1-5: 5min, 10min, 15min, 20min, 25min
FIG. 4 shows the results of electrophoresis for the detection of different strains by the method of the invention using RPA amplification, wherein M: marker, 1-6: shigella, EHEC O157: H7, salmonella, vibrio parahaemolyticus, enterococcus faecalis, and strain mixture
FIG. 5 shows a method of the present inventionMethod the results of electrophoresis using RPA amplification to detect different concentrations of EHEC O157: H7 nucleic acids, where M: marker, 1-8: 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 10-1ng/μL、10-2ng/μL、10-3ng/μL、10-4ng/μL、10-5ng/μL
FIG. 6 shows the results of electrophoresis for detecting PCR amplification sensitivity using different concentrations of nucleic acids, where M: marker, 1-8: 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 10-1ng/μL、10-2ng/μL、10-3ng/μL、10-4ng/μL、10-5ng/μL
FIG. 7 shows the results of the detection of EHEC O157: H7 with different concentrations of SYBR Green I according to the method of the present invention, where N: negative control, 1-5: 1.25 μ L SYBR Green I (100 ×), 0.5 μ L SYBR Green I (1000 ×), 1 μ L SYBR Green I (1000 ×), 2 μ L SYBR Green I (1000 ×), 4 μ L SYBR Green I (1000 ×).
FIG. 8 shows the results of the detection of different strains according to the method of the invention, wherein N: negative control, 1-6: shigella, EHEC O157: H7, salmonella, vibrio parahaemolyticus, enterococcus faecalis, and strain mixture
FIG. 9 shows the results of detection of different concentrations of EHEC O157: H7 nucleic acids for the methods of the invention, where N: negative control, 1-8: 10-5ng/μL、10-4ng/μL、10-3ng/μL、10-2ng/μL、10-1ng/μL、1ng/μL、10ng/μL、100ng/μL。
FIG. 10 is a schematic view of the detection process of the EHEC O157: H7 sample by the method of the present invention
Detailed Description
The following description of the embodiments of the present invention is provided for illustrative purposes, and other advantages and effects of the present invention will become apparent to those skilled in the art from the present disclosure.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In order to achieve the above objects and other related objects, the present invention provides a detection kit for rapidly detecting Escherichia coli O157: H7 by RPA visualization, comprising: a reference tube with the concentration of EHEC O157: H7, a positive control standard substance with the concentration of EHEC O157: H7, a negative control standard substance with the concentration of EHEC O157: H7, a sample processing tube, a detection tube, a buffer solution I, a buffer solution II and a color developing agent;
specifically, the reference tube for the concentration of EHEC O157: H7 is a PCR 8-linked calandria tube filled with liquids with different colors;
specifically, the reference tube for EHEC O157: H7 concentration is a PCR 8-linked calandria tube filled with 5 tubes of liquid with different colors;
specifically, the EHEC O157: H7 positive control standard is a solution of EHEC O157: H7 standard strain genome DNA, and the concentration of the solution is 10-2 ng/muL-100 ng/muL, preferably 1 ng/muL;
specifically, the EHEC O157: H7 negative control standard is sterile deionized water containing no nucleic acid;
specifically, the sample processing tube is a sterile EP tube containing 100-500 μ L of EHEC O157: H7 nucleic acid extraction reagent;
specifically, the detection tube is a twist Amp reaction tube containing an RPA universal reaction reagent freeze-dried preparation;
specifically, the buffer I is RPA universal reaction buffer containing an EHEC O157: H7 detection upstream primer F2 and an EHEC O157: H7 detection downstream primer R2, and the buffer I contains an EHEC O157: H7 detection upstream primer F2 with the concentration of 0.28 mu M-0.85 mu M; the concentration of the downstream primer R2 detected by the EHEC O157: H7 is 0.28-0.85 muM;
specifically, the buffer solution II is a solution containing 280mM magnesium acetate;
specifically, the color developing agent is SYBR Green I solution, and the concentration of the color developing agent is 100 x-10000 x;
specifically, the color developing agent is a nucleic acid dye, and does not influence RPA amplification, the activity of enzyme in a reaction system and the color developing process;
specifically, the method for acquiring the EHEC O157: H7 concentration reference tube comprises the following steps: detection of EHEC O157: H7 genomic DNA concentration according to the procedure for detecting EHEC O157: H7 in a test sample of the present inventionDegree of 10-3ng/μL、10-4ng/μL、10-5ng/mu L of three samples, EHEC O157: H7 negative control standard and EHEC O157: H7 positive control standard, 5 colored detection tubes are obtained after detection, 5 parts of staining solution with corresponding consistent colors are prepared by orange and green dyes according to the colors of the detection tubes, the 5 parts of staining solution are sequentially added into 5 holes of the PCR 8-linked comb tube, 8-linked comb tube covers are covered, and the outer side of the cover or the 8-linked comb tube is sequentially marked with 10-3ng/μL、10-4ng/μL、10-5ng/mu L, negative and positive, and taking the whole as a reference tube of EHEC O157: H7 concentration; the liquid capacity in the EHEC O157: H7 concentration reference tube is 50-200 muL, preferably 200 muL;
specifically, the result colors of the 5 colored detection tubes are green, yellow-green, light yellow-green, orange-red and green in sequence;
specifically, the steps of detecting EHEC O157: H7 in the sample to be detected in the invention are as follows: (1) extracting nucleic acid of a sample to be detected; (2) preparing a detection system of a sample to be detected, which is disclosed by the invention: adding 33.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 mu L of buffer solution II into a detection tube, and blowing, beating and uniformly mixing; dropping 2 microliter of color developing agent on the inner side of the cover of the detection tube; (3) covering a cover of the detection tube, and placing the detection tube at 25-45 ℃ for reaction for 10-25 min; (4) and (4) detecting a result: turning the detection tube upside down, and fully and uniformly mixing the liquid in the detection tube; placing the detection tube at room temperature for 1-5 min; synchronously detecting one part of an EHEC O157: H7 positive control standard and one part of an EHEC O157: H7 negative control standard in each detection reaction; (5) and (4) interpretation of results: firstly, detecting a detection tube of an EHEC O157: H7 positive control standard substance, comparing the EHEC O157: H7 concentration reference tube, wherein the color of the detection tube is consistent with that of a tube marked as 'positive' in the EHEC O157: H7 concentration reference tube and is green, and comparing the color of the detection tube of the EHEC O157: H7 negative control standard substance with that of a tube marked as 'negative' in the EHEC O157: H7 concentration reference tube and is orange-red, judging that the detection result is credible, otherwise, judging that the result is not credible and needing to detect again; detecting tube for detecting sample to be detectedComparing the EHEC O157: H7 concentration reference tube, judging the tube to be negative, wherein the color is orange red and is consistent with the color of the tube marked as negative; the color of the detection tube for detecting the sample to be detected is green, is similar to the color of the tube marked as 'positive' in the EHEC O157: H7 concentration reference tube, the detection tube is judged to be positive, and simultaneously, the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be more than 10-3ng/mu L; the color of the detection tube for detecting the sample to be detected is yellow green, and the reference tube of the EHEC O157: H7 concentration is marked as 10-4ng/microliter' tubes are similar in color and are judged to be positive, and meanwhile, the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be close to 10-4ng/mu L; the color of the detection tube for detecting the sample to be detected is light yellow green, and the reference tube for detecting the concentration of EHEC O157: H7 is marked as 10-5The tube with the ng/mu L color is judged to be positive, and the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be close to 10-5ng/μL；
Specifically, in the process that the detection tube is placed at room temperature in the step (4) of detecting EHEC O157: H7 in the sample to be detected, because the amplification reaction of the RPA is still continued, the detection process is still carried out, and the green color shown by the positive detection result is more obvious along with the extension of the time of placing at room temperature; the negative detection result does not change along with the prolonging of time;
specifically, the specific operation of the step (1) of extracting nucleic acid from the sample to be detected is as follows: 1) when the sample to be detected is liquid, 100-500 mu L of each sample is taken, and when the sample to be detected is colloidal, paste, semisolid or solid, 100-500 mg of each sample to be detected is taken and fully sheared into pieces; 2) adding the sample to be detected into a sample processing tube, and fully and uniformly mixing; 3) placing the sample processing tube at 90-100 ℃ and incubating for 5-25 min; 4) 12000g of the sample processing tube is centrifuged for 1min-5min, and supernatant fluid is reserved, namely the extracted nucleic acid to be detected.
Specifically, in step 1) of nucleic acid extraction of a sample to be tested according to the present invention, preferably, the sample volume of the sample to be tested is the same as the amount of the EHEC O157: H7 nucleic acid extraction reagent in the sample treatment tube; more preferably, the sample processing tube contains 200. mu.L of EHEC O157: H7 nucleic acid extraction reagent; more preferably, 200 μ L of the sample is taken for each sample when the sample to be tested is liquid, and 200mg of the sample to be tested is taken for each sample when the sample to be tested is colloidal, pasty, semisolid or solid.
Specifically, in the step 3) of extracting nucleic acid from the sample to be detected, incubation at 100 ℃ for 10min is more preferable; in the step 4) of extracting nucleic acid from the sample to be tested, the sample to be tested is more preferably centrifuged at 12000g for 3 min.
Specifically, the more preferable temperature in the step (3) of detecting EHEC O157: H7 in the sample to be detected is 35 ℃; wherein the preferable reaction time is 10min-25 min.
Specifically, more preferably, the reaction time of step (3) and the standing time of the detection tube of step (4) for detecting EHEC O157: H7 in the sample to be detected are 15min in total.
Specifically, the EHEC O157H 7 positive control standard acquisition method comprises the following steps: extracting genome DNA of EHEC O157: H7 standard strain according to general bacterial genome DNA extraction kit, obtaining DNA from bacterial sample, dissolving in TE solution or sterile deionized water, detecting nucleic acid concentration, diluting EHEC O157: H7 standard strain genome DNA with TE solution or sterile deionized water to 10-2ng/. mu.L-100 ng/. mu.L, and more preferably the concentration of genomic DNA of the diluted EHEC O157: H7 standard strain is 1 ng/. mu.L.
Specifically, the reagent for extracting the nucleic acid by using the EHEC O157: H7 in the sample processing tube comprises: 0.5mol/L NaOH, 2 percent Chelex-100 and 0.05mol/L TrisHCl, and the three are prepared according to the volume ratio of 1:1: 1.
Specifically, the sequence of an upstream primer F2 detected by the EHEC O157: H7 is 5'-AACTATTACTACAGGTGAAGGTGGAATGGT-3' (SEQ ID NO3), and the sequence of a downstream primer R2 detected by the EHEC O157: H7 is 5'-AATCATCAGCTTGTTCTAACTGGGCTAATC-3' (SEQ ID NO 4).
Specifically, the detection system of the sample to be detected is preferably a 50 μ L system, and the system is as follows: 12 mu L of nucleic acid to be detected obtained after the sample to be detected is extracted, 33.5 mu L of buffer solution I, 2.5 mu L of buffer solution II and 2 mu L of color developing agent; 50 mu.L of buffer I, the buffer I is RPA universal reaction buffer containing 0.7 mu M of EHEC O157: H7 detection upstream primer F2 and 0.7 mu M of EHEC O157: H7 detection downstream primer R2, and the color developing agent is SYBR Green I solution containing 1000X.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example one
Referring to FIG. 1, the present invention screens amplification primers with high specificity and high sensitivity to EHEC O157: H7
The rfbE gene sequence of EHEC O157: H7 was retrieved from NCBI database, by aligning the existing sequences in the database, relatively conserved regions were selected, primers were designed using Primer Premier 5.0 software, referring to the Primer design principles in the TwistDx company's instructions, and by blast alignment in NCBI database, 5 pairs of primers (Table 1) were selected that could amplify the rfbE gene sequence, which was synthesized by Shanghai Biotechnology, Inc.
A sample processing tube: preparing 0.5mol/LNaOH, preparing Chelex-100 with the concentration of 2%, preparing 0.05mol/L TrisHCl, mixing the 3 solutions according to the volume ratio of 1:1:1, and fully and uniformly mixing to prepare an EHEC O157: H7 nucleic acid extraction reagent; 200. mu.L of the above-mentioned EHEC O157: H7 nucleic acid isolation reagent was added to a sterile EP tube to prepare a sample processing tube of EHEC O157: H7.
Nucleic acid preparation: taking a culture bacterial liquid of an EHEC O157: H7 standard strain as a sample to be detected, taking 200 mu L of bacterial liquid, adding the bacterial liquid into a sample processing tube, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging for 12000g for 3min, and reserving supernatant, wherein the supernatant is extracted EHEC O157: H7 genomic DNA. Diluting EHEC O157: H7 genome DNA, and adjusting the nucleic acid concentration to be 100 ng/mu L to obtain the nucleic acid to be detected.
And (2) buffer solution II: is a solution containing 280mM magnesium acetate.
Preparing a total of 5 parts of 50 mu L of an RPA amplification reaction system: adding 30.5 mu LRPA universal reaction buffer solution into a detection tube, and uniformly mixing by blowing; respectively adding 2.5 mu L of 10 mu M upstream primer and 2.5 mu L of 10 mu M downstream primer, and blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed.
RPA amplification reaction: amplifying at 37 deg.C for 40 min;
and (4) detecting a result: preparing 2% agarose gel, detecting 5 μ L amplification product, performing electrophoresis at constant voltage of 3V/cm for 20min, and recording and storing the electrophoresis detection result by using a gel imaging analysis system.
Synchronously detecting one part of an EHEC O157: H7 negative control standard;
the electrophoresis result of the 5 pairs of designed primers amplified by using the culture solution of the EHEC O157: H7 standard strain as a sample to be detected is shown in figure 1, the amplification band of the designed primers is consistent with the target band in size, and the amplification band is single, clear, free of dispersion and trailing. The results show that the amplification efficiency of the primers is high, and the primers can be accurately detected. According to the RPA specification of TwistDX company, the RT-PCR product length is recommended to be not more than 200bp, and the experiment is continued for the subsequent design of a primer probe, so that the 2 nd pair of primer EQ ID NO3 and SEQ ID NO 4 are selected as the specific primers of the invention, and the amplification product length is 191 bp.
TABLE 1 RPA amplification primers for EHEC O157: H7
Figure BDA0003606122850000091
Example two
Referring to FIGS. 2-3, the establishment and optimization of the RPA amplification reaction according to the present invention
1. Optimization of optimal reaction temperature for RPA amplification
Taking a culture bacterial liquid of an EHEC O157: H7 standard strain as a sample to be detected, adding 200 mu l of the bacterial liquid into a sample processing tube containing 200 mu l of an EHEC O157: H7 nucleic acid extraction reagent, fully mixing uniformly, incubating for 5min at 100 ℃, centrifuging for 12000g for 3min, and reserving a supernatant, wherein the supernatant is the extracted EHEC O157: H7 genomic DNA. Diluting EHEC O157: H7 genome DNA, and adjusting the nucleic acid concentration to be 100 ng/mu L to obtain the nucleic acid to be detected.
Preparing a buffer solution I: firstly, adding a proper amount of RPA universal reaction buffer solution into EHEC O157: H7 detection upstream primer F2(SEQ ID NO3) powder to prepare RPA universal reaction buffer solution with 0.7 mu M of upstream primer, then taking a proper amount of the solution to dissolve EHEC O157: H7 detection downstream primer R2(SEQ ID NO 4) powder, finally preparing RPA universal reaction buffer solution containing 0.7 mu M of EHEC O157: H7 detection upstream primer F2 and 0.7 mu M of EHEC O157: H7 detection downstream primer R2, namely buffer solution I in the invention, and subpackaging for later use.
Preparing a total of 5 parts of 50 mu L of RPA detection system: adding 35.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed.
RPA amplification reaction: placing a detection tube at 4 deg.C, 15 deg.C, 25 deg.C, 35 deg.C, and 45 deg.C respectively, and performing amplification reaction for 40 min;
and (4) detecting a result: preparing 2% agarose gel, detecting 5 μ L amplification product, performing electrophoresis at constant voltage of 3V/cm for 20min, and recording and storing the electrophoresis detection result by using a gel imaging analysis system.
The genome DNA of the EHEC O157: H7 standard strain is taken as a template, the amplification results of an RPA reaction tube at 4 ℃, 15 ℃, 25 ℃, 35 ℃ and 45 ℃ are respectively shown in figure 2, the detection result shows that a weak target amplification band appears at the amplification temperature of 15 ℃, the target band is gradually bright along with the temperature increase, the target band is clearest and bright at 35 ℃ and becomes dark at 45 ℃, which indicates that the reaction can occur by the method at the temperature of 15 ℃, and the method can achieve a relatively ideal amplification effect when detecting the EHEC O157: H7 standard strain within the range of 25 ℃ to 45 ℃, and the amplification efficiency is highest at 35 ℃.
Minimum effective reaction time for RPA amplification
Nucleic acid preparation: taking a culture bacterial liquid of an EHEC O157: H7 standard strain as a sample to be detected, adding 200 mu l of the bacterial liquid into a sample processing tube containing 200 mu l of an EHEC O157: H7 nucleic acid extraction reagent, fully mixing uniformly, incubating for 5min at 100 ℃, centrifuging for 12000g for 3min, and reserving a supernatant, wherein the supernatant is the extracted EHEC O157: H7 genomic DNA. Diluting EHEC O157: H7 genome DNA, and adjusting the nucleic acid concentration to be 100 ng/mu L to obtain the nucleic acid to be detected.
Preparing a total of 5 parts of 50 mu L of RPA detection system: adding 35.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and mixing uniformly; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed.
RPA amplification reaction: 5 portions of the detection tube are placed at 35 ℃ and amplified for 5min, 10min, 15min, 20min and 25min respectively.
And (4) detecting a result: preparing 2% agarose gel, detecting 5 μ L amplification product, performing electrophoresis at constant voltage of 3V/cm for 20min, and recording and storing the electrophoresis detection result by using a gel imaging analysis system.
As a result: the detection results of 5min, 10min, 15min, 20min and 25min of amplification at 35 ℃ are shown in FIG. 3, the results show that the reaction is not obvious in 5min, clear products can be observed in 10min, and the RPA amplification effect is better along with the extension of the reaction time, which indicates that the method of the invention can achieve ideal detection results at least when the amplification is required for more than 10 min.
EXAMPLE III
Referring to FIG. 4, the specificity of the RPA reaction of the present invention was examined
Nucleic acid preparation: preparing shigella standard strain solution, EHEC O157: H7 standard strain solution, salmonella standard strain solution, vibrio parahaemolyticus standard strain solution, enterococcus faecalis standard strain solution and five standard strains according to the volume of 1:1: 1:1:1 ratio of the mixed bacteria solution. The 6 bacterial solutions are respectively samples to be detected, 200 mul of the bacterial solutions are respectively taken and added into a sample processing tube containing 200 mul of EHEC O157: H7 nucleic acid extraction reagent, the samples are fully and evenly mixed, the mixture is incubated for 5min at 100 ℃, 12000g of the mixture is centrifuged for 3min, and supernatant is reserved, namely the extracted genome DNA. Diluting 6 kinds of genome DNA, and regulating nucleic acid concentration to 100 ng/microliter to obtain the nucleic acid to be detected.
Preparing a total of 6 parts of 50 mu L of RPA detection system: adding 35.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; respectively adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed.
RPA amplification reaction: 6 test tubes were placed at 35 ℃ and amplified for 15 min.
And (4) detecting a result: preparing 2% agarose gel, detecting 5 μ L amplification product, performing electrophoresis at constant voltage of 3V/cm for 20min, and recording and storing the electrophoresis detection result by using a gel imaging analysis system.
As a result: the genome DNA of shigella standard strain, EHEC O157: H7 standard strain, salmonella standard strain, vibrio parahaemolyticus standard strain, enterococcus faecalis standard strain and strain mixed liquor are respectively used as templates, the RPA reaction amplification result is shown in figure 4, except that EHEC O157: H7 and mixed bacterial liquor are amplified to obtain a target fragment, other strains are not amplified, and the RPA detection method is proved to have good specificity and can not be interfered by other intestinal pathogenic bacteria.
Example four
Referring to FIGS. 5-6, sensitivity of the detection of the RPA response of the methods of the present invention
Nucleic acid preparation: taking a culture bacterial liquid of an EHEC O157: H7 standard strain as a sample to be detected, taking 200 mu l of the bacterial liquid, adding the bacterial liquid into a sample processing tube containing 200 mu l of an EHEC O157: H7 nucleic acid extraction reagent, fully mixing the bacterial liquid and the sample processing tube, incubating the mixture for 5min at 100 ℃, centrifuging the mixture for 12000g and 3min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid. Diluting EHEC O157: H7 genomic DNA to adjust the nucleic acid concentration to 100 ng/. mu.L, then diluting EHEC O157: H7 genomic DNA by 10 times, and finally obtaining the genomic DNA concentrations of 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L-1ng/μL、10-2ng/μL、10-3ng/μL、10- 4ng/μL、10-5ng/microliter, namely the nucleic acid to be detected.
Preparing a total of 8 parts of 50 mu L of RPA detection system: adding 35.5 mu L of buffer solution I into a detection tube, and blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed.
RPA amplification reaction: 8 test tubes were placed at 35 ℃ and amplified for 15 min.
PCR amplification reaction: according to the WS 271-2007 infectious diarrhea diagnosis standard, the sensitivity of EHEC O157: H7 is detected by a standard PCR method, the PCR reaction condition and the system reference standard are adopted, and the detection primers are as follows: O157-F: 5'-TTCAAACAGAGGACCATC-3', O157-R: 5'-CCCAGCCACTAAGTATTG-3', the length of the target amplification product is 636 bp.
And (4) detecting a result: preparing 2% agarose gel, detecting 5 μ L amplification product, performing electrophoresis at constant voltage of 3V/cm for 20min, and recording and storing the electrophoresis detection result by using a gel imaging analysis system.
As a result: at a concentration of 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 10-1ng/μL、10-2ng/μL、10-3ng/μL、10- 4ng/μL、10-5The result of RPA amplification carried out by ng/mu L of EHEC O157: H7 genomic DNA as a template is shown in FIG. 5, and the result shows that the concentration of nucleic acid for detecting EHEC O157: H7 by RPA is 10-5The amplified band is clearly visible at ng/. mu.L; the PCR detection result is shown in FIG. 6, which shows that the minimum detection limit of nucleic acid detected by PCR is 10-2ng/. mu.L, the band of interest becomes progressively brighter as the concentration of nucleic acid increases. Since the RPA method uses DNA in an amount close to 10 times that of the PCR method, the sensitivity of the PRA detection method established in the present invention is at least 100 times that of the standard PCR method.
EXAMPLE five
Referring to FIG. 7, the establishment and optimization of the RPA reaction visualization detection method of the present invention
Nucleic acid preparation: taking a culture bacterial liquid of an EHEC O157: H7 standard strain as a sample to be detected, adding 200 mu l of the bacterial liquid into a sample processing tube containing 200 mu l of an EHEC O157: H7 nucleic acid extraction reagent, fully mixing uniformly, incubating for 5min at 100 ℃, centrifuging for 12000g for 3min, and reserving a supernatant, wherein the supernatant is the extracted EHEC O157: H7 genomic DNA. Diluting EHEC O157: H7 genome DNA, and adjusting the nucleic acid concentration to 1 ng/mu L to obtain the nucleic acid to be detected.
Preparing a total of 5 parts of 48 mu L of a partial detection system of the invention: adding 33.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed.
Preparing a detection system of the invention: on the basis of the above 48. mu.L of the detection system of the present invention, 1.25. mu.L of SYBR Green I (100X), 0.5. mu.L of SYBR Green I (1000X), 1. mu.L of SYBR Green I (1000X), 2. mu.L of SYBR Green I (1000X), and 4. mu.L of SYBR Green I (1000X) were dropped onto the inner sides of the caps of 5 test tubes, respectively.
1 part of negative control detection tube: 35.5. mu.L of buffer I, 2.5. mu.L of buffer II, 12. mu.L of EHEC O157: H7 negative control standard, 2. mu.L of SYBR Green I (1000X) were added to the test tube, and the mixture was pipetted and mixed well.
RPA amplification reaction: 6 test tubes were placed at 35 ℃ and amplified for 10 min.
And (4) detecting the result: after the RPA amplification reaction is finished, the detection tube is inverted up and down, and the liquid is fully and uniformly mixed; the mixture was left at room temperature for 5min, and color change was observed.
As a result: SYBR Green I with different concentrations is added into the detection system after 10min of amplification, the visual detection result is shown in figure 7, the result shows that the detection tube of the negative control is orange red, other 5 detection tubes are observed, more than 1 mu L of SYBR Green I (1000 x) is added into 50 mu L of the detection system, the color of the detection tube is changed from orange red to Green, and the color change of 2 mu L of SYBR Green I (1000 x) is very obvious.
EXAMPLE six
Referring to FIG. 8, the specificity of the method of the present invention was examined
Nucleic acid preparation: preparing shigella standard strain solution, EHEC O157: H7 standard strain solution, salmonella standard strain solution, vibrio parahaemolyticus standard strain solution, enterococcus faecalis standard strain solution and five standard strains according to the volume of 1:1: 1:1:1 ratio of the mixed bacteria solution. The 6 bacterial solutions are respectively samples to be detected, 200 mul of the bacterial solutions are respectively taken and added into a sample processing tube containing 200 mul of EHEC O157: H7 nucleic acid extraction reagent, the samples are fully and uniformly mixed, the mixture is incubated for 5min at 100 ℃, 12000g is centrifuged for 3min, and supernatant is reserved, namely the extracted genome DNA. Diluting 6 kinds of genome DNA, and regulating nucleic acid concentration to 1 ng/microliter to obtain the nucleic acid to be detected.
Color developing agent: containing 1000 XSYBR Green I solution.
The detection system prepared by the method of the invention comprises 50 mu L of 6 parts in total: adding 33.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed. mu.L of developer was dropped onto the inside of the cap of the detection tube.
1 part of negative control detection tube: 35.5 mul of buffer solution I, 2.5 mul of buffer solution II, 12 mul of EHEC O157: H7 negative control standard substance and 2 mul of color developing agent are added into a detection tube and evenly blown and beaten.
RPA amplification reaction: 6 test tubes were placed at 35 ℃ and amplified for 10 min.
And (4) detecting a result: after the RPA amplification reaction is finished, the detection tube is inverted up and down, and the mixed liquid is fully inverted; the mixture was left at room temperature for 5min, and color change was observed.
As a result: the genome DNA of shigella standard strain, EHEC O157: H7 standard strain, salmonella standard strain, vibrio parahaemolyticus standard strain, enterococcus faecalis standard strain and strain mixed liquid are respectively used as nucleic acid templates to be detected, the detection result of the method is shown in figure 8, except that a yellow-green reaction occurs in a detection tube for detecting EHEC O157: H7 and mixed bacterial liquid, other detection tubes are orange-yellow negative results, and the result shows that the EHEC O157: H7 detected by the method has good specificity and cannot be interfered by other intestinal pathogenic bacteria.
EXAMPLE seven
Referring to FIG. 9, the sensitivity of the method of the present invention was tested
Preparation of nucleic acid: taking a culture bacterial liquid of an EHEC O157: H7 standard strain as a sample to be detected, adding 200 mu l of the bacterial liquid into a sample processing tube containing 200 mu l of an EHEC O157: H7 nucleic acid extraction reagent, fully mixing uniformly, incubating for 5min at 100 ℃, centrifuging for 12000g, centrifuging for 1min-5min, and reserving a supernatant, wherein the supernatant is the extracted nucleic acid. Diluting EHEC O157: H7 genome DNA, adjusting the nucleic acid concentration to be 100 ng/. mu.L, then diluting EHEC O157: H7 genome DNA by 10 times, and finally obtaining the genome DNA concentrations of 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L and 10 g/. mu.L-1ng/μL、10-2ng/μL、10-3ng/μL、10-4ng/μL、10-5ng/microliter, namely the nucleic acid to be detected.
The detection system prepared by the method of the invention comprises 50 mu L and 8 parts in total: adding 33.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed. mu.L of developer was dropped onto the inside of the cap of the detection tube.
1 part of negative control detection tube: 35.5 mul of buffer solution I, 2.5 mul of buffer solution II, 12 mul of EHEC O157: H7 negative control standard substance and 2 mul of color developing agent are added into a detection tube and are evenly blown and mixed.
RPA amplification reaction: 8 test tubes were placed at 35 ℃ and amplified for 10 min.
And (4) detecting the result: after the RPA amplification reaction is finished, the detection tube is inverted up and down, and the mixed liquid is fully inverted; standing at room temperature for 1min, and observing color change.
As a result: at a concentration of 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 10-1ng/μL、10-2ng/μL、10-3ng/μL、10- 4ng/μL、10-5The detection result of the method is shown in figure 9 when ng/mu L of EHEC O157: H7 genomic DNA is used as a template, and the result shows that when the DNA concentration of EHEC O157: H7 is 10-5ng/mu L, the reaction tube still has obvious color change reaction compared with the negative control, the detection result is consistent with the result of the fourth embodiment, the visual RPA detection EHEC O157: H7 method established in the research has the sensitivity of 10-5ng/. mu.L, is at least 100 times more sensitive than standard PCR methods.
Example eight
Referring to FIG. 10, the detection process of detecting EHEC O157: H7 in the detection sample according to the present invention detects the repeatability of the method according to the present invention
Nucleic acid preparation: taking the culture bacterial liquid of the EHEC O157: H7 standard strain as a sample to be detected, taking 200 mul of the bacterial liquid, adding the bacterial liquid into a sample processing tube containing 200 mul of the EHEC O157: H7 nucleic acid extraction reagent, fully mixing the bacterial liquid and the sample processing tube, incubating the mixture for 5min at 100 ℃, centrifuging the mixture for 12000g and 3min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid. Diluting EHEC O157: H7 genome DNA to adjust the nucleic acid concentration to 100 ng/mu L, and diluting EHEC O157: H7 genome DNA to obtain the genome DNA concentrations of 20 ng/mu L and 2 × 10 respectively-1ng/μL、2×10-3ng/microliter, namely 3 parts of nucleic acid to be detected; diluting and obtaining the liquid with the genome DNA concentration of 1 ng/mu L, namelyEHEC O157: H7 positive control standard.
Preparing 50 mu L of the detection system of the invention: adding 33.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed. mu.L of developer was dropped onto the inside of the cap of the detection tube.
EHEC O157H 7 concentration reference tube: the reference nucleic acid concentration to be tested is 100 ng/. mu.L, 10 ng/. mu.L, 1 ng/. mu.L, 10 ng/. mu.L- 1ng/μL、10-2ng/μL、10-3ng/μL、10-4ng/μL、10-5ng/microliter, the color of the detecting tube of the method of the invention is more than 10-3ng/mu L, the color of the detection tube is green, and the detection tube has no obvious change with the increase of the concentration, so the concentration of the nucleic acid to be detected is 10-3ng/μL、10-4ng/μL、10-5ng/muL, detecting EHEC O157: H7 negative reference standard and EHEC O157: H7 positive reference standard, preparing translucent orange red dye solution with water-soluble dye, preparing translucent green dye solution with fluorescent green dye, preparing 5 parts of orange red dye solution and green dye solution, and preparing the test nucleic acid with the concentration of 10-3ng/μL、10-4ng/μL、10- 5ng/mu L, detecting EHEC O157: H7 negative control standard and EHEC O157: H7 positive control standard, the color of the detection tube after detection by the method of the invention is corresponding to, same and semitransparent liquid, the color sequence is green, yellow green, light yellow green, orange red and green, taking 5 parts of the liquid 200 mu L, adding the liquid into 5 holes of the PCR 8-linked comb tube in sequence, covering 8-linked comb tubes, and marking the outer side of the cover or the 8-linked comb tube as 10 in sequence-3ng/μL、10-4ng/μL、10-5ng/uL, negative and positive; reference tube color order is shown in table 2 as EHEC O157: H7 concentrations.
And (3) detecting the repeatability among batches: repeating detection for 3 parts of nucleic acid to be detected for 3 consecutive days under the same reaction system condition, and synchronously detecting one part of EHEC O157: H7 positive control standard and one part of EHEC O157: H7 negative control standard in each detection reaction;
and (3) carrying out repeatability detection in batches: 3 parts of nucleic acid to be detected are subjected to repeated determination for 3 times in the same reaction system, and each part of EHEC O157: H7 positive control standard and EHEC O157: H7 negative control standard is synchronously detected in each detection reaction;
RPA amplification reaction: the detection tube was placed at 35 ℃ and amplified for 10 min.
And (4) detecting a result: after the RPA amplification reaction is finished, the detection tube is inverted up and down, and the liquid is fully and uniformly mixed; the mixture was left at room temperature for 5min, and color change was observed.
Quantitative interpretation: and (3) comparing the concentrations of EHEC O157 and H7 with a reference tube, if the colors are similar, the concentrations of EHEC O157 and H7 are similar, and preliminarily judging the nucleic acid concentrations of EHEC O157 and H7.
As a result: the results of the batch-to-batch repeatability are shown in Table 3, and the results show that the results of the continuous 3-day detection of the nucleic acids to be detected with the same concentration are consistent, the results of the batch repeatability are shown in Table 4, and the results show that the results of the simultaneous 3-time repeated detection of the nucleic acids to be detected with the same concentration are consistent in the same reaction system. The method is proved to have good detection repeatability.
TABLE 2 EHEC O157H 7 concentration reference tube color vs. concentration control
Figure BDA0003606122850000151
TABLE 3 results of the repeatability tests between batches
Figure BDA0003606122850000152
Figure BDA0003606122850000161
TABLE 4 results of in-batch reproducibility measurements
Figure BDA0003606122850000162
Example eight
The method of the invention is used for detecting laboratory samples
Nucleic acid preparation: 10 samples which are detected to be positive by the reference standard WS 271-2007 diagnosis standard for infectious diarrhea are stored in a laboratory, 5 samples which are detected to be negative again after treatment recovery, and 15 samples in total are detected by the invention. Adding 200 mul of sample into a sample processing tube, fully and uniformly mixing, incubating for 5min at 100 ℃, centrifuging for 12000g for 3min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid to be detected.
Preparing 50 mu L of the detection system of the invention: adding 33.5 mu L of buffer solution I into a detection tube, and uniformly mixing by blowing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; 2.5. mu.L of buffer II was added to the test tube and the mixture was pipetted and mixed. mu.L of developer was dropped onto the inside of the cap of the detection tube.
Synchronously detecting one part of an EHEC O157: H7 positive control standard and one part of an EHEC O157: H7 negative control standard in each detection reaction;
RPA amplification reaction: the detection tube was placed at 35 ℃ and amplified for 10 min.
And (4) detecting a result: after the RPA amplification reaction is finished, the detection tube is inverted up and down, and the mixed liquid is fully inverted; the mixture was left at room temperature for 5min, and color change was observed.
Quantitative interpretation: and (3) comparing the concentrations of the EHEC O157 and the H7 with the reference tube, if the colors are similar, the concentrations of the EHEC O157 and the H7 are similar, and preliminarily judging the nucleic acid concentrations of the EHEC O157 and the H7.
As a result: the results of the detection of the laboratory samples are shown in Table 5, and the results show that 10 positive samples are also positive in the detection of the method, wherein 5 negative samples are negative in 4 parts, 1 positive in 1 part and the concentration of the nucleic acid is 10-4ng/μL-10-5ng/. mu.L. The method of the invention has higher sensitivity than the standard PCR method, and the sample has no symptom after being taken, but pathogenic bacteria or pathogenic bacteria nucleic acid in excrement is not necessarily negative, so the sample with the number 1 is judged to be positive.
TABLE 5 comparison of the results of the PCR and the method of the present invention
Figure BDA0003606122850000163
Figure BDA0003606122850000171
In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Sequence listing
<110> Qingdao international travel health care center (Qingdao customs port outpatient service department)
<120> detection kit for rapidly detecting Escherichia coli O157: H7 through RPA visualization
<130> 04142022
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 33
<212> DNA
<213> Artificial Synthesis ()
<400> 1
attctaacta ggaccgcaga ggaaagagag gaa 33
<210> 2
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 2
agtacattgg catcgtgtgg acagggtaaa 30
<210> 3
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 3
aactattact acaggtgaag gtggaatggt 30
<210> 4
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 4
aatcatcagc ttgttctaac tgggctaatc 30
<210> 5
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 5
aactattact acaggtgaag gtggaatggt 30
<210> 6
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 6
ttcctctgcg gtcctagtta gaattgagac 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 7
tggcatgacg ttataggcta caattatagg 30
<210> 8
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 8
ttcctctgcg gtcctagtta gaattgagac 30
<210> 9
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 9
tggcatgacg ttataggcta caattatagg 30
<210> 10
<211> 30
<212> DNA
<213> Artificial Synthesis ()
<400> 10
ggtgattcct taattcctct ctttcctctg 30

Claims (10)

1. A detection kit for rapidly detecting Escherichia coli O157: H7 through RPA visualization is characterized by comprising: a reference tube with the concentration of EHEC O157 of H7, a reference tube with the concentration of EHEC O157 of H7, a negative reference tube with the concentration of EHEC O157 of H7, a sample processing tube, a detection tube, a buffer solution I, a buffer solution II and a color developing agent;
the EHEC O157H 7 concentration reference tube is a PCR 8 cascade tube filled with 5 tubes of liquid with different colors;
the EHEC O157: H7 positive control standard is a solution of EHEC O157: H7 standard strain genome DNA, and the concentration of the solution is 10-2ng/. mu.L-100 ng/. mu.L, preferably 1 ng/. mu.L;
the EHEC O157: H7 negative control standard is sterile deionized water containing no nucleic acid;
the sample processing tube is a sterile EP tube containing 100-500 μ L of EHEC O157: H7 nucleic acid extraction reagent;
the detection tube is a Twist Amp reaction tube containing an RPA universal reaction reagent freeze-dried preparation;
the buffer solution I is RPA universal reaction buffer solution containing an EHEC O157: H7 detection upstream primer F2 and an EHEC O157: H7 detection downstream primer R2, and the buffer solution I contains an EHEC O157: H7 detection upstream primer F2 with the concentration of 0.28 mu M-0.85 mu M; the concentration of the downstream primer R2 detected by the EHEC O157: H7 is 0.28-0.85 muM;
the buffer solution II is a solution containing 280mM magnesium acetate;
the color developing agent is SYBR Green I solution, and the concentration of the color developing agent is between 100 x and 10000 x.
2. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 1, wherein the kit comprises: the EHEC O157H 7 concentration reference tube obtaining method comprises the following steps: the method for detecting EHEC O157: H7 in the sample to be detected detects that the concentration of EHEC O157: H7 genome DNA is 10-3ng/μL、10-4ng/μL、10- 5ng/mu L of three samples, EHEC O157: H7 negative control standard and EHEC O157: H7 positive control standard, 5 colored detection tubes are obtained after detection, 5 parts of staining solution with corresponding consistent colors are prepared by orange and green dyes according to the colors of the detection tubes, the 5 parts of staining solution are sequentially added into 5 holes of the PCR 8-linked comb tube, 8-linked comb tube covers are covered, and the outer side of the cover or the 8-linked comb tube is sequentially marked with 10-3ng/μL、10-4ng/μL、10-5ng/mu L, negative and positive, and taking the whole as a reference tube of EHEC O157: H7 concentration; the liquid capacity in the EHEC O157: H7 concentration reference tube is 50-200 muL, preferably 200 muL;
the result colors of the 5 colored detection tubes are green, yellow-green, light yellow-green, orange-red and green in sequence;
the method for detecting EHEC O157: H7 in the sample to be detected comprises the following steps: (1) extracting nucleic acid of a sample to be detected; (2) preparing a detection system of a sample to be detected, which is disclosed by the invention: adding 33.5 mu L of buffer solution I into a detection tube, and blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 mu L of buffer solution II into a detection tube, and blowing, beating and uniformly mixing; dropping 2 microliter of color developing agent on the inner side of the cover of the detection tube; (3) covering a cover of the detection tube, and placing the detection tube at 25-45 ℃ for reaction for 10-25 min; (4) and (4) detecting the result: turning the detection tube upside down, and fully and uniformly mixing the liquid in the detection tube; placing the detection tube at room temperature for 1-5 min; synchronously detecting one part of an EHEC O157: H7 positive control standard and one part of an EHEC O157: H7 negative control standard in each detection reaction; (5) and (4) interpretation of results: firstly, detecting a detection tube of the EHEC O157: H7 positive control standard, comparing the EHEC O157: H7 concentration reference tube, and determining that the color of the tube marked as 'positive' in the EHEC O157: H7 concentration reference tube is greenComparing the color of a detection tube for detecting the EHEC O157: H7 negative control standard substance with that of an EHEC O157: H7 concentration reference tube, wherein the color of the tube marked as 'negative' in the EHEC O157: H7 concentration reference tube is orange red, judging that the detection result is credible, otherwise, judging that the result is not credible and needing to detect again; comparing the detected sample with a reference tube of EHEC O157: H7 concentration, wherein the tube is orange red and has the same color with the tube marked as 'negative', and judging the tube is negative; the color of the detection tube for detecting the sample to be detected is green, is similar to the color of the tube marked as 'positive' in the EHEC O157: H7 concentration reference tube, the detection tube is judged to be positive, and simultaneously, the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be more than 10-3ng/mu L; the color of the detection tube for detecting the sample to be detected is yellow green, and the reference tube of the EHEC O157: H7 concentration is marked as 10-4ng/microliter' tubes are similar in color and are judged to be positive, and meanwhile, the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be close to 10-4ng/mu L; the color of the detection tube for detecting the sample to be detected is light yellow green, and the reference tube for detecting the concentration of EHEC O157: H7 is marked as 10-5The tube with the ng/mu L color is judged to be positive, and the concentration of the nucleic acid to be detected extracted from the sample to be detected is judged to be close to 10-5ng/μL；
The specific operation of the step (1) of extracting the nucleic acid of the sample to be detected is as follows: 1) when the sample to be detected is liquid, 100-500 mu L of each sample is taken, and when the sample to be detected is colloidal, paste, semisolid or solid, 100-500 mg of each sample to be detected is taken and fully sheared into pieces; 2) adding the sample to be detected into a sample processing tube, and fully and uniformly mixing; 3) placing the sample processing tube at 90-100 ℃ and incubating for 5-25 min; 4) and (3) centrifuging 12000g of the sample processing tube for 1min-5min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid to be detected.
3. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 2, wherein: in the step 1) of nucleic acid extraction from a test sample of the present invention, preferably, the sample volume of the test sample is the same as the volume of the reagent for nucleic acid extraction EHEC O157: H7 in the sample treatment tube; more preferably, the sample processing tube contains 200. mu.L of EHEC O157: H7 nucleic acid extraction reagent; more preferably, 200 μ L of the sample is taken for each sample when the sample to be tested is liquid, and 200mg of the sample to be tested is taken for each sample when the sample to be tested is colloidal, pasty, semisolid or solid.
4. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 2, wherein: in the step 3) of extracting nucleic acid from the sample to be detected, more preferably, incubation is carried out for 10min at 100 ℃; in the step 4) of extracting nucleic acid from the sample to be tested, the sample to be tested is more preferably centrifuged at 12000g for 3 min.
5. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 2, wherein: the more preferable temperature in the step (3) of detecting EHEC O157: H7 in the sample to be detected is 35 ℃; wherein the preferable reaction time is 10min-25 min.
6. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 2, wherein: more preferably, the reaction time of the step (3) and the standing time of the detection tube of the step (4) for detecting the EHEC O157: H7 in the sample to be detected are 15min in total.
7. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 1, wherein the kit comprises: the EHEC O157H 7 positive control standard acquisition method comprises the following steps: extracting genome DNA of EHEC O157: H7 standard strain according to general bacterial genome DNA extraction kit, obtaining DNA from bacterial sample, dissolving in TE solution or sterile deionized water, detecting nucleic acid concentration, diluting EHEC O157: H7 standard strain genome DNA with TE solution or sterile deionized water to 10-2ng/. mu.L-100 ng/. mu.L, and more preferably the concentration of genomic DNA of the diluted EHEC O157: H7 standard strain is 1 ng/. mu.L.
8. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 1, wherein the kit comprises: the reagent for extracting the nucleic acid of EHEC O157H 7 in the sample processing tube is as follows: 0.5mol/L NaOH, 2 percent Chelex-100 and 0.05mol/L TrisHCl, and the three are prepared according to the volume ratio of 1:1: 1.
9. The detection kit for the rapid detection of the Escherichia coli O157: H7 through RPA visualization according to claim 1, wherein the sequence of the upstream primer F2 for the EHEC O157: H7 detection is 5'-AACTATTACTACAGGTGAAGGTGGAATGGT-3' (SEQ ID NO3), and the sequence of the downstream primer R2 for the EHEC O157: H7 detection is 5'-AATCATCAGCTTGTTCTAACTGGGCTAATC-3' (SEQ ID NO 4).
10. The detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization according to claim 2, wherein: the detection system of the sample to be detected is preferably a 50 mu L system, and the system is as follows: 12 mu L of nucleic acid to be detected obtained after the sample to be detected is extracted, 33.5 mu L of buffer solution I, 2.5 mu L of buffer solution II and 2 mu L of color developing agent; 50 mu.L of buffer I, the buffer I is RPA universal reaction buffer containing 0.7 mu M of EHEC O157: H7 detection upstream primer F2 and 0.7 mu M of EHEC O157: H7 detection downstream primer R2, and the color developing agent is SYBR Green I solution containing 1000X.
CN202210419012.2A 2022-04-20 2022-04-20 Detection kit for rapidly detecting Escherichia coli O157H 7 through RPA visualization Pending CN114606330A (en)

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