CN114561481B - Shigella quick integrated detection kit - Google Patents

Shigella quick integrated detection kit Download PDF

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CN114561481B
CN114561481B CN202210419023.0A CN202210419023A CN114561481B CN 114561481 B CN114561481 B CN 114561481B CN 202210419023 A CN202210419023 A CN 202210419023A CN 114561481 B CN114561481 B CN 114561481B
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shigella
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reagent
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CN114561481A (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 shigella rapid integrated detection kit, which comprises: control system, positive quality control product, negative quality control product, pretreatment system, detection reagent I, detection reagent II, and detection reagent III; the invention has low instrument requirements in the nucleic acid extraction process and the nucleic acid amplification process, strong detection specificity and no cross reaction with other diarrhea pathogens, has the sensitivity 1000-10000 times of that of PCR detection, has simple result interpretation, can be directly interpreted by naked eyes, can meet the conditions and carry out detection in food supervision, quick detection sites and basic laboratories, and is suitable for popularization and use.

Description

Shigella quick integrated detection kit
Technical Field
The invention relates to an extraction and detection kit for diarrhea pathogenic bacteria, in particular to a detection kit for shigella.
Background
Shigella (Shigella) belongs to the family enterobacteriaceae and is the most common pathogen causing bacillary dysentery in humans. The bacillary dysentery caused by shigella is serious, is mainly characterized by systemic toxic symptoms and suppurative inflammation of large intestine, and can cause infectious shock and/or toxic encephalopathy for serious patients, so far, the bacillary dysentery is one of main intestinal infectious diseases affecting human health, and is an important public health problem facing the world.
At present, there are various detection methods for shigella, and common methods for detecting shigella include traditional bacterial detection methods, immunological analysis methods, molecular biological detection, microfluidic sensor detection methods and the like. Although these methods are widely used, the analysis time of the traditional bacterial detection method is usually 2-3 d, which is time-consuming and labor-consuming; although the immunological analysis method is shortened, the sample pretreatment time is long, the sample is easy to pollute, and false positive results are easy to occur; compared with the traditional detection method, the nucleic acid detection method has various advantages, such as good specificity and sensitivity of PCR detection, but the detection speed is still about 120min, and expensive instruments and equipment are needed.
The existing national standard GB4789 is characterized in that the method comprises the steps of adding bacteria to a sample, then carrying out multiple screening and biochemical test identification by a plate method, and has the defects of complex operation and long period, and the method requires an expensive culture medium and a perfect experimental environment, thereby limiting 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 ipaH gene conserved sequence of the shigella, establishes a visual and rapid shigella detection method, and detects the specificity and sensitivity of the shigella.
To achieve the above and other related objects, the present invention provides a shigella rapid integrated detection kit comprising: control system, positive quality control product, negative quality control product, pretreatment system, detection reagent I, detection reagent II, and detection reagent III; the control system is a dense liquid filled with green, yellowish green, orange red and green liquid respectivelySealing the PCR reaction tube; the positive quality control material is a solution of standard shigella genomic DNA, and the concentration of the positive quality control material is 10 -2 ng/. Mu.L to 100 ng/. Mu.L, preferably 1 ng/. Mu.L; the negative quality control product is sterile deionized water which does not contain 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-dried 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 in the detection reagent I is 0.28 mu M-0.85 mu M; the concentration of the downstream primer R2 for detecting shigella is 0.28 mu M-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 100X-10000X.
Preferably, the control system acquisition method is as follows: the genomic DNA concentration detected according to the method of the present invention was 10 -3 ng/μL、10 -4 ng/μL、10 -5 ng/mu L of three samples, negative quality control products and positive quality control products, obtaining 5 detection results after detection, wherein the colors of the results are green, yellow-green, light yellow-green, orange-red and green in sequence, preparing 5 parts of staining solutions with the same color with orange and green dyes according to the colors of the detection results, adding the 5 parts of staining solutions into a PCR reaction tube for sealing, and marking 10 on the cover or the outer side of the PCR reaction tube in sequence -3 ng/μL、10 -4 ng/μL、10 -5 ng/mu L, negative and positive, and the 5 sealed PCR reaction tubes are used as a control system; the liquid volume in the sealed PCR reaction tube of the control system is 50-200. Mu.L, preferably 200. Mu.L; the method for detecting shigella in the sample to be detected comprises the following steps: (1) nucleic acid extraction; (2) preparing the detection result of the sample to be detected: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowingBeating and mixing uniformly; 2.5 mu L of detection reagent II is added into the sealable PCR reaction tube, and is blown and evenly mixed; 2. Mu.L of detection reagent III is dripped on the inner side of a cover of a sealable PCR reaction tube; (3) Covering a sealable PCR reaction tube cover, and placing the PCR reaction tube at 25-45 ℃ for reaction for 15-25 min; (4) result detection: turning the PCR reaction tube upside down, and fully mixing the liquid in the PCR reaction tube; the PCR reaction tube is placed at room temperature for 1min-5min; each detection reaction synchronously detects one part of positive quality control product and one part of negative quality control product; (5) result interpretation: firstly, detecting a detection result of a positive quality control product, wherein the color of a tube marked positive in a comparison system is consistent with that of the tube marked negative in the comparison system, and the detection result of a negative quality control product is consistent with that of the tube marked negative in the comparison system, namely orange red, and judging that the detection result is reliable, otherwise, the result is unreliable and needs to be detected again; detecting a sample to be detected, comparing the detection result with a control system, wherein the color of the control system is orange red and is consistent with that of a tube marked as negative, and judging that the control system is negative; the color of the detection result of the detection sample is green and is similar to the color of a tube marked positive in a control system, the detection result is positive, and the concentration of the nucleic acid to be detected extracted from the detection sample is more than 10 - 3 ng/. Mu.L; the color of the detection result of the sample to be detected is yellow-green, and the detection result is marked as '10' in a comparison system -4 The colors of ng/mu L' tubes are similar, and the tube is positive, and the concentration of the nucleic acid to be detected extracted from the sample to be detected is close to 10 -4 ng/. Mu.L; the color of the detection result of the sample to be detected is light yellow-green, and is marked as 10 in a comparison system -5 The color of the ng/mu L tube 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 -5 ng/. Mu.L; the specific operation of the nucleic acid extraction in the step (1) is as follows: 1) Taking 100-500 mu L of each sample when the sample to be detected is liquid, taking 100-500 mg of each sample to be detected when the sample to be detected is colloid, paste, semisolid or solid, and fully cutting; 2) Adding the sample to be tested into a pretreatment system, and fully and uniformly mixing; 3) Placing the pretreatment system at 90-100 ℃ for incubation for 5-25 min; 4) Centrifuging 12000g of the pretreatment system for 1min-5min, and collecting supernatantExtracting the obtained nucleic acid to be detected.
Preferably, in the step 1) of extracting the nucleic acid from the sample to be tested according to the present invention, preferably, the sample to be tested is measured in the same amount as 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 200ul; more preferably, 200 μl of the sample to be tested is taken per sample when the sample to be tested is liquid, and 200mg of the sample to be tested is taken per sample when the sample to be tested is colloidal or pasty or semi-solid or solid.
Preferably, in the step 3) of extracting the nucleic acid of the sample to be detected, more preferably, incubating at 100 ℃ for 10min; in the nucleic acid extraction step 4) of the sample to be tested, more preferably, 12000g is centrifuged for 3min.
Preferably, the more preferred temperature in step (3) of the present invention for detecting shigella in a sample to be tested is 35 ℃; among them, the preferable reaction time is 15min-20min.
Preferably, the more preferable reaction time of the step (3) for detecting shigella in the sample to be detected is 15min.
Preferably, the positive quality control acquisition method comprises the following steps: extracting standard shigella genomic DNA according to general bacterial genomic DNA extraction kit operation, obtaining DNA from bacterial sample, dissolving in TE solution or sterile deionized water, detecting nucleic acid concentration, diluting the standard shigella genomic DNA with TE solution or sterile deionized water to 10 -2 The concentration of the genomic DNA of the Shigella is more preferably 1 ng/. Mu.L, which is a dilution standard, from ng/. Mu.L to 100 ng/. Mu.L.
Preferably, the extraction reagent I in the pretreatment system is 0.5mol/LNaOH; extracting reagent II in the pretreatment system is Chelex-100 with the concentration of 2%; extracting TrisHCl with the reagent III of 0.05mol/L from the pretreatment system; the three are prepared according to the volume ratio of 1:1:1.
Preferably, the sequence of the upstream primer F6 for shigella detection is 5'-GTCTTTCGCTGTTGCTGCTGATGCCACTGA-3' (SEQ ID NO 11), and the sequence of the downstream primer R6 for shigella detection is 5'-CTGAAGTTTCTCTGCGAGCATGGTCTGGAAGG-3' (SEQ ID NO 12).
Preferably, the detection system of the sample to be detected is a 50-mu L system, and the system is as follows: 12 mu L of nucleic acid to be detected obtained after extracting the sample to be detected, 33.5 mu L of detection reagent I, 2.5 mu L of detection reagent II and 2 mu L of detection reagent III; and 50 mu L of 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 1000 times.
As described above, the shigella rapid integrated detection kit of the invention has the following beneficial effects:
(1) The nucleic acid extraction process and the nucleic acid amplification process have low requirements on instruments, one water bath kettle or a hot water kettle and one centrifugal machine can be met, and the conditions can be met by food supervision, rapid detection sites and basic laboratories; (2) Shigella can be specifically detected, 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 -5 ng/. Mu.L, 1000-10000 times of PCR detection; (4) The method has the advantages of high nucleic acid extraction efficiency, no influence of fecal pollution during diarrhea on the nucleic acid extraction of shigella, high nucleic acid detection sensitivity and good detection efficiency (5) the method has the advantages of simple nucleic acid extraction, few detection steps, simple interpretation method and low requirement on operators; (6) The shigella in the sample is detected, the extraction from nucleic acid to result interpretation can be completed within 30-60 min, and the sensitivity and specificity are met and the method is faster and larger than other methods; (7) The result is simple to judge, the naked eyes directly judge, the longer the time is, the better the color development effect is, and the concentration of shigella in the sample can be judged by combining a contrast control system, so that the judgment is not accurate due to the too low concentration, and the judgment is false negative.
Drawings
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 product No. 1-5, 6: blank control.
FIG. 2 shows the electrophoresis results of the detection of Shigella bacteria with different amplification temperatures for the method of the present invention, wherein M: marker,1-5:4 ℃, 15 ℃, 25 ℃, 35 ℃, 45 ℃.
FIG. 3 shows the electrophoresis results of the detection of Shigella bacteria with different amplification times for the method of the present invention, wherein M: marker,1-5:5min, 10min, 15min, 20min, 25min
FIG. 4 shows the results of electrophoresis of different strains detected by RPA amplification for the method of the invention, wherein M: marker,1-6: shigella, EHECO157, H7, salmonella, vibrio parahaemolyticus, enterococcus faecalis and strain mixed solution
FIG. 5 shows the results of electrophoresis of different concentrations of Shigella nucleic acid detected by RPA amplification in the method of the invention, wherein M: marker,1-8:100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 10 -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 -4 ng/μL、10 - 5 ng/μL
FIG. 6 shows the results of electrophoresis for detection of PCR amplification sensitivity with different concentrations of nucleic acid, where M: marker,1-8:100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 10 -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 -4 ng/μL、10 -5 ng/μL
FIG. 7 shows the results of the detection of Shigella by the method of the invention with different concentrations of SYBRGreenI, where N: negative control, 1-5: 1.25. Mu. LSYBLRGrenI (100×), 0.5. Mu. LSYBLRGrenI (1000×), 1. Mu. LSYBLRGrenI (1000×), 2. Mu. LSYBLRGrenI (1000×), 4. Mu. LSYBLRGrenI (1000×).
FIG. 8 shows the results of the detection of different strains by the method of the invention, wherein N: negative control, 1-6: shigella, EHECO157, H7, salmonella, vibrio parahaemolyticus, enterococcus faecalis and strain mixed solution
FIG. 9 shows the detection results of different concentrations of Shigella nucleic acid by the method of the present invention, wherein N: negative control, 1-8:10 -5 ng/μL、10 -4 ng/μL、10 -3 ng/μL、10 -2 ng/μL、10 -1 ng/μL、1ng/μL、10ng/μL、100ng/μL。
FIG. 10 is a schematic diagram of a detection flow of a sample of Shigella detected by the method of the present invention
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples.
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
To achieve the above and other related objects, the present invention provides a shigella rapid integrated detection kit comprising: control system, positive quality control product, negative quality control product, pretreatment system, detection reagent I, detection reagent II, and detection reagent III;
specifically, the control system is a sealed PCR reaction tube which is respectively provided with green, yellowish green, orange red and green liquid;
specifically, the positive quality control is a solution of standard shigella genomic DNA, and the concentration of the positive quality control is 10-2 ng/. Mu.L to 100 ng/. Mu.L, preferably 1 ng/. Mu.L;
Specifically, the negative quality control product is sterile deionized water which does not contain 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-dried 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;
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, wherein the concentration of the shigella detection upstream primer F2 in the detection reagent I is 0.28 mu M-0.85 mu M; the concentration of the downstream primer R2 for detecting shigella is 0.28 mu M-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 100X-10000X;
specifically, the detection reagent III is a nucleic acid dye, and does not influence the RPA amplification, the enzyme activity in a reaction system and the color development process;
Specifically, the control system acquisition method comprises the following steps: the genomic DNA concentration detected according to the method of the present invention was 10 -3 ng/μL、10 -4 ng/μL、10 -5 ng/mu L of three samples, negative quality control products and positive quality control products, obtaining 5 detection results after detection, wherein the colors of the results are green, yellow-green, light yellow-green, orange-red and green in sequence, preparing 5 parts of staining solutions with the same color with orange and green dyes according to the colors of the detection results, adding the 5 parts of staining solutions into a PCR reaction tube for sealing, and marking 10 on the cover or the outer side of the PCR reaction tube in sequence -3 ng/μL、10 -4 ng/μL、10 -5 ng/mu L, negative and positive, and the 5 sealed PCR reaction tubes are used as a control system; the liquid volume in the sealed PCR reaction tube of the control system is 50-200. Mu.L, preferably 200. Mu.L;
specifically, the step of detecting shigella in a sample to be detected comprises the following steps: (1) nucleic acid extraction; (2) preparing the detection result of the sample to be detected: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into the sealable PCR reaction tube, and is blown and evenly mixed; 2. Mu.L of detection reagent III is dripped on the inner side of a cover of a sealable PCR reaction tube; (3) Covering a sealable PCR reaction tube cover, and placing the PCR reaction tube at 25-45 ℃ for reaction for 15-25 min; (4) result detection: turning the PCR reaction tube upside down, and fully mixing the liquid in the PCR reaction tube; the PCR reaction tube is placed at room temperature for 1min-5min; each detection reaction synchronously detects one part of positive quality control product and one part of negative quality control product; (5) result interpretation: firstly, detecting the detection result of the positive quality control, comparing the color consistency of the tube marked positive in the control system with green, and detecting the detection result of the negative quality control, comparing the color consistency of the tube marked negative in the control system with orange-red, Judging that the detection result is credible, otherwise, the result is not credible and needs to be detected again; detecting a sample to be detected, comparing the detection result with a control system, wherein the color of the control system is orange red and is consistent with that of a tube marked as negative, and judging that the control system is negative; the color of the detection result of the detection sample is green and is similar to the color of a tube marked positive in a control system, the detection result is positive, and the concentration of the nucleic acid to be detected extracted from the detection sample is more than 10 -3 ng/. Mu.L; the color of the detection result of the sample to be detected is yellow-green, and the detection result is marked as '10' in a comparison system -4 The colors of ng/mu L' tubes are similar, and the tube is positive, and the concentration of the nucleic acid to be detected extracted from the sample to be detected is close to 10 -4 ng/. Mu.L; the color of the detection result of the sample to be detected is light yellow-green, and is marked as 10 in a comparison system -5 The color of the ng/mu L tube 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 -5 ng/μL；
Specifically, in the room temperature standing process of the step (4) for detecting shigella in the sample to be detected, as the amplification reaction of the RPA is still continued, the detection process is still carried out, and along with the extension of the room temperature standing time, the positive detection result shows more obvious green; the negative detection result does not change with the time;
Specifically, the specific operation of the nucleic acid extraction in the step (1) is as follows: 1) Taking 100-500 mu L of each sample when the sample to be detected is liquid, taking 100-500 mg of each sample to be detected when the sample to be detected is colloid, paste, semisolid or solid, and fully cutting; 2) Adding the sample to be tested into a pretreatment system, and fully and uniformly mixing; 3) Placing the pretreatment system at 90-100 ℃ for incubation for 5-25 min; 4) Centrifuging 12000g of the pretreatment system for 1-5 min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid to be detected.
Specifically, in the step 1) of extracting the nucleic acid from the 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 200ul; more preferably, 200 μl of the sample to be tested is taken per sample when the sample to be tested is liquid, and 200mg of the sample to be tested is taken per sample when the sample to be tested is colloidal or pasty or semi-solid or solid.
Specifically, in the step 3) of extracting the nucleic acid of the sample to be detected, more preferably, the sample to be detected is incubated for 10min at 100 ℃; in the nucleic acid extraction step 4) of the sample to be tested, more preferably, 12000g is centrifuged for 3min.
Specifically, the more preferable temperature in the step (3) for detecting shigella in the sample to be detected is 35 ℃; among them, the preferable reaction time is 15min-20min.
Specifically, a more preferable reaction time is 15min.
Specifically, the method for acquiring the positive quality control comprises the following steps: extracting standard shigella genomic DNA according to general bacterial genomic DNA extraction kit operation, obtaining DNA from bacterial sample, dissolving in TE solution or sterile deionized water, detecting nucleic acid concentration, diluting the standard shigella genomic DNA with TE solution or sterile deionized water to 10 -2 The concentration of the genomic DNA of the Shigella is more preferably 1 ng/. Mu.L, which is a dilution standard, from ng/. Mu.L to 100 ng/. Mu.L.
Specifically, the extraction reagent I in the pretreatment system is 0.5mol/LNaOH; extracting reagent II in the pretreatment system is Chelex-100 with the concentration of 2%; extracting TrisHCl with the 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 the shigella is 5'-GTCTTTCGCTGTTGCTGCTGATGCCACTGA-3' (SEQ ID NO 11), and the sequence of the downstream primer R6 for detecting the shigella is 5'-CTGAAGTTTCTCTGCGAGCATGGTCTGGAAGG-3' (SEQ ID NO 12).
Specifically, the detection system of the sample to be detected is preferably a 50 μl system, and the system is as follows: 12 mu L of nucleic acid to be detected obtained after extracting the sample to be detected, 33.5 mu L of detection reagent I, 2.5 mu L of detection reagent II and 2 mu L of detection reagent III; and 50 mu L of 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 1000 times.
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 for illustrating the present invention and should not be construed as limiting the scope of the present invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
Referring to FIG. 1, the invention screens amplification primers with high specificity and sensitivity to shigella
The ipaH gene sequence of Shigella was retrieved from NCBI database, the relative conserved region was selected by comparing the existing sequences in database, primers were designed by using PrimerPremier5.0 software and referring to primer design rules on the TwistDx company specifications, and 5 pairs of primers capable of amplifying the ipaH gene sequence were selected by blast comparison in NCBI database (Table 1), and were synthesized by Shanghai Bioengineering Co Ltd.
Pretreatment system: preparing 0.5mol/LNaOH, preparing 2% concentration Chelex-100, 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 shigella nucleic acid extraction reagent; 200. Mu.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 standard shigella culture solution as a sample to be detected, taking 200 mu L of the culture solution, adding a pretreatment system, fully and uniformly mixing, incubating for 5min at 100 ℃, centrifuging 12000g for 3min, and reserving supernatant, wherein the supernatant is the extracted shigella genome DNA. Diluting shigella genome DNA, and adjusting the concentration of nucleic acid to 100 ng/. Mu.L to obtain the nucleic acid to be detected.
Detection reagent II: is a solution containing 280mM magnesium acetate.
50. Mu.L of the RPA amplification reaction system was prepared in total of 5 parts: adding 30.5 mu LRPA universal reaction detection reagent into the sealable PCR reaction tube, and blowing and mixing uniformly; 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 and mixing uniformly; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly.
RPA amplification reaction: performing reaction amplification for 40min at 37 ℃;
and (3) result detection: agarose gel with 2% concentration is prepared, 5 mu L of amplified product is taken for detection, 3V/cm constant pressure electrophoresis is carried out for 20min, and the electrophoresis detection result is recorded and stored by a gel imaging analysis system.
The method comprises the steps of taking a standard shigella culture solution as a sample to be detected, amplifying the designed 5 pairs of primers, wherein the electrophoresis result is shown in figure 1, the amplification strips of the designed primers 1, 4, 5, 6 and 7 are consistent with the size of a target strip, and the amplification strips are clear and have no dispersion and tailing; primers 2, 3, 9 did not appear as a target band and with a bright band primer 8 did not amplify. The results show that the 1 st, 4 th, 5 th, 6 th and 7 th primers have high amplification efficiency and can be accurately detected by an RPA detection system. According to the RPA primer design suggestion, the product length is not more than 200bp, but a primer probe is designed for the subsequent step so as to facilitate the experiment of fluorescent detection, therefore, the 6 th pair of primer pairs EQ ID NO 11 and SEQ ID NO 12 are selected as specific primers of the invention, and the amplified product length is 268bp.
Table 1 RPA amplification primers for Shigella
Example two
Referring to FIGS. 2-3, the method of the present invention establishes and optimizes RPA amplification reactions
1. Optimizing the optimal reaction temperature for RPA amplification
Taking a standard shigella culture solution as a sample to be detected, taking 200 mu l of the culture solution, adding a pretreatment system containing 200 mu l of shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and reserving supernatant, wherein the supernatant is the extracted shigella genome DNA. Diluting shigella genome DNA, and adjusting the concentration of nucleic acid to 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 NO 11) powder to prepare an RPA universal reaction detection reagent with 0.7 mu M upstream primer, dissolving shigella detection downstream primer R6 (SEQ ID NO 12) powder by a proper amount of the solution, and finally preparing an RPA universal reaction detection reagent containing 0.7 mu M shigella detection upstream primer F6 and 0.7 mu M shigella detection downstream primer R6, namely the detection reagent I in the invention, and packaging for later use.
50. Mu.L of RPA assay system was prepared in a total of 5 parts: 35.5 mu L of detection reagent I is added into the sealable PCR reaction tube, and is blown and evenly mixed; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly.
RPA amplification reaction: placing a detection system at 4 ℃, 15 ℃, 25 ℃, 35 ℃ and 45 ℃ respectively, and performing reaction amplification for 20min;
and (3) result detection: agarose gel with 2% concentration is prepared, 5 mu L of amplified product is taken for detection, 3V/cm constant pressure electrophoresis is carried out for 20min, and the electrophoresis detection result is recorded and stored by a gel imaging analysis system.
The detection results show that faint target amplified bands appear at the amplification temperature of 15 ℃ and are gradually brighter along with the temperature rise, the target bands are clearer and brighter at 35 ℃, the bands darken at 45 ℃ and the temperature is in the range of 25-45 ℃, and the detection method can achieve ideal amplification effect when detecting the standard shigella, and has highest amplification efficiency at 35 ℃.
Minimum effective reaction time for RPA amplification
Nucleic acid preparation: taking a standard shigella culture solution as a sample to be detected, taking 200 mu l of the culture solution, adding a pretreatment system containing 200 mu l of shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g,1min-5min, and reserving supernatant, wherein the supernatant is the extracted shigella genome DNA. Diluting shigella genome DNA, and adjusting the concentration of nucleic acid to 100 ng/. Mu.L to obtain the nucleic acid to be detected.
50. Mu.L of RPA assay system was prepared in a total of 5 parts: 35.5 mu L of detection reagent I is added into the sealable PCR reaction tube, and is blown and evenly mixed; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly.
RPA amplification reaction: 5 parts of the detection system are placed at 35 ℃ to be amplified for 5min, 10min, 15min, 20min and 25min respectively.
And (3) result detection: agarose gel with 2% concentration is prepared, 5 mu L of amplified product is taken for detection, 3V/cm constant pressure electrophoresis is carried out for 20min, and the electrophoresis detection result is recorded and stored by a gel imaging analysis system.
Results: the detection results of the amplification for 5min, 10min, 15min, 20min and 25min at 35 ℃ are shown in figure 3, and the better the RPA amplification effect is along with the extension of the reaction time, the more than 15min of the RPA amplification effect is needed, so that the obvious and clear amplification strips can be seen by the method of the invention, and the ideal detection result is achieved.
Example III
Referring to FIG. 4, the specificity of the RPA reaction of the method of the invention is examined
Nucleic acid preparation: preparing standard shigella solution, EHECO157, H7 standard strain solution, salmonella standard strain solution, vibrio parahaemolyticus standard strain solution and enterococcus faecalis standard strain solution, wherein the volumes of the five standard strains are 1:1:1:1:1 ratio of the mixed bacterial liquid. The 6 bacterial liquids are respectively samples to be detected, 200 mu l of each bacterial liquid is taken and added into a pretreatment system containing 200 mu l of shigella nucleic acid extraction reagent, the mixture is fully and uniformly mixed, incubated for 5min at 100 ℃, centrifuged for 12000g and 3min, and the supernatant is reserved, thus obtaining the extracted genome DNA. Diluting 6 kinds of genome DNA, and regulating the concentration of nucleic acid to 100 ng/. Mu.L to obtain the nucleic acid to be detected.
50. Mu.L of RPA assay system was prepared in 6 parts: 35.5 mu L of detection reagent I is added into the sealable PCR reaction tube, and is blown and evenly mixed; respectively adding 12 mu L of nucleic acid to be detected, and blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly.
RPA amplification reaction: 6 parts of the detection system are placed at 35 ℃ for amplification for 20min.
And (3) result detection: agarose gel with 2% concentration is prepared, 5 mu L of amplified product is taken for detection, 3V/cm constant pressure electrophoresis is carried out for 20min, and the electrophoresis detection result is recorded and stored by a gel imaging analysis system.
Results: the genome DNA of standard shigella, EHECO 157:H27 standard strain, salmonella standard strain, vibrio parahaemolyticus standard strain, enterococcus faecalis standard strain and strain mixed solution is used as a template, the RPA reaction amplification result is shown in figure 4, and other strains are not amplified except shigella and mixed bacterial solution to amplify target fragments, so that the RPA detection method has good specificity and cannot be interfered by other intestinal pathogens.
Example IV
Referring to FIGS. 5-6, the sensitivity of the detection of RPA response of the methods of the invention
Nucleic acid preparation: taking a standard shigella culture solution as a sample to be detected, taking 200 mu l of the culture solution, adding a pretreatment system containing 200 mu l of shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid. Diluting Shigella genome DNA to adjust the nucleic acid concentration to 100 ng/. Mu.L, and diluting Shigella genome DNA by 10 times to obtain genome DNA with concentrations of 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L and 10 -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 -4 ng/μL、10 -5 ng/. Mu.L, namely the nucleic acid to be detected.
50. Mu.L of RPA assay system was prepared in 8 parts: 35.5 mu L of detection reagent I is added into the sealable PCR reaction tube, and is blown and evenly mixed; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly.
RPA amplification reaction: 8 parts of the detection system are placed at 35 ℃ for amplification for 20min.
PCR amplification reaction: according to the PCR method, the sensitivity of detecting shigella by using a standard PCR method is detected by referring to the SN/T1869-2007 rapid detection method of various pathogenic bacteria in food, and the PCR reaction conditions and the system reference standard are as follows: f-shig:5'-GTTCCTTGACCGCCTTTCCGATACCGTC-3', R-shig:5'-GCCGGTCAGCCACCCTCTGAGAGTAC-3' the target amplified fragment length is 629bp.
And (3) result detection: agarose gel with 2% concentration is prepared, 5 mu L of amplified product is taken for detection, 3V/cm constant pressure electrophoresis is carried out for 20min, and the electrophoresis detection result is recorded and stored by a gel imaging analysis system.
Results: at a concentration of 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 10 -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 - 4 ng/μL、10 -5 As shown in FIG. 5, the result of RPA amplification using ng/. Mu.L shigella genomic DNA as a template shows that the concentration of nucleic acid for detecting shigella by RPA is 10 -5 At ng/. Mu.L, the amplified bands are clearly visible; the PCR detection result is shown in FIG. 6, and the result shows that the minimum detection limit of the nucleic acid in the PCR detection is 10 -1 ng/. Mu.L, the target band becomes increasingly brighter as the nucleic acid concentration increases. Since the amount of DNA used in the RPA method is approximately 10 in the PCR method 4 The sensitivity of the PRA detection method established by the invention is at least 1000 times that of the standard PCR method.
Example five
Referring to FIG. 7, the method of the present invention RPA reaction visualization detection method is established and optimized
Nucleic acid preparation: taking a standard shigella culture solution as a sample to be detected, taking 200 mu l of the culture solution, adding a pretreatment system containing 200 mu l of shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g for 3min, and reserving supernatant, wherein the supernatant is the extracted shigella genome DNA. Diluting shigella genome DNA, and adjusting the concentration of nucleic acid to 1 ng/. Mu.L to obtain the nucleic acid to be detected.
48. Mu.L of the partial detection system of the present invention was prepared in total 5 parts: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly.
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 GreenI (100X), 0.5. Mu.L of GreenI (1000X), 1. Mu.L of GreenI (1000X), 2. Mu.L of GreenI (1000X), 4. Mu.L of GreenI (1000X) were added dropwise to the inside of the lid of 5 parts of the detection system, respectively.
Negative control detection system 1 part: 35.5. Mu.L of detection reagent I, 2.5. Mu.L of detection reagent II, 12. Mu.L of negative quality control material and 2. Mu.L of LSYBRGrenI (1000X) are added into a sealable PCR reaction tube, and are blown and mixed uniformly.
RPA amplification reaction: 6 parts of the detection system are placed at 35 ℃ and amplified for 15min.
And (3) result detection: after the RPA amplification reaction is finished, the detection system is turned upside down, and the uniformly mixed liquid is fully turned upside down; the mixture was left at room temperature for 5min, and the color change was observed.
Results: the detection system of the invention is added with SYBRGreenI with different concentrations after amplification for 10min, the visual detection result is shown in fig. 7, the result shows that the detection system of the negative control is orange red, other 5 detection systems are observed to find that more than 1 mu L of SYBRGreenI (1000×) is added into 50 mu L of detection system, the color of the detection system changes from orange red to green, and the color change of 2 mu L of SYBRGreenI (1000×) is very obvious.
Example six
Referring to FIG. 8, the specificity of the method of the present invention is detected
Nucleic acid preparation: preparing standard shigella solution, EHECO157, H7 standard strain solution, salmonella standard strain solution, vibrio parahaemolyticus standard strain solution and enterococcus faecalis standard strain solution, wherein the volumes of the five standard strains are 1:1:1:1:1 ratio of the mixed bacterial liquid. The 6 bacterial liquids are respectively samples to be detected, 200 mu l of each bacterial liquid is taken and added into a pretreatment system containing 200 mu l of shigella nucleic acid extraction reagent, the mixture is fully and uniformly mixed, incubated for 5min at 100 ℃, centrifuged for 12000g and 3min, and the supernatant is reserved, thus obtaining the extracted genome DNA. Diluting 6 kinds of genome DNA, and regulating the concentration of nucleic acid to 1 ng/. Mu.L to obtain the nucleic acid to be detected.
Detection reagent III: contains 1000 XSYBRGreenI solution.
50 mu L of the detection system of the method of the invention is prepared into 6 parts: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly. 2. Mu.L of the detection reagent III was dropped onto the inside of the lid of the detection system.
Negative control detection system 1 part: 35.5 mu L of detection reagent I, 2.5 mu L of detection reagent II, 12 mu L of negative quality control material and 2 mu L of detection reagent III are added into a sealable PCR reaction tube, and are blown and uniformly mixed.
RPA amplification reaction: 6 parts of the detection system are placed at 35 ℃ and amplified for 15min.
And (3) result detection: after the RPA amplification reaction is finished, the detection system is turned upside down, and the uniformly mixed liquid is fully turned upside down; the mixture was left at room temperature for 5min, and the color change was observed.
Results: the detection results of the method are shown in figure 8, and besides the yellow-green reaction of the detection system for detecting shigella and mixed bacterial liquid, other detection systems are orange-yellow negative results, which indicate that the shigella detected by the method has good specificity and cannot be interfered by other intestinal pathogens.
Example seven
Referring to FIG. 9, the sensitivity of the method of the present invention is detected
Nucleic acid preparation: taking a standard shigella culture solution as a sample to be detected, taking 200 mu l of the culture solution, adding a pretreatment system containing 200 mu l of shigella nucleic acid extraction reagent, fully and uniformly mixing, incubating at 100 ℃ for 5min, centrifuging 12000g,1min-5min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid. Diluting Shigella genome DNA, adjusting nucleic acid concentration to 100 ng/. Mu.L, and diluting Shigella genome DNA by 10 times to obtain genome DNA concentrations of 10 respectively 0ng/μL、10ng/μL、1ng/μL、10 -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 -4 ng/μL、10 -5 ng/. Mu.L, namely the nucleic acid to be detected.
50 mu L of the detection system of the method of the invention is prepared into 8 parts: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly. 2. Mu.L of the detection reagent III was dropped onto the inside of the lid of the detection system.
Negative control detection system 1 part: 35.5 mu L of detection reagent I, 2.5 mu L of detection reagent II, 12 mu L of negative quality control material and 2 mu L of detection reagent III are added into a sealable PCR reaction tube, and are blown and uniformly mixed.
RPA amplification reaction: 8 parts of the detection system are placed at 35 ℃ for amplification for 15min.
And (3) result detection: after the RPA amplification reaction is finished, the detection system is turned upside down, and the uniformly mixed liquid is fully turned upside down; the mixture was left at room temperature for 5min, and the color change was observed.
Results: at a concentration of 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 10 -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 - 4 ng/μL、10 -5 ng/. Mu.L of Shigella genomic DNA as a template, the detection result of the method of the invention is shown in FIG. 9, and the result shows that when the DNA concentration of Shigella is 10 -5 When ng/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, and the sensitivity of the visualized RPA shigella detection method established in the study reaches 10 -5 ng/. Mu.L, is at least 1000 times the sensitivity of standard PCR methods.
Example eight
Referring to FIG. 10, the detection flow of the present invention for detecting Shigella in a sample detects the repeatability of the method of the present invention
Nucleic acid preparation: taking standard shigella culture solution as a sample to be detected, adding 200 μl of the culture solution into a pretreatment system containing 200 μl of shigella nucleic acid extraction reagent, mixing thoroughly, incubating at 100deg.C for 5min, centrifuging 12000g for 3min,and (4) keeping supernatant, wherein the supernatant is the extracted nucleic acid. Diluting Shigella genomic DNA to adjust nucleic acid concentration to 100 ng/. Mu.L, and diluting Shigella genomic DNA to obtain genomic DNA with concentration of 20 ng/. Mu.L and 2×10 respectively -1 ng/μL、2×10 -3 ng/mu L, namely 3 parts of nucleic acid to be detected; diluting and obtaining the liquid with the concentration of the genome DNA of 1 ng/. Mu.L, namely the positive quality control product.
Preparation of the detection system of the invention 50 μl: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly. 2. Mu.L of the detection reagent III was dropped onto the inside of the lid of the detection system.
Control system: the reference nucleic acid concentration to be measured was 100 ng/. Mu.L, 10 ng/. Mu.L, 1 ng/. Mu.L, 10 -1 ng/μL、10 -2 ng/μL、10 -3 ng/μL、10 -4 ng/μL、10 -5 The color of the detection system of the method is higher than 10 in the concentration of the nucleic acid to be detected at ng/. Mu.L - 3 At ng/. Mu.L, the color of the detection system is green, and the concentration of the nucleic acid to be detected is 10 -3 ng/μL、10 -4 ng/μL、10 -5 When ng/mu L and negative quality control and positive quality control are detected, the color of the detection system after detection is finished by the method is prepared by firstly preparing semitransparent orange-red dye liquor by using sunset yellow dye with water-soluble dye, preparing semitransparent green dye liquor by using fluorescent green dye, and preparing 5 parts of color and 10 parts of nucleic acid concentration to be detected by using the orange-red dye liquor and the green dye liquor -3 ng/μL、10 -4 ng/μL、10 -5 When ng/mu L and negative quality control and positive quality control are detected, the color of the detection system after detection by the method is corresponding, identical and semitransparent liquid, the color sequence is sequentially green, yellowish green, orange red and green, 200 mu L of 5 parts of liquid is taken and sequentially added into 5 holes of a PCR8 row pipe, an 8 row cover is covered, and 10 is marked on the cover or the outer side of the PCR reaction pipe in sequence -3 ng/μL、10 -4 ng/μL、10 -5 ng/μLNegative, positive; the color sequence is shown in Table 2 as a control system.
Batch-to-batch repeatability detection: for 3 parts of nucleic acid to be detected, continuously and repeatedly detecting for 3 days under the same condition of a reaction system, and synchronously detecting one part of positive quality control material and one part of negative quality control material in each detection reaction;
Batch repeatability detection: 3 parts of nucleic acid to be detected are subjected to repeated measurement for 3 times simultaneously in the same reaction system, and each detection reaction synchronously detects one part of positive quality control product and one part of E negative quality control product;
RPA amplification reaction: the detection system was placed at 35℃and amplified for 15min.
And (3) result detection: after the RPA amplification reaction is finished, the detection system is turned upside down, and the uniformly mixed liquid is fully turned upside down; the mixture was left at room temperature for 5min, and the color change was observed.
Quantitative interpretation: and (3) comparing the detection system with a control system, and preliminarily judging the concentration of shigella nucleic acid if the colors are similar.
Results: the results of the batch-to-batch repeatability are shown in Table 3, and the results show that the continuous 3-day detection results of the nucleic acids to be detected with the same concentration are consistent, the results of the batch-to-batch repeatability are shown in Table 4, and the results show that the detection results of the nucleic acids to be detected with the same concentration are consistent in the same reaction system after 3 times of repeated detection. The method is proved to have good detection repeatability.
Table 2 control system color and concentration control
TABLE 3 results of batch-to-batch reproducibility test
TABLE 4 within-batch repeatability test results
Example nine
The method of the invention detects laboratory samples
Nucleic acid preparation: 10 samples tested positive by reference standard WS271-2007 infectious diarrhea diagnostic standard were stored in the laboratory, 5 samples tested negative again after recovery from treatment, and 15 samples were tested using the present invention. 200 μl of the sample is taken and added into a pretreatment system, the mixture is fully and uniformly mixed, incubated for 5min at 100 ℃, centrifuged for 12000g and 3min, and the supernatant is reserved, thus obtaining the extracted nucleic acid to be detected.
Preparation of the detection system of the invention 50 μl: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into a sealable PCR reaction tube, and is blown and mixed uniformly. 2. Mu.L of the detection reagent III was dropped onto the inside of the lid of the detection system.
Each detection reaction synchronously detects one part of positive quality control product and one part of negative quality control product;
RPA amplification reaction: the detection system was placed at 35℃and amplified for 15min.
And (3) result detection: after the RPA amplification reaction is finished, the detection system is turned upside down, and the uniformly mixed liquid is fully turned upside down; the mixture was left at room temperature for 5min, and the color change was observed.
Quantitative interpretation: and (3) comparing the detection system with a control system, and preliminarily judging the concentration of shigella nucleic acid if the colors are similar.
Results: the results of the laboratory samples are shown in Table 5, and the results show that 10 positive samples are positive, wherein 5 negative samples are negative, and the detection result is consistent with the PCR detection result.
TABLE 5 comparison of PCR with the detection results of the method of the invention
In summary, the present invention effectively overcomes the disadvantages of the prior art and has high industrial utility value.
The above embodiments are merely illustrative of the principles of the present invention and its effectiveness, and are not intended to limit the invention. Modifications and variations may be made to the above-described embodiments by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is intended that all equivalent modifications and variations of the invention be covered by the claims, which are within the ordinary skill of the art, be within the spirit and scope of the present disclosure.
Sequence listing
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Claims (7)

1. The shigella integrated detection kit 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 which is respectively filled with green, yellowish green, orange red and green liquid;
the positive quality control material is a solution of standard shigella genomic DNA, and the concentration of the positive quality control material is 10 -2 ng/μL-100ng/μL；
The negative quality control product is sterile deionized water which does not contain nucleic acid;
the pretreatment system comprises a sterile EP tube, an extraction reagent I, an extraction reagent II and an extraction reagent III;
extracting reagent I from the pretreatment system is 0.5mol/L NaOH; extracting reagent II in the pretreatment system is Chelex-100 with the concentration of 2%; extracting TrisHCl with the reagent III of 0.05mol/L from the pretreatment system; the three components are prepared according to the volume ratio of 1:1:1;
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-dried 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 system is a 50 mu L system, and the system is as follows: 12 mu L of nucleic acid to be detected obtained after extracting the sample to be detected, 33.5 mu L of detection reagent I, 2.5 mu L of detection reagent II and 2 mu L of detection reagent III; a total of 50 mu L, wherein the detection reagent I is an RPA universal reaction detection reagent containing 0.7 mu M of shigella detection upstream primer F6 and 0.7 mu M of shigella detection downstream primer R6; the detection reagent II is a solution containing 280mM magnesium acetate; the detection reagent III is SYBR Green I solution containing 1000 multiplied by the number of the detection reagent;
The sequence of the upstream primer F6 for detecting the shigella is 5'-GTCTTTCGCTGTTGCTGCTGATGCCACTGA-3' (SEQ ID NO 11), and the sequence of the downstream primer R6 for detecting the shigella is 5'-CTGAAGTTTCTCTGCGAGCATGGTCTGGAAGG-3' (SEQ ID NO 12).
2. The shigella integrated detection kit of claim 1, wherein: the control system acquisition method comprises the following steps: the genomic DNA concentration was measured to be 10 -3 ng/μL、10 -4 ng/μL、10 -5 ng/mu L of three samples, negative quality control products and positive quality control products, obtaining 5 detection results after detection, wherein the colors of the results are green, yellow-green, light yellow-green, orange-red and green in sequence, preparing 5 parts of staining solutions with the same color with orange and green dyes according to the colors of the detection results, adding the 5 parts of staining solutions into a PCR reaction tube for sealing, and marking 10 on the cover or the outer side of the PCR reaction tube in sequence - 3 ng/μL、10 -4 ng/μL、10 -5 ng/mu L, 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;
the step of detecting shigella in the sample to be detected comprises the following steps: (1) nucleic acid extraction; (2) preparing detection of a sample to be detected: adding 33.5 mu L of detection reagent I into the sealable PCR reaction tube, and blowing and uniformly mixing; adding 12 mu L of nucleic acid to be detected, blowing and uniformly mixing; 2.5 mu L of detection reagent II is added into the sealable PCR reaction tube, and is blown and evenly mixed; 2. Mu.L of detection reagent III is dripped on the inner side of a cover of a sealable PCR reaction tube; (3) Covering a sealable PCR reaction tube cover, and placing the PCR reaction tube at 25-45 ℃ for reaction for 15-25 min; (4) result detection: turning the PCR reaction tube upside down, and fully mixing the liquid in the PCR reaction tube; the PCR reaction tube is placed at room temperature for 1min-5min; each detection reaction synchronously detects one part of positive quality control product and one part of negative quality control product; (5) result interpretation: firstly, detecting the detection result of the positive quality control substance, and comparing the detection result with a control substance The color of the tube marked as positive in the system is consistent with green, and the detection result of the negative quality control is consistent with orange red compared with the color of the tube marked as negative in the control system, so that the detection result is judged to be credible, otherwise, the result is not credible and needs to be detected again; detecting a sample to be detected, comparing the detection result with a control system, wherein the color of the control system is orange red and is consistent with that of a tube marked as negative, and judging that the control system is negative; the color of the detection result of the detection sample is green and is similar to the color of a tube marked positive in a control system, the detection result is positive, and the concentration of the nucleic acid to be detected extracted from the detection sample is more than 10 -3 ng/. Mu.L; the color of the detection result of the sample to be detected is yellow-green, and the detection result is marked as '10' in a comparison system -4 The colors of ng/mu L' tubes are similar, and the tube is positive, and the concentration of the nucleic acid to be detected extracted from the sample to be detected is close to 10 -4 ng/. Mu.L; the color of the detection result of the sample to be detected is light yellow-green, and is marked as 10 in a comparison system -5 The color of the ng/mu L tube 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 -5 ng/μL；
The specific operation of the nucleic acid extraction in the step (1) is as follows: 1) Taking 100-500 mu L of each sample when the sample to be detected is liquid, taking 100-500 mg of each sample to be detected when the sample to be detected is colloid, paste or solid, and fully cutting; 2) Adding the sample to be tested into a pretreatment system, and fully and uniformly mixing; 3) Placing the pretreatment system at 90-100 ℃ for incubation for 5-25 min; 4) Centrifuging 12000g of the pretreatment system for 1-5 min, and reserving supernatant, wherein the supernatant is the extracted nucleic acid to be detected.
3. The shigella integrated detection kit of claim 2, wherein: in the step 1) of nucleic acid extraction, the volume of the extraction reagent I, the volume of the extraction reagent II and the volume of the extraction reagent III in the pretreatment system are combined to be 200ul, 200 mu L of each sample is taken when the sample to be detected is liquid, and 200mg of each sample to be detected is taken when the sample to be detected is colloid, paste or solid.
4. The shigella integrated detection kit of claim 2, wherein: nucleic acid extraction step 3) incubation at 100℃for 10min; step 4) nucleic acid extraction of the sample to be detected, wherein 12000g is centrifuged for 3min.
5. The shigella integrated detection kit of claim 2, wherein: detecting shigella in a sample to be detected, wherein the temperature of the step (3) is 35 ℃; the reaction time is 15min-20min.
6. The shigella integrated detection kit of claim 5, wherein: the reaction time was 15min.
7. The shigella integrated detection kit of claim 1, wherein: the positive quality control product acquisition method comprises the following steps: extracting standard shigella genomic DNA according to general bacterial genomic DNA extraction kit operation, obtaining DNA from bacterial sample, dissolving in TE solution or sterile deionized water, detecting nucleic acid concentration, diluting the standard shigella genomic DNA with TE solution or sterile deionized water to 10 -2 ng/μL-100ng/μL。
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