CN114561481A - Rapid integrated detection kit for shigella - Google Patents

Rapid integrated detection kit for shigella Download PDF

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CN114561481A
CN114561481A CN202210419023.0A CN202210419023A CN114561481A CN 114561481 A CN114561481 A CN 114561481A CN 202210419023 A CN202210419023 A CN 202210419023A CN 114561481 A CN114561481 A CN 114561481A
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shigella
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CN114561481B (en
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徐颖
王岩
侯伟
闫吉辉
张娟
滕新栋
刘海江
梁洁
张瑾
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 rapid integrated detection kit for shigella, which comprises: a control system, a positive quality control product, a negative quality control product, a pretreatment system, a detection reagent I, a detection reagent II and a detection reagent III; 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 10000 times of that of PCR detection, the result interpretation is simple, the naked eye can directly interpret, the conditions can be met and the detection can be carried out in food supervision, rapid detection sites and basic laboratories, and the invention is suitable for popularization and use.

Description

Rapid integrated detection kit for shigella
Technical Field
The invention relates to an extraction detection kit for diarrhea pathogenic bacteria, in particular to a detection kit for shigella.
Background
Shigella (Shigella) is a gram-negative bacterium of the family enterobacteriaceae and is the most common pathogen causing bacillary dysentery in humans. Shigella-induced bacillary dysentery is serious, and is mainly characterized by systemic poisoning symptoms and large intestine suppurative inflammation, severe patients can generate septic shock and/or toxic encephalopathy, so far, the shigella-induced bacillary dysentery is one of main intestinal infectious diseases affecting human health, and is an important public health problem facing the world.
At present, the detection methods for Shigella are various, and common methods for detecting Shigella comprise a traditional bacteria detection method, an immunological analysis method, a molecular biology detection method, a microfluidic sensor detection method 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 is a method of increasing bacteria for a sample, then carrying out multiple screening and biochemical test identification by using a flat plate method, has complex operation and long period, needs expensive culture medium and perfect experimental environment, and limits the application in basic laboratories and sites.
Disclosure of Invention
In view of the defects of the prior art, the invention designs the RPA detection primer according to the conserved sequence of the ipaH gene of the Shigella, establishes a visual and rapid detection method for the Shigella, and detects the specificity and the sensitivity of the Shigella.
In order to achieve the above and other related objects, the present invention provides a rapid integrated detection kit for shigella comprising: a control system, a positive quality control product, a negative quality control product, a pretreatment system, a detection reagent I, a detection reagent II and a detection reagent III; the control system is a sealed PCR reaction tube respectively filled with green, yellow-green, light yellow-green, orange-red and green liquids; the positive quality control product is a solution of standard Shigella genome DNA with the concentration of 10-2ng/. mu.L-100 ng/. mu.L, preferably 1 ng/. mu.L; the negative quality control product is sterile deionized water without nucleic acid; the pretreatment system comprises a sterile EP tube, an extraction reagent I, an extraction reagent II and an extraction reagent III; the detection system comprises a sealable PCR reaction tube, a detection reagent I, a detection reagent II, a detection reagent III and an RPA universal reaction reagent freeze-drying preparation; the RPA universal reaction reagent freeze-dried preparation is arranged in a sealable PCR reaction tube; the detection reagent I, the detection reagent II and the detection reagent III are respectively and independently sealed and packaged; the detection reagent I is an RPA universal reaction detection reagent containing a Shigella detection upstream primer F2 and a Shigella detection downstream primer R2, and the concentration of the Shigella detection upstream primer F2 contained in the detection reagent I is 0.28-0.85 muM; the concentration of the shigella detection downstream primer R2 is 0.28-0.85. mu.M; the detection reagent II is a solution containing 280mM magnesium acetate; the detection reagent III is SYBRGreenI solution, and the concentration of the detection reagent III is between 100 x and 10000 x.
Preferably, the control system obtaining method comprises the following steps: the concentration of genomic DNA detected according to the method of the present invention was 10-3ng/μL、10-4ng/μL、10-5ng/mu L of three samples, a negative quality control product and a positive quality control product, 5 detection results are obtained after the detection is finished, the colors of the results are green, yellow-green, light yellow-green, orange-red and green in turn, according to the colors of the detection results,preparing 5 parts of dyeing solution with corresponding colors by using orange and green dyes, adding the 5 parts of dyeing solution into a PCR reaction tube for sealing, and sequentially marking 10 parts of dyeing solution on the cover or the outer side of the PCR reaction tube-3ng/μL、10-4ng/μL、10-5ng/microliter, negative and positive, the 5 sealed PCR reaction tubes are used as a control system; the liquid capacity in the sealed PCR reaction tube of the control system is 50-200 mu L, preferably 200 mu L; the method for detecting the shigella in the sample to be detected comprises the following steps: (1) extracting nucleic acid; (2) preparing a detection result of the sample to be detected of the invention: adding 33.5 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of detection reagent II into the sealable PCR reaction tube, blowing, beating and uniformly mixing; dripping 2 mu L of detection reagent III on the inner side of a cover of the sealable PCR reaction tube; (3) covering a cap capable of sealing the PCR reaction tube, and placing the PCR reaction tube at 25-45 ℃ for reaction for 15-25 min; (4) and (4) detecting a result: turning the PCR reaction tube upside down, and fully and uniformly mixing the liquid in the PCR reaction tube; placing the PCR reaction tube at room temperature for 1-5 min; synchronously detecting one part of positive quality control material and one part of negative quality control material in each detection reaction; (5) and (4) interpretation of results: firstly, detecting the detection result of a positive quality control product, comparing the color of a tube marked as 'positive' in a comparison system to be green, and comparing the color of a tube marked as 'negative' in the comparison system to be orange-red, judging that the detection result is credible, otherwise, judging that the result is not credible and needing to be detected again; comparing the detection result of the sample to be detected with a comparison system, wherein the color is orange red and is consistent with the color of the tube marked as negative, and judging the tube to be negative; the color of the detection result of the sample to be detected is green and is similar to the color of the tube marked as 'positive' in the comparison system, the sample to be detected 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 result of the sample to be detected is yellow green, and the color is marked as 10 in the comparison system-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; for testing samples to be testedThe color of the detection result is light yellow green, and the color is marked as 10 in a comparison system-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 nucleic acid extraction in the step (1) 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 pretreatment system, and fully and uniformly mixing; 3) the pretreatment system is placed at 90-100 ℃ and incubated for 5-25 min; 4) and (3) centrifuging 12000g of the pretreatment system 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 sample to be detected of the present invention, the amount of the sample to be detected is preferably the same as the amount of the pretreatment system; more preferably, the volume of the extraction reagent I, the extraction reagent II and the extraction reagent III in the pretreatment system is 200 ul; 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 nucleic acid extraction step 3) of the sample to be tested, more preferably, incubation is carried out at 100 ℃ for 10 min; 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 shigella in the sample to be detected is 35 ℃; wherein the preferable reaction time is 15min-20 min.
Preferably, the reaction time of the step (3) for detecting Shigella in the sample to be detected is 15 min.
Preferably, the positive quality control substance acquisition method comprises the following steps: extracting standard Shigella genome DNA according to the operation of the universal bacterial genome DNA extraction kit, obtaining DNA from a bacterial sample, dissolving the DNA in TE solution or sterile deionized water, detecting the concentration of nucleic acid, and diluting the standard Shigella genome DNA with the TE solution or the sterile deionized water to reach the concentration of 10-2ng/. mu.L-100 ng/. mu.L, more preferably diluted standard shigaThe concentration of the bacterial genome DNA is 1 ng/. mu.L.
Preferably, the extraction reagent I in the pretreatment system is 0.5 mol/LNaOH; the extraction reagent II in the pretreatment system is Chelex-100 with the concentration of 2%; TrisHCl with a reagent III of 0.05mol/L is extracted from the pretreatment system; the three are prepared according to the volume ratio of 1:1: 1.
Preferably, the sequence of the Shigella detection upstream primer F6 is 5'-GTCTTTCGCTGTTGCTGCTGATGCCACTGA-3' (SEQ ID NO11), and the sequence of the Shigella detection downstream primer R6 is 5'-CTGAAGTTTCTCTGCGAGCATGGTCTGGAAGG-3' (SEQ ID NO 12).
Preferably, the detection system of the sample to be detected is preferably a 50 μ L system, which is: 12 microliter of nucleic acid to be detected obtained after extracting a sample to be detected, 33.5 microliter of detection reagent I, 2.5 microliter of detection reagent II and 2 microliter of detection reagent III; and the total amount is 50 mu L, the detection reagent I is an RPA universal reaction detection reagent containing 0.7 mu M of shigella detection upstream primer F2 and 0.7 mu M of shigella detection downstream primer R2, and the detection reagent III is a SYBRGreenI solution containing 1000X.
As mentioned above, the rapid integrated detection kit for Shigella of the invention 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 Shigella and has no cross reaction with other diarrhea pathogens; (3) the sensitivity for detecting Shigella is good, and the lower limit of detection can reach 10-5ng/muL is 10000 times of that of PCR detection of 1000-; (4) the nucleic acid extraction efficiency is high, the nucleic acid extraction of Shigella can not be influenced by excrement pollution during diarrhea, the nucleic acid detection sensitivity is high, and the detection efficiency is good (5) the invention has the advantages of simple nucleic acid extraction, few detection steps, simple interpretation method and low requirement on operators; (6) the method can detect the shigella in the sample, and the interpretation can be completed within 30-60 min from nucleic acid extraction to result interpretation, so that the method is quicker and faster than other methods while meeting the sensitivity and specificity; (7) simple interpretation of results, direct interpretation with naked eyes, and time settingThe longer the length, the better the color development effect, and the combination of a contrast system can judge the concentration of shigella in the sample, and the result that the judgment is not accurate due to too low concentration and the judgment is false negative is avoided.
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FIG. 1 shows the electrophoresis results of the detection of Shigella by using different amplification primers according to the method of the present invention, wherein M is Marker, 1-5: primer amplification products 1-5, 6: blank control.
FIG. 2 shows the electrophoresis results of the detection of Shigella using different amplification temperatures according to the method of the present invention, wherein 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 for detecting Shigella using different amplification times according to the method of the present invention, wherein 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, EHECO157: H7, salmonella, vibrio parahaemolyticus, enterococcus faecalis, strain mixture
FIG. 5 shows the results of electrophoresis of the method of the present invention for detecting Shigella nucleic acids at different concentrations using RPA amplification, wherein 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 Shigella by the method of the present invention using different concentrations of SYBRGreenI, where N: negative control, 1-5: 1.25 μ LSYBRGreenI (100 ×), 0.5 μ LSYBRGreenI (1000 ×), 1 μ LSYBRGreenI (1000 ×), 2 μ LSYBRGreenI (1000 ×), 4 μ LSYBRGreenI (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, EHECO157: H7, salmonella, vibrio parahaemolyticus, enterococcus faecalis, strain mixture
FIG. 9 shows the results of detection of Shigella nucleic acid at different concentrations by the method of the invention, wherein 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 for detecting Shigella samples according to 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 rapid integrated detection kit for shigella comprising: a control system, a positive quality control product, a negative quality control product, a pretreatment system, a detection reagent I, a detection reagent II and a detection reagent III;
specifically, the control system is a sealed PCR reaction tube which is respectively filled with green, yellow-green, light yellow-green, orange-red and green liquids;
specifically, the positive quality control product is a solution of standard Shigella genome DNA, and the concentration of the positive quality control product is 10-2 ng/muL-100 ng/muL, preferably 1 ng/muL;
specifically, the negative quality control product is sterile deionized water containing no nucleic acid;
specifically, the pretreatment system comprises a sterile EP tube, an extraction reagent I, an extraction reagent II and an extraction reagent III;
specifically, the detection system comprises a sealable PCR reaction tube, a detection reagent I, a detection reagent II, a detection reagent III and an RPA universal reaction reagent freeze-drying preparation; the RPA universal reaction reagent freeze-dried preparation is arranged in a sealable PCR reaction tube; the detection reagent I, the detection reagent II and the detection reagent III are respectively and independently packaged in a sealing way;
specifically, the detection reagent I is an RPA universal reaction detection reagent containing a Shigella detection upstream primer F2 and a Shigella detection downstream primer R2, and the concentration of the Shigella detection upstream primer F2 contained in the detection reagent I is 0.28-0.85. mu.M; the concentration of the shigella detection downstream primer R2 is 0.28-0.85. mu.M;
specifically, the detection reagent II is a solution containing 280mM magnesium acetate;
specifically, the detection reagent III is SYBRGreenI solution, and the concentration of the detection reagent III is between 100 x and 10000 x;
specifically, the detection reagent III is a nucleic acid dye, and does not influence RPA amplification, the activity of enzyme in a reaction system and a color development process;
specifically, the method for obtaining the control system comprises the following steps: the concentration of genomic DNA detected according to the method of the present invention was 10-3ng/μL、10-4ng/μL、10-5ng/mu L of three samples, a negative quality control product and a positive quality control product, obtaining 5 detection results after the detection is finished, wherein the colors of the results are green, yellow green, light yellow green, orange red and green in turn, preparing 5 parts of dyeing solution with orange and green dyes according to the colors of the detection results, adding the 5 parts of dyeing solution into a PCR reaction tube for sealing, and marking 10 parts of dyeing solution on the cover or the outer side of the PCR reaction tube in turn-3ng/μL、10-4ng/μL、10-5ng/uL, negative and positive, and the 5 sealed PCR reaction tubes are used as a control system; the liquid capacity in the sealed PCR reaction tube of the control system is 50-200 mu L, preferably 200 mu L;
specifically, the steps of detecting shigella in a sample to be detected in the invention are as follows: (1) extracting nucleic acid; (2) preparing a detection result of the sample to be detected of the invention: adding 33.5 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of detection reagent II into the sealable PCR reaction tube, and blowing, beating and mixingHomogenizing; dripping 2 mu L of detection reagent III on the inner side of a cover of the sealable PCR reaction tube; (3) covering a cap capable of sealing the PCR reaction tube, and placing the PCR reaction tube at 25-45 ℃ for reaction for 15-25 min; (4) and (4) detecting a result: turning the PCR reaction tube upside down, and fully and uniformly mixing the liquid in the PCR reaction tube; placing the PCR reaction tube at room temperature for 1-5 min; synchronously detecting one part of positive quality control material and one part of negative quality control material in each detection reaction; (5) and (4) interpretation of results: firstly, detecting the detection result of a positive quality control product, comparing the color of a tube marked as 'positive' in a comparison system to be green, and comparing the color of a tube marked as 'negative' in the comparison system to be orange-red, judging that the detection result is credible, otherwise, judging that the result is not credible and needing to be detected again; comparing the detection result of the sample to be detected with a comparison system, wherein the color is orange red and is consistent with the color of the tube marked as negative, and judging the tube to be negative; the color of the detection result of the sample to be detected is green and is similar to the color of the tube marked as 'positive' in the comparison system, the sample to be detected 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 result of the sample to be detected is yellow green, and the color is marked as 10 in the comparison system-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 result of the sample to be detected is light yellow green, and the color is marked as 10 in the comparison system-5The tube with ng/mu L is judged to be positive if the colors are similar, 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 room temperature placing process of the detection system in the step (4) of detecting the shigella 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 of the positive detection result is more obvious along with the prolonging of the room temperature placing time; the negative detection result does not change along with the prolonging of time;
specifically, the nucleic acid extraction in the step (1) comprises the following specific operations: 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 pretreatment system, and fully and uniformly mixing; 3) the pretreatment system is placed at 90-100 ℃ and incubated for 5-25 min; 4) and (3) centrifuging 12000g of the pretreatment system for 1min-5min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid to be detected.
Specifically, in the step 1) of extracting nucleic acid from a sample to be detected, preferably, the amount of the sample to be detected is the same as the amount of the pretreatment system; more preferably, the volume of the extraction reagent I, the extraction reagent II and the extraction reagent III in the pretreatment system is 200 ul; 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 shigella in the sample to be detected is 35 ℃; wherein the preferable reaction time is 15min-20 min.
Specifically, the reaction time is more preferably 15 min.
Specifically, the positive quality control material acquisition method comprises the following steps: extracting standard Shigella genome DNA according to the operation of the universal bacterial genome DNA extraction kit, obtaining DNA from a bacterial sample, dissolving the DNA in TE solution or sterile deionized water, detecting the concentration of nucleic acid, and diluting the standard Shigella genome DNA with the TE solution or the sterile deionized water to reach the concentration of 10-2ng/. mu.L-100 ng/. mu.L, more preferably the standard Shigella genomic DNA is diluted to a concentration of 1 ng/. mu.L.
Specifically, the extraction reagent I in the pretreatment system is 0.5 mol/LNaOH; the extraction reagent II in the pretreatment system is Chelex-100 with the concentration of 2%; extracting TrisHCl with a reagent III of 0.05mol/L from the pretreatment system; the three are prepared according to the volume ratio of 1:1: 1.
Specifically, the sequence of the upstream primer F6 for detecting Shigella is 5'-GTCTTTCGCTGTTGCTGCTGATGCCACTGA-3' (SEQ ID NO11), and the sequence of the downstream primer R6 for detecting Shigella is 5'-CTGAAGTTTCTCTGCGAGCATGGTCTGGAAGG-3' (SEQ ID NO 12).
Specifically, the detection system of the sample to be detected is preferably a 50 μ L system, and the system is as follows: 12 microliter of nucleic acid to be detected obtained after extracting a sample to be detected, 33.5 microliter of detection reagent I, 2.5 microliter of detection reagent II and 2 microliter of detection reagent III; and the total amount is 50 mu L, the detection reagent I is an RPA universal reaction detection reagent containing 0.7 mu M of shigella detection upstream primer F2 and 0.7 mu M of shigella detection downstream primer R2, and the detection reagent III is a SYBRGreenI 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 the amplification primers with high Shigella specificity and high sensitivity
The ipaH gene sequence of Shigella is retrieved from NCBI database, relative conservative region is selected by comparing the existing sequences in the database, primer design principle on primer design rule of TwistDx company is used by PrimerPremier5.0 software, 5 pairs of primers (table 1) capable of amplifying ipaH gene sequence are selected by blast comparison in NCBI database, and the primers are synthesized by Shanghai biological engineering Limited company.
A pretreatment system: 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 the Shigella nucleic acid extracting reagent; 200 μ L of the shigella nucleic acid extraction reagent was added to a sterile EP tube as a pretreatment system for shigella.
Nucleic acid preparation: taking a culture bacterial liquid of standard Shigella as a sample to be detected, taking 200 mu L of bacterial liquid, adding a pretreatment system, fully and uniformly mixing, incubating for 5min at 100 ℃, centrifuging for 12000g for 3min, and retaining supernatant, wherein the supernatant is the extracted Shigella genome DNA. Diluting the Shigella genome DNA, and adjusting the nucleic acid concentration to be 100 ng/mu L to obtain the nucleic acid to be detected.
And (3) detection reagent 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 detection reagent into a sealable PCR reaction tube, blowing, beating and uniformly mixing; 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; adding 2.5 microliter of detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly.
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.
The electrophoresis result of the 5 pairs of designed primers is shown in figure 1 by taking the culture bacterial liquid of the standard Shigella as a sample to be detected and amplifying, wherein the amplification bands of the designed primers 1, 4, 5, 6 and 7 are consistent with the target band in size, and the amplification bands are clear without dispersion and tailing; primer 2, 3, 9 did not show the band of interest and with a bright band of impurities, primer 8 did not amplify. The results show that the primers 1, 4, 5, 6 and 7 have high amplification efficiency and can be accurately detected by an RPA detection system. According to the RPA primer design suggestion, the length of the product does not exceed 200bp, but a primer probe is designed for the subsequent process so as to continue the experiment for designing fluorescence detection, therefore, the 6 th pair of primer pairs EQ ID NO11 and SEQ ID NO 12 are selected as the specific primers of the invention, and the length of the amplification product is 268 bp.
TABLE 1 RPA amplification primers of Shigella
Figure BDA0003606132190000081
Figure BDA0003606132190000091
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 standard Shigella as a sample to be detected, adding 200 mul of the bacterial liquid into a pretreatment system containing 200 mul of the Shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and retaining a supernatant, wherein the supernatant is the extracted Shigella genome DNA. Diluting the Shigella genome DNA, and adjusting the nucleic acid concentration to be 100 ng/mu L to obtain the nucleic acid to be detected.
Preparing a detection reagent I: firstly, adding a proper amount of RPA universal reaction detection reagent into Shigella detection upstream primer F6(SEQ ID NO11) powder to prepare an RPA universal reaction detection reagent with an upstream primer of 0.7 mu M, then taking a proper amount of the solution to dissolve Shigella detection downstream primer R6(SEQ ID NO 12) powder, and finally preparing the RPA universal reaction detection reagent containing the Shigella detection upstream primer F6 of 0.7 mu M and the Shigella detection downstream primer R6 of 0.7 mu M, namely the detection reagent I of the invention, and subpackaging for later use.
Preparing a total of 5 parts of 50 mu L of RPA detection system: adding 35.5 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly.
RPA amplification reaction: placing a part of detection system at 4 deg.C, 15 deg.C, 25 deg.C, 35 deg.C and 45 deg.C respectively, and performing reaction amplification for 20 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 standard Shigella is taken as a template, the amplification result of an RPA reaction tube at 4 ℃, 15 ℃, 25 ℃, 35 ℃ and 45 ℃ is shown in figure 2, the detection result shows that weak target amplification bands appear at the amplification temperature of 15 ℃, the target bands are gradually bright along with the temperature rise, the target bands are most clear and bright at 35 ℃, the bands are darkened at 45 ℃, and the temperature is indicated to be in the range of 25 ℃ to 45 ℃.
Minimum effective reaction time for RPA amplification
Nucleic acid preparation: taking a culture bacterial liquid of standard Shigella as a sample to be detected, adding 200 mul of the bacterial liquid into a pretreatment system containing 200 mul of the Shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 1min-5min, and retaining a supernatant, wherein the supernatant is the extracted Shigella genome DNA. Diluting the Shigella 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 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly.
RPA amplification reaction: 5 parts of the detection system is 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 figure 3, the RPA amplification effect is better along with the extension of the reaction time, and obvious and clear amplification bands can be seen by the method of the invention at least needing to be amplified for more than 15min, so that an ideal detection result is achieved.
EXAMPLE III
Referring to FIG. 4, the specificity of the RPA reaction according to the method of the present invention was examined
Nucleic acid preparation: preparing a standard shigella solution, an EHECO157: H7 standard strain solution, a salmonella standard strain solution, a vibrio parahaemolyticus standard strain solution, an enterococcus faecalis standard strain solution and five standard strains according to the volume ratio of 1:1: 1:1:1 ratio of the mixed bacteria solution. And respectively taking the 6 bacterial solutions as samples to be detected, respectively adding 200 mu l of the bacterial solutions into a pretreatment system containing 200 mu l of the shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and retaining supernatant, wherein the supernatant is the extracted genomic DNA. Diluting 6 kinds of genome DNA, and regulating the 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 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; respectively adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly.
RPA amplification reaction: 6 parts of the detection system were placed at 35 ℃ and amplified for 20 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 standard Shigella, EHECO157: H7 standard strains, salmonella standard strains, Vibrio parahaemolyticus standard strains, enterococcus faecalis standard strains and strain mixed liquor are respectively used as templates, the RPA reaction amplification result is shown in figure 4, target fragments are amplified except for Shigella and mixed bacterial liquor, and other strains are not amplified, so that the RPA detection method is good in specificity and cannot 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 standard Shigella as a sample to be detected, adding 200 mul of the bacterial liquid into a pretreatment system containing 200 mul of the Shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and retaining supernatant, wherein the supernatant is extracted nucleic acid. Diluting Shigella genome DNA, and adjusting nucleic acid concentration100 ng/mu L, then diluting Shigella genome DNA by 10 times, finally obtaining the genome DNA with the concentration of 100 ng/mu L, 10 ng/mu L, 1 ng/mu L, 10 ng/mu L respectively-1ng/μL、10-2ng/μL、10-3ng/μL、10-4ng/μL、10-5ng/muL is the nucleic acid to be detected.
Preparing a total of 8 parts of 50 mu L of RPA detection system: adding 35.5 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly.
RPA amplification reaction: 8 parts of the detection system were placed at 35 ℃ and amplified for 20 min.
PCR amplification reaction: according to the standard SN/T1869-2007 rapid detection method-PCR method for multiple pathogenic bacteria in food, the detection of the sensitivity of Shigella detection by the standard PCR method is carried out, the PCR reaction conditions and the system reference standard are adopted, and the detection primers are as follows: f-shig: 5'-GTTCCTTGACCGCCTTTCCGATACCGTC-3', R-shig: 5'-GCCGGTCAGCCACCCTCTGAGAGTAC-3', the length of the target amplified fragment is 629 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 by using ng/microliter of Shigella genome DNA as a template is shown in figure 5, and the result shows that the concentration of nucleic acid for detecting Shigella by using 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 10-1ng/. mu.L, the band of interest becomes progressively brighter as the concentration of nucleic acid increases. The usage amount of DNA is close to 10 of that of PCR method by RPA method4The sensitivity of the detection method of PRA established in the present invention is thus at least 1000 times greater than 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 standard Shigella as a sample to be detected, adding 200 mul of the bacterial liquid into a pretreatment system containing 200 mul of the Shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and retaining a supernatant, wherein the supernatant is the extracted Shigella genome DNA. Diluting the Shigella 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 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly.
Preparing a detection system of the invention: on the basis of the above 48. mu.L detection system of the present invention, 1.25. mu.L of LSYBR GreenI (100X), 0.5. mu.L of LSYBRGreenI (1000X), 1. mu.L of LSYBRGreenI (1000X), 2. mu.L of LSYBR GreenI (1000X), 4. mu.L of LSYBRGreenI (1000X) were dropped on the inside of the lid of 5 parts of the detection system, respectively.
1 part of negative control detection system: 35.5 mul of detection reagent I, 2.5 mul of detection reagent II, 12 mul of negative quality control material and 2 mul of LSYBRGreenI (1000X) are added into a sealable PCR reaction tube and are blown and beaten evenly.
RPA amplification reaction: 6 parts of the detection system were placed at 35 ℃ and amplified for 15 min.
And (4) detecting a result: after the RPA amplification reaction is finished, the detection system 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: SYBRGreenI 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 system with negative control is orange red, other 5 detection systems observe that more than 1 microliter of SYBRGreenI (1000 x) is added into 50 microliter of detection system, the color of the detection system is changed from orange red to green, and the color change of 2 microliter of SYBRGreenI (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 a standard shigella solution, an EHECO157: H7 standard strain solution, a salmonella standard strain solution, a vibrio parahaemolyticus standard strain solution, an enterococcus faecalis standard strain solution and five standard strains according to the volume ratio of 1:1: 1:1:1 ratio of the mixed bacteria solution. And respectively taking the 6 bacterial solutions as samples to be detected, respectively adding 200 mu l of the bacterial solutions into a pretreatment system containing 200 mu l of the shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and retaining supernatant, wherein the supernatant is the extracted genomic DNA. Diluting 6 kinds of genome DNA, and regulating nucleic acid concentration to 1 ng/microliter to obtain the nucleic acid to be detected.
And (3) detection reagent III: containing 1000 XSYBRGreenI solution.
The detection system prepared by the method of the invention comprises 50 mu L of 6 parts in total: adding 33.5 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly. mu.L of detection reagent III was added drop-wise to the inside of the lid of the detection system.
1 part of negative control detection system: adding 35.5 mul of detection reagent I, 2.5 mul of detection reagent II, 12 mul of negative quality control product and 2 mul of detection reagent III into a sealable PCR reaction tube, blowing, beating and mixing evenly.
RPA amplification reaction: 6 parts of the detection system were placed at 35 ℃ and amplified for 15 min.
And (4) detecting a result: after the RPA amplification reaction is finished, the detection system 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 a standard Shigella, an EHECO157: H7 standard strain, a salmonella standard strain, a Vibrio parahaemolyticus standard strain, an enterococcus faecalis standard strain and a strain mixed solution 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 system for detecting the Shigella and the mixed bacterial solution, other detection systems are orange-yellow negative results, and the result shows that the Shigella 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
Nucleic acid preparation: taking a culture bacterial liquid of standard Shigella as a sample to be detected, adding 200 mul of the bacterial liquid into a pretreatment system containing 200 mul of the Shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 1min-5min, and retaining supernatant, wherein the supernatant is extracted nucleic acid. Diluting Shigella genome DNA, adjusting nucleic acid concentration to 100 ng/muL, then diluting Shigella genome DNA by 10 times, finally obtaining genome DNA concentrations of 100 ng/muL, 10 ng/muL, 1 ng/muL, 10 ng/muL, etc-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 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly. mu.L of detection reagent III was added dropwise onto the inside of the lid of the detection system.
1 part of negative control detection system: adding 35.5 mul of detection reagent I, 2.5 mul of detection reagent II, 12 mul of negative quality control product and 2 mul of detection reagent III into a sealable PCR reaction tube, blowing, beating and mixing evenly.
RPA amplification reaction: 8 parts of the detection system are placed at 35 ℃ for amplification for 15 min.
And (4) detecting a result: after the RPA amplification reaction is finished, the detection system 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: 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/muL shigella genome DNA is used as a template, and the result shows that when the concentration of the shigella DNA is 10-5ng/muL, 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, and the sensitivity of the visual RPA method for detecting Shigella, which is established in the research, reaches 10-5ng/. mu.L, at least 1000 times the sensitivity of standard PCR methods.
Example eight
Referring to FIG. 10, the detection process of Shigella in the detection sample of the present invention, the repeatability of the method of the present invention
Nucleic acid preparation: taking a culture bacterial liquid of standard Shigella as a sample to be detected, adding 200 mul of the bacterial liquid into a pretreatment system containing 200 mul of the Shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and retaining supernatant, wherein the supernatant is extracted nucleic acid. Diluting Shigella genome DNA, adjusting nucleic acid concentration to 100 ng/muL, diluting the Shigella genome DNA, and finally obtaining genome DNA concentrations of 20 ng/muL and 2 × 10-1ng/μL、2×10-3ng/microliter, namely 3 parts of nucleic acid to be detected; diluting and obtaining liquid with the genome DNA concentration of 1 ng/mu L, namely the positive quality control product.
Preparing 50 mu L of the detection system of the invention: adding 33.5 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly. mu.L of detection reagent III was added dropwise onto the inside of the lid of the detection system.
Control system: 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 detection system of the method of the invention is more than 10 when the concentration of the nucleic acid to be detected is- 3ng/mu L, the color of the detection system is green, and the detection system 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 negative quality control material and positive quality control material, mixing water soluble dye with sunset yellow dye to obtain semi-transparent orange red dye solution, and using fluorescent green dye to obtain the color of the detection systemPreparing translucent green dye liquor, preparing 5 portions of dye liquor with orange red dye liquor and green dye liquor and nucleic acid concentration to be tested being 10-3ng/μL、10-4ng/μL、10-5ng/mu L, and detecting negative quality control products and positive quality control products, the color of the detection system after the detection of the method is corresponding to, same and semitransparent liquid, the color sequence is green, yellow green, light yellow green, orange red and green, 200 mu L of the 5 parts of liquid are taken and sequentially added into 5 holes of a PCR8 row-by-row pipe, 8 row-by-row covers are covered, and 10 parts of liquid are sequentially marked on the cover or the outer side of the PCR reaction pipe-3ng/μL、10-4ng/μL、10-5ng/uL, negative and positive; the color sequence is shown in Table 2 as a control system.
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, and synchronously detecting one part of positive quality control material and one part of negative quality control material in each detection reaction;
and (3) carrying out repeatability detection in batches: 3 parts of nucleic acid to be detected are repeatedly detected for 3 times in the same reaction system, and a positive quality control material and an E negative quality control material are synchronously detected in each detection reaction;
RPA amplification reaction: the detection system is placed at 35 ℃ for amplification for 15 min.
And (4) detecting the result: after the RPA amplification reaction is finished, the detection system 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 detection system with the control system, and judging the concentration of the shigella nucleic acid preliminarily when the color is similar.
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 color vs. concentration control of control system
Figure BDA0003606132190000151
TABLE 3 results of the repeatability tests between batches
Figure BDA0003606132190000152
TABLE 4 results of in-batch reproducibility measurements
Figure BDA0003606132190000153
Example nine
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 WS271-2007 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. And adding 200 mu l of sample into the pretreatment system, 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 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of the detection reagent II into the sealable PCR reaction tube, blowing, beating and mixing evenly. mu.L of detection reagent III was added dropwise onto the inside of the lid of the detection system.
Synchronously detecting one part of positive quality control material and one part of negative quality control material in each detection reaction;
RPA amplification reaction: the detection system is placed at 35 ℃ for amplification for 15 min.
And (4) detecting a result: after the RPA amplification reaction is finished, the detection system 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 comparing the detection system with the control system, wherein the concentration of the shigella is similar when the colors are similar, and preliminarily judging the concentration of the nucleic acid of the shigella.
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, and 5 negative samples are also negative in the detection of the method, and are consistent with the PCR detection results.
TABLE 5 comparison of the results of the PCR and the method of the present invention
Figure BDA0003606132190000161
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.
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Claims (10)

1. The rapid integrated detection kit for the shigella is characterized by comprising: a control system, a positive quality control product, a negative quality control product, a pretreatment system and a detection system;
the control system is a sealed PCR reaction tube respectively filled with green, yellow-green, light yellow-green, orange-red and green liquids;
the positive quality control product is a solution of standard Shigella genome DNA with the concentration of 10-2ng/. mu.L-100 ng/. mu.L, preferably 1 ng/. mu.L;
the negative quality control material is sterile deionized water containing no nucleic acid;
the pretreatment system comprises a sterile EP tube, an extraction reagent I, an extraction reagent II and an extraction reagent III;
the detection system comprises a sealable PCR reaction tube, a detection reagent I, a detection reagent II, a detection reagent III and an RPA universal reaction reagent freeze-drying preparation; the RPA universal reaction reagent freeze-dried preparation is arranged in a sealable PCR reaction tube; the detection reagent I, the detection reagent II and the detection reagent III are respectively and independently sealed and packaged;
the detection reagent I is an RPA universal reaction detection reagent containing a Shigella detection upstream primer F2 and a Shigella detection downstream primer R2, and the concentration of the Shigella detection upstream primer F2 contained in the detection reagent I is 0.28-0.85 muM; the concentration of the shigella detection downstream primer R2 is 0.28-0.85. mu.M;
the detection reagent II is a solution containing 280mM magnesium acetate;
the detection reagent III is SYBR Green I solution, and the concentration of the detection reagent III is between 100 x and 10000 x.
2. The rapid integrated shigella detection kit according to claim 1, wherein: the control system acquisition method comprises the following steps: the concentration of genomic DNA detected according to the method of the present invention was 10-3ng/μL、10-4ng/μL、10-5ng/mu L of three samples, a negative quality control product and a positive quality control product, obtaining 5 detection results after the detection is finished, wherein the colors of the results are green, yellow green, light yellow green, orange red and green in turn, preparing 5 parts of dyeing solution with orange and green dyes according to the colors of the detection results, adding the 5 parts of dyeing solution into a PCR reaction tube for sealing, and marking 10 parts of dyeing solution on the cover or the outer side of the PCR reaction tube in turn-3ng/μL、10-4ng/μL、10-5ng/microliter, negative and positive, the 5 sealed PCR reaction tubes are used as a control system; the liquid capacity in the sealed PCR reaction tube of the control system is 50-200 mu L, preferably 200 mu L;
the method for detecting the shigella in the sample to be detected comprises the following steps: (1) extracting nucleic acid; (2) preparing a detection result of the sample to be detected of the invention: adding 33.5 microliter of detection reagent I into the sealable PCR reaction tube, blowing, beating and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing, beating and uniformly mixing; adding 2.5 microliter of detection reagent II into the sealable PCR reaction tube, blowing, beating and uniformly mixing; dripping 2 mu L of detection reagent III on the inner side of a cover of the sealable PCR reaction tube; (3) covering a cap of the PCR reaction tube which can be sealed, and placing the PCR reaction tube at 25-45 ℃ for reaction for 15-25 min; (4) resultsAnd (3) detection: turning the PCR reaction tube upside down, and fully and uniformly mixing the liquid in the PCR reaction tube; placing the PCR reaction tube at room temperature for 1-5 min; synchronously detecting one part of positive quality control material and one part of negative quality control material in each detection reaction; (5) and (4) interpretation of results: firstly, detecting the detection result of a positive quality control product, comparing the color of a tube marked as 'positive' in a comparison system to be green, and comparing the color of a tube marked as 'negative' in the comparison system to be orange-red, judging that the detection result is credible, otherwise, judging that the result is not credible and needing to be detected again; comparing the detection result of the sample to be detected with a comparison system, wherein the color is orange red and is consistent with the color of the tube marked as negative, and judging the tube to be negative; the color of the detection result of the sample to be detected is green and is similar to the color of the tube marked as 'positive' in the comparison system, the sample to be detected is 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 more than 10-3ng/mu L; the color of the detection result of the sample to be detected is yellow-green, and the color of the detection result is marked with 10 in a comparison system-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 result of the sample to be detected is light yellow green, and the color is marked as 10 in the comparison system-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 nucleic acid extraction in the step (1) 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 pretreatment system, and fully and uniformly mixing; 3) the pretreatment system is placed at 90-100 ℃ and incubated for 5-25 min; 4) and (3) centrifuging 12000g of the pretreatment system for 1min-5min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid to be detected.
3. The rapid integrated detection kit for shigella according to claim 2, wherein: in the step 1) of extracting nucleic acid according to the present invention, preferably, the amount of the sample to be tested is the same as the amount of the pretreatment system; more preferably, the volume of the extraction reagent I, the extraction reagent II and the extraction reagent III in the pretreatment system is 200 ul; 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 rapid integrated shigella detection kit according to claim 2, wherein: in the nucleic acid extraction step 3), more preferably, incubation is performed at 100 ℃ for 10 min; 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 rapid integrated shigella detection kit according to claim 2, wherein: the more preferable temperature in the step (3) of detecting the shigella in the sample to be detected is 35 ℃; wherein the preferable reaction time is 15min-20 min.
6. The rapid integrated shigella detection kit according to claim 5, wherein: more preferably, the reaction time is 15 min.
7. The rapid integrated shigella detection kit according to claim 1, wherein: the positive quality control product acquisition method comprises the following steps: extracting standard Shigella genome DNA according to the operation of the universal bacterial genome DNA extraction kit, obtaining DNA from a bacterial sample, dissolving the DNA in TE solution or sterile deionized water, detecting the concentration of nucleic acid, and diluting the standard Shigella genome DNA with the TE solution or the sterile deionized water to 10-2The concentration of the genomic DNA of the standard Shigella is 1 ng/mu L, and the concentration of the genomic DNA of the standard Shigella is more preferably 1 ng/mu L.
8. The rapid integrated shigella detection kit according to claim 1, wherein: extracting reagent I in the pretreatment system is 0.5mol/L NaOH; the extraction reagent II in the pretreatment system is Chelex-100 with the concentration of 2%; extracting TrisHCl with a reagent III of 0.05mol/L from the pretreatment system; the three are prepared according to the volume ratio of 1:1: 1.
9. The rapid integrated detection kit for Shigella according to claim 1, wherein the sequence of the upstream primer F6 for Shigella detection is 5'-GTCTTTCGCTGTTGCTGCTGATGCCACTGA-3' (SEQ ID NO11), and the sequence of the downstream primer R6 for Shigella detection is 5'-CTGAAGTTTCTCTGCGAGCATGGTCTGGAAGG-3' (SEQ ID NO 12).
10. The rapid integrated shigella detection kit 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 microliter of nucleic acid to be detected obtained after extracting a sample to be detected, 33.5 microliter of detection reagent I, 2.5 microliter of detection reagent II and 2 microliter of detection reagent III; and the total amount is 50 mu L, the detection reagent I is an RPA universal reaction detection reagent containing 0.7 mu M of Shigella detection upstream primer F2 and 0.7 mu M of Shigella detection downstream primer R2, and the detection reagent III is a SYBR Green I solution containing 1000X.
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