CN114891903A - Kit and method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA - Google Patents
Kit and method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA Download PDFInfo
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Abstract
The invention relates to the technical field of microbial detection, in particular to a kit and a detection method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA. The kit comprises PMAxx, LAMP amplification reaction liquid and a nucleic acid detection test strip. The PMAxx dye is adopted to treat bacteria and then is subjected to LAMP amplification, so that the death and activity of the bacteria can be effectively distinguished, false positive interference of the dead bacteria on a detection result can be eliminated, the detection time can be effectively shortened, the cost is reduced, the detection precision is improved, and the rapid detection on site is realized.
Description
Technical Field
The invention relates to the technical field of microbial detection, in particular to a kit and a detection method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA.
Background
Salmonella (Salmonella) is one of the most widely distributed food-borne pathogenic bacteria, and is the chief culprit in causing most food poisoning events in countries around the world. It is often detected in eggs and products thereof, meat, dairy products and other food products. In areas with limited resources and lack of laboratories and detection equipment, the rapid and accurate salmonella field detection method is provided, which has important significance for food safety prevention and control.
The detection method for salmonella mainly comprises a national standard method, a PCR method and the like. The national standard method is the gold standard of detection, but the method is time-consuming, labor-consuming, high in cost, strong in specificity and incapable of realizing timely and effective detection. The PCR method has high detection precision and short time, but is dependent on a PCR instrument and difficult to meet the requirement of actual field detection. The immunochromatography technology is convenient and fast, is suitable for field detection, but has low detection sensitivity and low detection precision caused by the fact that dead bacteria and live bacteria cannot be distinguished.
Loop-mediated isothermal amplification (LAMP) is a DNA amplification method other than PCR. The method can amplify DNA under the condition of constant temperature, and has the advantages of strong specificity, high speed and high efficiency. The detection time is shorter than that of PCR, and the amplification can be realized by only a few simple small heaters without depending on a thermal cycler during amplification. The LAMP-NALFA (LAMP-NALFA) technology combined with the chromatography technology has the high sensitivity of the nucleic acid amplification technology and the visibility, portability and easy operability of the immunochromatography technology, and is a rapid and visual field detection technology. However, like the LAMP technology and the immunochromatography technology, the LAMP-NALFA technology cannot distinguish dead bacteria, and the detection precision of the technology is affected by the existence of the dead bacteria. The nucleic acid dyes such as ethidium azide bromide (EMA) and propidium azide bromide (PMA) are used, so that the defect that live bacteria cannot be distinguished by the LAMP-NALFA technology is overcome, and the salmonella is accurately detected.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a PMAX nucleic acid dye based on improvement, namely PMAxx, combined with a PMAxx-LAMP-NALFA viable bacteria detection kit based on LAMP technology and a detection method thereof, which can effectively shorten the detection time, reduce the cost, improve the detection precision and realize the rapid detection on site.
The invention firstly provides a detection kit for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA, which comprises PMAxx, LAMP amplification reaction liquid and a nucleic acid detection test strip.
Preferably, the LAMP amplification reaction solution is 23 μ L, and comprises: 0.2. mu.M primer F3, 0.2. mu.M primer B3, 1.6. mu.M primer FIP, 1.6. mu.M primer BIP, 0.8. mu.M primer LF, 0.8. mu.M primer LB, 2.5. mu.L buffer, 6mM Mg 2+ 0.6M betaine, 6U Bst DNA polymerase, 1.4mM dNTP, in addition to 20U L paraffin oil to play a liquid seal.
The sequences of the primers FIP, BIP, F3, B3, LF and LB are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
Further preferably, the 5 'end of the primer FIP is labeled with FITC and the 5' end of LF is labeled with biotin.
Preferably, the concentration of PMAxx is 2 mM.
Preferably, the kit further comprises a running buffer solution of the nucleic acid detection test strip and a negative control sample, wherein the running buffer solution is a phosphate buffer solution containing 2% of S9, and the negative control sample is DEPC water without nucleic acid.
Preferably, the nucleic acid detection test strip comprises a sample pad, a combination pad, an NC membrane, a water absorption pad and a bottom plate, wherein a detection line T line coated with streptavidin and a quality control line C line coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody are respectively arranged on the NC membrane, the NC membrane is arranged on the bottom plate, the sample pad and the combination pad are sequentially connected with one end of the NC membrane close to the detection line, and the water absorption pad is connected with one end of the NC membrane close to the quality control line.
Further preferably, the preparation steps of the binding pad of the nucleic acid detection test strip are as follows: preparing a FITC antibody marked by color microspheres, diluting the FITC antibody marked by the color microspheres in a treatment solution of a bonding pad, dropwise adding the solution onto the bonding pad, and drying for 3-4 hours at 45 ℃.
Preferably, the detection line and the quality control line of the nucleic acid detection test strip are prepared by the following steps: and respectively scribing streptavidin and goat anti-mouse IgG antibodies diluted by phosphate buffer solution on the NC membrane, wherein the coating concentration is 0.5-1.5 mg/mL, and placing in an oven at 37 ℃ for overnight.
Preferably, the nucleic acid detection test strip is provided with a detection cavity, a sample adding slot and an observation window which are communicated with the detection cavity, and the nucleic acid detection test strip is arranged in the detection cavity.
The invention also provides a detection method of the detection kit for rapidly detecting the live salmonella based on the PMAxx-LAMP-NALFA, which comprises the following steps:
s1, mixing a sample to be detected with the PMAxx uniformly to enable the final concentration of the PMAxx in the system to be 2-20 mu M, dyeing for 5-20 min in a dark place, carrying out photoactivation treatment for 5-15 min in a nucleic acid marker, centrifuging at 10000rpm for 2min, then carrying out resuspension by using a phosphate buffer solution, then centrifuging, carrying out resuspension by using TE water, heating at 100 ℃ for 10min, carrying out ice bath for 5min, and centrifuging at 12000rpm for 2 min;
s2, sucking 2 mu L of supernatant obtained after centrifugation in the step S1 by using a pipette tip, adding the supernatant into the LAMP amplification reaction solution, amplifying at 60-65 ℃ for 30-60 min, and heating at 85 ℃ for 5 min;
s3, adding 2-10 mu L of amplification sample into the nucleic acid detection test strip, adding 70-98 mu L of running buffer solution, reacting for 5-15 min, and judging the detection result according to the color development band of the nucleic acid detection test strip.
Compared with the prior art, the invention has the beneficial effects that:
(1) the PMAxx dye adopted by the invention can not pass through cell membranes, and only selectively permeates cells with broken membranes (dead bacteria). After the dye is embedded into double-stranded DNA, covalent connection with the DNA can be formed under the strong visible light exposure, the DNA cannot be amplified after being combined with the dye, and the living bacterium DNA cannot be influenced. After the dye is used for treating bacteria, LAMP amplification is carried out, so that the death and activity of the bacteria can be effectively distinguished, and false positive interference caused by the death and bacteria can be eliminated;
(2) the invention is used without depending on large-scale instruments and is suitable for field detection.
Drawings
FIG. 1 is a schematic structural diagram of a PMAxx-LAMP-NALFA-based test strip for rapidly detecting live bacteria of salmonella
FIG. 2 is a diagram showing the result of fast detecting the viable bacteria of Salmonella by PMAxx-LAMP-NALFA of the present invention, note that: 1: blank; 2: 1X 10 6 CFU/mL；3：1×10 5 CFU/mL；4：1×10 4 CFU/mL；5：1×10 3 CFU/mL；6：1×10 2 CFU/mL；7：1×10 1 CFU/mL。
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The test methods used in the examples of the present invention are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1:
the embodiment provides a nucleic acid detection test strip for rapidly detecting live bacteria of salmonella based on PMAxx-LAMP-NALFA.
As shown in fig. 1, includes: the device comprises a sample pad, an NC membrane, a water absorption pad and a bottom plate; a detection line coated with streptavidin and a quality control line coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody are respectively arranged on the NC membrane; the NC membrane is arranged on the bottom plate, the sample pad and the combination pad are sequentially connected with one end, close to the detection line, of the NC membrane, and the water absorption pad is connected with one end, close to the quality control line, of the NC membrane.
Example 2:
the embodiment provides a preparation method of a nucleic acid detection test strip for rapidly detecting live bacteria of salmonella based on PMAxx-LAMP-NALFA.
(1) Preparation of conjugate pad
Preparing a color microsphere probe, adding color microspheres into HEPES coupling buffer solution, adding an FITC antibody to be marked, wherein the ratio of the FITC antibody to the color microspheres is 20-50 mu g/100 mu L of microspheres, carrying out ultrasonic coupling for 5-30 min, adding a sealing solution for sealing, carrying out high-speed centrifugation for 5-30 min, washing and resuspending for 2-4 times by using a preservation solution, and preserving in a refrigerator at 4 ℃.
And (3) selecting a proper microsphere diluent to dilute the color microsphere probe, wherein the ratio of the microspheres to the diluent is 1.25-2.5 mu L/mL, dripping the diluted solution on a bonding pad, and drying the bonding pad for 3-4 hours at 45 ℃.
(2) Preparation of detection line on NC membrane
And (3) balancing the streptavidin diluted by the PBS solution in an environment with room temperature and humidity of 30-60%, scribing on an NC membrane with the coating concentration of 0.5-1.5 mg/mL, and placing in an oven at 37 ℃ for overnight.
(3) Preparation of quality control line on NC film
After the goat anti-mouse IgG antibody or the rabbit anti-mouse IgG antibody diluted by the PBS solution is balanced in an environment with room temperature and 30% -60% of humidity, the antibody is scribed on the NC membrane, the coating concentration is 0.5-1.5 mg/mL, and the NC membrane is placed in an oven with the temperature of 37 ℃ for overnight.
Example 3:
the embodiment provides a detection kit for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA, which comprises: comprises PMAxx, LAMP amplification reaction liquid and a nucleic acid detection test strip. The nucleic acid detection test strip is provided with a detection cavity, a sample adding groove and an observation window which are communicated with the detection cavity, and the nucleic acid detection test strip is arranged in the detection cavity.
Wherein, the PMAxx is used for staining salmonella in a sample to be detected, and the PMAxx can enter the cell of the salmonella and be combined with the DNA of the killed bacteria due to the damaged cell membrane of the killed bacteria. The combined dead bacteria DNA cannot be amplified in the subsequent LAMP, and only the live bacteria DNA can be amplified, so that the interference of the dead bacteria DNA is eliminated. The bacteria after PMAxx treatment are centrifuged to resuspend and then subjected to DNA extraction, and then DNA is added to the LAMP amplification reaction solution. The LAMP amplification reaction solution contains primers marked with FITC and biotin, so that the two substances are marked by the amplification target. And dripping the amplification product with the label onto a nucleic acid detection test strip, and combining the FITC end of the amplification product with a FITC antibody color microsphere probe on the binding pad when the amplification product passes through the binding pad. When the product reaches the detection line, biotin on the amplification product is combined with streptavidin on the detection line to form a corresponding red strip on the test strip, and the excessive color microsphere probe is continuously chromatographed to the quality control line to form a red strip.
Example 4:
the embodiment provides a method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA, which comprises the following steps:
(1) and uniformly mixing the sample to be detected and the PMAxx solution in proportion to enable the final concentration of the PMAxx in the system to be 2-20 mu M, dyeing for 5-20 min in a dark place, and performing photoactivation treatment for 5-15 min in a nucleic acid marker. After centrifugation at 10000rpm for 2min, the suspension was resuspended in PBS. The mixture was centrifuged again and resuspended in TE water. Heating at 100 deg.C for 10min, ice-cooling for 5min, and centrifuging at 12000rpm for 2 min.
Preferably, the final concentration of PMAxx in the sample to be detected is 6 mu M, the sample is dyed for 15min in a dark place, and photoactivation treatment is carried out for 8min in a nucleic acid marker;
(2) and (3) sucking 2 mu L of supernatant by using a pipette tip, adding the supernatant into the LAMP amplification reaction solution, amplifying at 60-65 ℃ for 30-60 min, and then at 85 ℃ for 5 min.
Preferably, 2. mu.L of the supernatant is pipetted into the LAMP amplification reaction and amplified at 64 ℃ for 40 min.
(3) Adding 2-10 mu L of amplification sample into the nucleic acid detection test strip, adding 70-98 mu L of running buffer solution, reacting for 5-15 min, and judging the detection result according to the color development band of the nucleic acid detection test strip.
Preferably, 5 μ L of the amplified sample is added to the nucleic acid detection test strip, 75 μ L of the running buffer solution is added, the reaction is carried out for 6min, and the detection result is judged according to the color development band of the nucleic acid detection test strip.
Test examples
In this embodiment, the detection kit and the detection method for rapidly detecting the live salmonella based on the PMAxx-LAMP-NALFA disclosed by the invention are used for detecting the live salmonella.
(1) The salmonella standard strain ATCC14028 preserved at-80 ℃ is streaked on a TSA solid culture medium and is cultured in a constant temperature incubator at 37 ℃ for 24 h. A typical single colony was picked and inoculated into TSB broth, placed on a shaker, and cultured overnight at 37 ℃ at 180 r/min. And (3) carrying out gradient dilution on the bacterial liquid by using sterile PBS, selecting a proper concentration to carry out plate coating on a TSA solid culture medium, carrying out constant temperature overnight culture at 37 ℃, and counting bacterial colonies to obtain the viable count.
(2) Preparation of 1.0X 10 Using sterile PBS 1 ～1.0×10 6 CFU/mL live Salmonella bacteria. PMAxX was added to 1mL of the bacterial solution to a final concentration of 6. mu.M, and the mixture was stained in the dark for 15min and photoactivated in a nucleic acid labeling instrument for 8 min. After centrifugation at 10000rpm for 2min, the suspension was resuspended in PBS. The mixture was centrifuged again and resuspended in 50. mu.L of TE water. Heating at 100 deg.C for 10min, ice-cooling for 5min, and centrifuging at 12000rpm for 2 min.
(3) And (3) sucking 2 mu L of supernatant liquid by using a pipette gun, adding the supernatant liquid into the LAMP amplification reaction liquid, and amplifying for 40min at 64 ℃. The 23. mu.L LAMP amplification reaction solution contained: 0.2. mu.M primer F3, 0.2. mu.M primer B3, 1.6. mu.M primer FIP, 1.6. mu.M primer BIP, 0.8. mu.M primer LF, 0.8. mu.M primer LB, 2.5. mu.L Buffer, 6mM Mg 2+ 0.6M betaine, 6U Bst DNA polymerase, 1.4mM dNTP. In addition, 20. mu.L of paraffin oil can be used for liquid sealing.
The primer is designed based on salmonella virulence gene invA, FITC and biotin are respectively marked at the 5' ends of FIP and LF, and the specific sequence is shown in a sequence table.
The sequences of the primers FIP, BIP, F3, B3, LF and LB are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
(4) And adding 5 mu L of amplified sample into the nucleic acid detection test strip, adding 75 mu L of running buffer solution, reacting for 6min, and judging the detection result according to the color development band of the nucleic acid detection test strip. As a result, as shown in FIG. 2, the detection limit of this technique was 1.0X 10 2 CFU/mL。
The above-mentioned embodiments of the present invention are merely examples for clearly illustrating the technical solutions of the present invention, and are not intended to limit the specific embodiments of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention claims should be included in the protection scope of the present invention claims.
Sequence listing
<110> southern China university of agriculture
<120> kit and detection method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA
<130> ZM21139WM
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 39
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
gcgaggtttg gcggctgata tcgcgacgac aaaatctgg 39
<210> 2
<211> 42
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
aactttagcg caaggtgcct ctgccggtaa actacacgat ga 42
<210> 3
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
ctgggcgttt ttttgtcctg 20
<210> 4
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
gggaaggtta aggagggtga 20
<210> 5
<211> 18
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
aggcttcgcg tacagagg 18
<210> 6
<211> 19
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
cacgtcagca aagcgtacc 19
Claims (10)
1. A detection kit for rapidly detecting salmonella viable bacteria based on PMAxx-LAMP-NALFA is characterized by comprising PMAxx, LAMP amplification reaction liquid and a nucleic acid detection test strip.
2. The PMAxx-LAMP-NALFA-based kit for rapidly detecting live Salmonella bacteria according to claim 1, wherein the LAMP amplification reaction solution is 23 μ L and comprises: 0.2. mu.M primer F3, 0.2. mu.M primer B3, 1.6. mu.M primer FIP, 1.6. mu.M primer BIP, 0.8. mu.M primer LF, 0.8. mu.M primer LB, 2.5. mu.L buffer, 6mM Mg 2+ 0.6M betaine, 6U Bst DNA polymerase, 1.4mM dNTP, in addition to 20L paraffin oil liquid seal;
the sequences of the primers FIP, BIP, F3, B3, LF and LB are respectively shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5 and SEQ ID No. 6.
3. The kit for rapidly detecting the viable salmonella based on PMAxx-LAMP-NALFA according to claim 2, wherein FITC is labeled at the 5 'end of the primer FIP, and biotin is labeled at the 5' end of LF.
4. The PMAxx-LAMP-NALFA-based rapid detection kit for viable Salmonella bacteria according to claim 1, wherein the concentration of PMAxx is 2 mM.
5. The PMAxx-LAMP-NALFA-based rapid detection kit for the viable bacteria of Salmonella according to claim 1, further comprising a running buffer solution of a nucleic acid detection test strip and a negative control sample, wherein the running buffer solution is a phosphate buffer solution containing 2% S9, and the negative control sample is DEPC water without nucleic acid.
6. The PMAxx-LAMP-NALFA-based rapid detection kit for viable salmonella of claim 1, wherein the nucleic acid detection test strip comprises a sample pad, a binding pad, an NC membrane, a water absorption pad and a bottom plate, the NC membrane is respectively provided with a detection line T line coated with streptavidin and a quality control line C line coated with goat anti-mouse IgG antibody or rabbit anti-mouse IgG antibody, the NC membrane is arranged on the bottom plate, the sample pad and the binding pad are sequentially connected with one end of the NC membrane close to the detection line, and the water absorption pad is connected with one end of the NC membrane close to the quality control line.
7. The PMAxx-LAMP-NALFA-based rapid detection kit for viable Salmonella bacteria according to claim 6, wherein the preparation steps of the binding pad of the nucleic acid detection test strip are as follows: preparing a FITC antibody marked by color microspheres, diluting the FITC antibody marked by the color microspheres in a treatment solution of a bonding pad, dropwise adding the solution onto the bonding pad, and drying for 3-4 hours at 45 ℃.
8. The PMAxx-LAMP-NALFA-based rapid detection kit for the viable bacteria of Salmonella according to claim 6, wherein the detection line and the quality control line of the nucleic acid detection test strip are prepared by the following steps: and respectively scribing streptavidin and goat anti-mouse IgG antibodies diluted by phosphate buffer solution on the NC membrane, wherein the coating concentration is 0.5-1.5 mg/mL, and placing the membrane in an oven at 37 ℃ for overnight.
9. The PMAxx-LAMP-NALFA-based rapid detection kit for the viable bacteria of Salmonella according to claim 1, wherein the nucleic acid detection test strip has a detection cavity, and is provided with a sample adding slot and an observation window which are communicated with the detection cavity, and the nucleic acid detection test strip is installed in the detection cavity.
10. The detection method of the PMAxx-LAMP-NALFA-based rapid detection kit for viable salmonella of claim 1, characterized by comprising the following steps:
s1, mixing a sample to be detected with the PMAxx uniformly to enable the final concentration of the PMAxx in the system to be 2-20 mu M, dyeing for 5-20 min in a dark place, carrying out photoactivation treatment for 5-15 min in a nucleic acid marker, centrifuging at 10000rpm for 2min, then carrying out resuspension by using a phosphate buffer solution, then centrifuging, carrying out resuspension by using TE water, heating at 100 ℃ for 10min, carrying out ice bath for 5min, and centrifuging at 12000rpm for 2 min;
s2, sucking 2 mu L of supernatant obtained after centrifugation in the step S1 by using a pipette tip, adding the supernatant into the LAMP amplification reaction solution, amplifying at 60-65 ℃ for 30-60 min, and heating at 85 ℃ for 5 min;
s3, adding 2-10 mu L of amplified sample into the nucleic acid detection test strip, adding 70-98 mu L of running buffer solution, reacting for 5-15 min, and judging the detection result according to the color development zone of the nucleic acid detection test strip.
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| CN202210438502.7A CN114891903A (en) | 2022-04-25 | 2022-04-25 | Kit and method for rapidly detecting salmonella live bacteria based on PMAxx-LAMP-NALFA |
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| CN102329790A (en) * | 2011-09-28 | 2012-01-25 | 河南省农业科学院 | Constant-temperature amplification method of double-labeled nucleic acid and detection test strip |
| CN107340389A (en) * | 2017-06-22 | 2017-11-10 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection salmonella |
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| CN112760212A (en) * | 2021-01-21 | 2021-05-07 | 上海海洋大学 | Fast live bacteria DNA screening instrument, and use method and application thereof |
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| CN102329790A (en) * | 2011-09-28 | 2012-01-25 | 河南省农业科学院 | Constant-temperature amplification method of double-labeled nucleic acid and detection test strip |
| CN107340389A (en) * | 2017-06-22 | 2017-11-10 | 中国农业大学 | Method based on nucleic acid chromatography biosensor technique detection salmonella |
| US20190127778A1 (en) * | 2017-10-26 | 2019-05-02 | Arizona Board Of Regents On Behalf Of Arizona State University | Methods for Detection and Quantification of Infectious Carbapenem Resistant Enterobacteriaceae (CRE) |
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