CN111378717A - Nucleic acid immune gold-labeled test strip, kit and method for detecting campylobacter jejuni - Google Patents
Nucleic acid immune gold-labeled test strip, kit and method for detecting campylobacter jejuni Download PDFInfo
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- CN111378717A CN111378717A CN201911414215.7A CN201911414215A CN111378717A CN 111378717 A CN111378717 A CN 111378717A CN 201911414215 A CN201911414215 A CN 201911414215A CN 111378717 A CN111378717 A CN 111378717A
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C12Q1/6804—Nucleic acid analysis using immunogens
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/56922—Campylobacter
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Abstract
The invention discloses a nucleic acid immune gold-labeled test strip, a kit and a method for detecting campylobacter jejuni. The kit comprises a PCR reaction system, wherein the PCR reaction system comprises a primer pair, the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2. The kit for detecting Campylobacter jejuni by using the test strip has strong specificity and high sensitivity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit and a method for detecting Campylobacter jejuni (Campylobacter jejuni) by using a test strip.
Background
The incidence of food-borne diseases is the front of the overall incidence of various diseases, and becomes one of the most prominent public health problems. Current systematic microbiological testing, pathogen isolation, treatment and control have not been able to meet the needs of current food monitoring efforts. Therefore, establishing a new pathogenic microorganism detection technology which is simple, easy to use and high in sensitivity has important significance for effectively solving the problems and the technical difficulties.
Campylobacter causes one of the major causes of food-borne diarrhea diseases in humans, and is also the most common bacterium causing gastroenteritis worldwide. In some developed and developing countries, the bacterium causes more cases of diarrhea than Salmonella foodborne. It is mainly found in food animals such as poultry, cattle, pigs, sheep, ostriches and shellfish, and infection of human beings with Campylobacter is mainly caused by contaminated food, water and environment. Campylobacter causing human diseases are mainly campylobacter jejuni (c.jejuni), campylobacter coli (c.coli), campylobacter fetus (c.fetus), campylobacter gull (c.upsamplilensis), and campylobacter rubescens (c.lari). Campylobacter jejuni is most common and is often associated with complications such as human Green-Barry.
Disclosure of Invention
The invention aims to solve the technical problems that in the prior art, a method or a kit for detecting campylobacter jejuni is high in cost, complex to operate, low in sensitivity and the like, and provides a nucleic acid immune gold-labeled test strip, a kit and a method for detecting campylobacter jejuni.
The inventor creatively combines the PCR with the immune gold-labeled test strip and designs the primer particularly suitable for the test strip method, thereby greatly reducing the cost, simplifying the operation steps and improving the sensitivity of detecting the campylobacter jejuni.
The invention mainly solves the technical problems through the following technical scheme.
The invention provides a kit for detecting Campylobacter jejuni (Campylobacter jejuni) by a test strip, which comprises a PCR reaction system, wherein the PCR reaction system comprises a primer pair, the nucleotide sequence of an upstream primer in the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer in the primer pair is shown as SEQ ID NO. 2.
Preferably, the forward primer is digoxigenin-labeled and the reverse primer is isothiocyanate-labeled.
Preferably, the amplification reaction system of the kit comprises: 0.25 μ L of the upstream primer at a concentration of 10 μ M, 0.25 μ L of the downstream primer at a concentration of 10 μ M, 12.5 μ L of Premix Ex Taq, and 7 μ L of double distilled water.
In the invention, the kit preferably further comprises a test strip, wherein the test strip comprises a water absorption filter paper pad, a conjugate pad, a cross-linked body attached with FITC antibody and colloidal gold particles, a lateral chromatography matrix, a detection band, a quality control band, a substrate and a water absorption pad; the detection band is attached with digoxin antibody; the quality control band is attached with a goat anti-mouse secondary antibody, and the secondary antibody comprises an anti-FITC antibody and an anti-digoxin antibody.
In the present invention, the concentration of the digoxin antibody is preferably 1 mg/mL.
In the present invention, the concentration of the FITC antibody is preferably 1 mg/mL.
In the present invention, the concentration of the anti-digoxin antibody is preferably 1 mg/mL.
In the present invention, the lateral chromatography matrix is preferably a nitrocellulose membrane.
In the present invention, the kit preferably further comprises a positive control and a negative control; wherein, the positive control is preferably campylobacter jejuni DNA; the negative control is preferably double distilled water.
The invention also provides a nucleic acid immune gold-labeled test strip for detecting campylobacter jejuni, which comprises a water absorption filter paper pad, a conjugate pad, an crosslinked body attached with FITC antibody and colloidal gold particles, a lateral chromatography matrix, a detection band, a quality control band, a substrate and a water absorption pad, and is characterized in that the detection band is attached with digoxin antibody; the quality control band is attached with a goat anti-mouse secondary antibody, and the secondary antibody comprises an anti-FITC antibody and an anti-digoxin antibody; preferably:
the concentration of the digoxin antibody is 1 mg/mL;
and/or the concentration of the FITC antibody is 1 mg/mL;
and/or, the concentration of the anti-digoxin antibody is 1 mg/mL.
The structure of the test strip is shown in figure 13. Wherein the meanings of the figures are as follows:
1: a water absorbing filter paper pad; 2: conjugate pad, conjugate 3 attached with FITC antibody and colloidal gold particles: lateral chromatography matrices (nitrocellulose membranes); 4: a detection band to which a digoxin antibody is attached; 5: a quality control band attached with a goat anti-mouse secondary antibody; 6: a substrate; 7: an absorbent pad.
The test paper strip detection principle is as follows:
and (3) breaking sample cells by using a lysis solution, and extracting the genomic DNA by a boiling method or a magnetic bead method. The extracted DNA is taken as a template, upstream primers and downstream primers are adopted to carry out PCR amplification on a specific lpxA gene fragment by using specific detection primers of campylobacter jejuni marked by digoxin (Digoxigenin) and isothiocyanate (FITC), and the two ends of a positive amplification product are respectively provided with digoxin and FITC. The gold-labeled FITC monoclonal antibody is contained on the water absorption pad on the test strip for detection, can be combined with FITC labeled molecules on one chain of a PCR product, and the anti-digoxin antibody is fixed on the position of the T detection line and can be combined with digoxin labeled molecules on the other chain of the PCR product. Under the action of a developing solution, a PCR amplification product is firstly combined with a gold-labeled FITC antibody, colloidal gold is driven to move towards the front end through capillary action, when the PCR amplification product passes through the position of a detection line fixed with a digoxin antibody, the digoxin antibody is captured at the position due to the digoxin label, the red brown color is displayed, and an excessive gold conjugate continuously moves upwards to the position of a C quality control line fixed with a goat anti-mouse secondary antibody to be combined with the secondary antibody, so that two color bands are displayed. If the template is not amplified, no PCR product binds to digoxin antibody and FITC antibody, and thus color cannot be displayed at the detection line (T line). The unreacted colloidal gold conjugate diffused upward until the control line (line C) was captured by the goat anti-mouse secondary antibody and developed.
The invention also provides a primer pair for detecting campylobacter jejuni by the test strip, wherein the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2.
The invention also provides a method for detecting campylobacter jejuni for non-diagnostic purposes, which comprises the following steps:
(1) extracting total DNA in a sample to be detected by using a DNA extraction reagent;
(2) taking the total DNA extracted in the step (1) as a template, and carrying out reaction by using the reaction system in the kit to obtain a PCR product;
(3) spotting the PCR product obtained in the step (2) on a test strip in the kit for detection; and analyzing the detection result.
Preferably, the reaction conditions in step (2) are: a.95 ℃ pre-denaturation for 5 min; b.95 ℃ denaturation for 30sec, 60 ℃ annealing for 30sec, 72 ℃ extension for 1 min; c. c, performing 35 cycles on the b and b, and then performing heating at 72 ℃ for 7 min; d.4 ℃ storage.
On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.
The reagents and starting materials used in the present invention are commercially available.
The positive progress effects of the invention are as follows:
the method combines PCR with a general nucleic acid detection immuno-gold-labeled chromatography test strip, and aims at specific fragments of campylobacter jejuni by comparing sequence differences of lpxA genes (encoding UDP-N-acetylglucosamine acyltransferase) of campylobacter jejuni and other campylobacter coli, campylobacter fetus, campylobacter gull, campylobacter erythrocaudatus and the like with close relativityDesigning an upstream primer and a downstream primer, marking a nucleic acid amplification primer, optimizing a reaction system, taking a universal nucleic acid detection immune gold-labeled chromatography test strip as a carrier, and judging a color development result by specific combination of a PCR amplification product and the immune gold-labeled chromatography test strip to realize sensitive detection of campylobacter jejuni, wherein the lowest detection limit can reach 3.1 × 103CFU/mL; the kit and the detection method are simple, convenient, quick, nontoxic and low in cost.
Drawings
FIG. 1 shows the specific PCR-electrophoresis detection result of Campylobacter jejuni; m: DL2000 marker; 1: campylobacter jejuni NCTC 11322; 2: campylobacter jejuni NCTC 11168; 3: campylobacter jejuni NCTC 13367; 4: campylobacter jejuni ATCC 49943; 5: campylobacter jejuni ATCC 49349; 6: campylobacter jejuni ATCCBAA 1153; 7: campylobacter jejuni ATCC 49350; 8: campylobacter jejuni NCTC 11351; 9: campylobacter larii ATCC 35221; 10, campylobacter coli ATCC 33559; 11: campylobacter foetidus DSM 5361; 12: campylobacter uppsalana DSM 5365; 13: campyylobacter hygointestinalis ATCC 35217; 14: helicobacter pylori ATCC 43504; 15: campylobacter coli ATCC 43478; 16: sterile water.
FIG. 2 shows the specific detection results of Campylobacter jejuni PCR-test strip (numbers 1-16 are the same as in FIG. 1).
FIG. 3 shows the result of PCR-electrophoresis specific detection of Campylobacter jejuni; m is DL2000 marker; 1, campylobacter jejuni NCTC 11322; 2: campylobacter jejuni ATCC 49349; 3: shigella flexneri CMCC 51573; 4: salmonella typhimurium CMCC 50013; 5: escherichia coli ATCC 25922; 6: cronobacter 21944; 7: vibrio parahaemolyticus ATCC 17802; 8, pseudomonas aeruginosa ZCIC 0034; 9: staphylococcus aureus CMCC 26003; 10: bacillus cereus ATCC 33019; 11: klebsiella oxytoca ATCC 700324; 12: proteus mirabilis ZCIC 0058; 13: listeria monocytogenes ATCC BAA 751; 14: streptococcus pyogenes; 15: vibrio parahaemolyticus ATCC 17802.
FIG. 4 shows the result of PCR-test strip specific detection of Campylobacter jejuni (numbered 1-15 in the same way as in FIG. 3).
FIG. 5 shows the PCR-electrophoresis sensitivity detection result of Campylobacter jejuni; m: DL2000 marker; 1E8 is Campylobacter jejuni NCTC11322 with different concentrations, 1: 3.1 × 108cfu/mL；2：3.1×107cfu/mL；3：3.1×106cfu/mL；4：3.1×105cfu/mL；5：3.1×104cfu/mL；6：3.1×103cfu/mL；7：3.1×102cfu/mL；8：3.1×101cfu/mL。
FIG. 6 shows the results of the PCR-test strip sensitivity assay for Campylobacter jejuni (numbers 1-8 are the same as those in FIG. 5).
FIG. 7 shows PCR-electrophoresis detection results of Campylobacter jejuni in artificially contaminated chicken samples, wherein 1-7 are Campylobacter jejuni NCTC11322 with different concentrations, and the ratio of 1: 3.1 × 10 is 1-77cfu/mL；2：3.1×106cfu/mL；3：3.1×105cfu/mL；4：3.1×104cfu/mL；5：3.1×103cfu/mL；6：3.1×102cfu/mL；7：3.1×101cfu/mL。
FIG. 8 shows the PCR-test strip test results of Campylobacter jejuni in artificially contaminated chicken (numbers 1-7 are the same as those in FIG. 7).
FIG. 9 shows the results of PCR electrophoresis (a) and PCR paper (b) at the Cj323 primer concentration of 0.2. mu.M annealing temperature of 60 ℃; 1: campylobacter jejuni NCTC 11351; 2: campylobacter coli ATCC 43478; 3: campylobacter coli ATCC 33559; 4: campylobacter lari ATCC 33559; 5: sterile water.
FIG. 10 shows the results of PCR electrophoresis (a) and PCR paper (b) at 62 ℃ with the Cj323 primer concentration of 0.1. mu.M annealing temperature; 1: campylobacter jejuni NCTC 11351; 2: campylobacter jejuni ATCC 49349; 3: campylobacter coli ATCC 43478; 4: campylobacter larii ATCC 35221; 5: sterile water.
FIG. 11 shows the results of PCR electrophoresis (a) and PCR paper (b) at the condition of 0.1. mu.M primer concentration and 60 ℃ annealing temperature; 1: campylobacter jejuni NCTC 11351; 2: campylobacter jejuni ATCC 49349; 3: campylobacter larii ATCC 35221; 4: campylobacter coli ATCC 43478; 5: sterile water.
FIGS. 12A and 12B show the results of PCR-electrophoresis of 4 pairs of primers on Campylobacter jejuni (M: 50bp DNA ladder marker; 1: Campylobacter jejuni ATCC 49943; 2: Campylobacter jejuni ATCC 49349; 3: Campylobacter coli ATCC 43478; 4: sterile water; P1: primer Cj 161; P2: primer hipO 344; P3: primer mapA; P4: cjlpxA (the present invention)).
FIG. 13 shows a test strip structure.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.
Example 1 sample preparation
Sample preparation was carried out according to GB 4789.9-2014.
Example 2 Pre-enrichment and enrichment
Culturing at 36 + -1 deg.C for 4h under microaerophilic conditions, and shaking at 100r/min if allowed. If necessary, measuring the pH value of the enrichment fluid, adjusting the pH value to 7.4 +/-0.2, and continuously culturing for 24-48h at the temperature of 42 +/-1 ℃.
Example 3DNA template extraction
Sucking 1mL of enrichment medium by a liquid transfer machine, placing the enrichment medium in a 1.5mL centrifuge tube, centrifuging for 2min at 12,000g, discarding the supernatant as much as possible, adding 1mL of PBS buffer solution for washing once, adding 500 mu L of sterile water, fully mixing, boiling for 15 min at 100 ℃ (water bath and metal bath can be used), centrifuging for 3min at 12,000g, and taking the supernatant to a new centrifuge tube. The extracted nucleic acid can be stored at 4 deg.C for short term, and can be frozen at-20 deg.C for long term storage.
Example 4PCR primers and reaction conditions
The specific primers for detecting campylobacter jejuni are as follows:
TABLE 1 Campylobacter jejuni-specific primers
TABLE 2 PCR amplification System
Reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 deg.C for 30sec, annealing at 60 deg.C for 30sec, extension at 72 deg.C for 1min, and entering at 72 deg.C for 7min after 35 cycles; storing at 4 ℃.
And (4) observing results: and uniformly mixing 6 mu L of PCR product with 54 mu L of developing solution, spotting on a test strip sample pad for detection, and observing the result for about 3 minutes.
Example 5 Campylobacter jejuni PCR test strip method for specific detection
The PCR-test strip reaction system and the program are used for specificity detection, the genomic DNA of a campylobacter jejuni standard strain such as campylobacter jejuni NCTC11322 is taken as a positive control, the genomic DNA of other campylobacter and other food-borne pathogenic bacteria is taken as a negative control, sterile water is taken as a blank control, the test strip detection result shows that the specificity reaches 100 percent, and the electrophoresis and test strip results are consistent with the electrophoresis result and are shown in figures 1 to 4.
Example 6 Campylobacter jejuni PCR test strip method sensitivity detection
In order to verify the sensitivity of the method to the campylobacter jejuni, the campylobacter jejuni NCTC11322 is inoculated and streaked on colombia blood agar for 24-48h in a microaerobic environment at 42 ℃, and the colibinia blood agar is prepared into a solution with the concentration of 3.1 × 10 by adopting 0.85 percent NaCl8And (3) taking CFU/mL bacterial suspension as a mother solution, then carrying out gradient dilution on the mother solution by 10 times, taking 1mL of bacterial suspension with each concentration to extract DNA, carrying out PCR, and carrying out electrophoresis and test strip detection respectively. Meanwhile, each gradient diluent is inoculated on a Columbia blood agar plate, and is cultured for 24 hours in a microaerophilic environment at 42 ℃, and bacterial liquids with different gradients are counted.
The PCR-test paper strip test result shows that when the concentration of the sample bacterial liquid is 3.1 × 103In CFU/mL, the test strip quality control line and the detection line can both display red strips, and the test strip result is consistent with the electrophoresis result, which indicates that the minimum detection limit of the test strip can reach 3.1 × 103CFU/mL。
Example 7 detection of Campylobacter jejuni Artificial pollution sample by PCR test strip method
In order to verify the detection sensitivity of the method to actual samples of the contaminated campylobacter jejuni, frozen chicken meat was randomly sampled from farmer markets, 7 samples were taken in an aseptic operation, 1g of each sample was placed in an aseptic bag, and 9mL of bolton broth, numbered 1-7, was added to each sample.
The bacterial liquid of Campylobacter jejuni NCTC11322 is diluted to 7 gradients of ① 3.1.1 3.1 × 102CFU/mL，②3.1×103CFU/mL，③3.1×104CFU/mL，④3.1×105CFU/mL，⑤3.1×106CFU/mL，⑥3.1×107CFU/m，⑦3.1×108CFU/mL, corresponding to sample No. 1-7, adding 1mL of bacterial liquid into sterile bag, wherein gradient of No. 1-7 is ① 3.1.1 3.1 × 101CFU/mL，②3.1×102CFU/mL，③3.1×103CFU/mL，④3.1×104CFU/mL，⑤3.1×105CFU/mL，⑥3.1×106CFU/mL，⑦3.1×107CFU/mL。
The PCR-test paper strip test result shows that when the concentration of the bacterial liquid of the mixed sample is 3.1 × 103In CFU/mL, the test strip can display red strips on the quality control line and the detection line of campylobacter jejuni lpxA gene amplification, and the test strip result is consistent with the electrophoresis result, which shows that the minimum detection limit of the test strip mixed sample can reach 3.1 × 103The result of CFU/mL is consistent with the sensitivity detection result of the PCR test strip method of the campylobacter jejuni in example 6.
EXAMPLE 8 practical detection of Campylobacter jejuni in food
The method comprises the steps of carrying out pre-enrichment on food samples from import and export inspection samples, domestic farmer markets and supermarkets, extracting genome DNA, and detecting actual samples by using an established PCR-test strip method and a method adopted by the existing national standard GB4789.9-2014, wherein the detection result shows that the detection result of the PCR-test strip method is consistent with the detection result of the existing national standard.
TABLE 3 actual sample test results
The established method has strong specificity, high sensitivity, simple and convenient operation, rapidness, environmental protection and low limit of detection (LOD) of 3.1 × 103CFU/mL, test paper detection time is about 3 minutes, is applicable to the rapid detection of campylobacter jejuni in food.
The technology fully utilizes the high sensitivity of PCR detection, combines the dual specificity of the PCR primer and the gold-labeled antibody, greatly enhances the specificity, and simultaneously has the advantages of simple and convenient operation, quickness and low cost. Compared with the conventional detection, the technology avoids the unfavorable conditions that the electrophoresis detection of the PCR result is time-consuming, easy to pollute, complex in procedure, and the like, and operators need to train, enables the detection result to be visual, simple and easy to operate, shortens the detection time, and provides a new safe and convenient means for developing the molecular biology field detection for a first-line detection laboratory, a laboratory in the grassroots area and a remote underdeveloped area.
Comparative example
PCR primer screening and condition optimization
The primers designed by the method are compared with other 4 sets of common PCR primers in the literature (Table 4), and PCR reaction is carried out at the annealing temperatures of 56 ℃, 58 ℃, 60 ℃ and 62 ℃ respectively.
TABLE 4 Campylobacter jejuni PCR candidate primers
2. Primer Cj323
The Cj323 test was first performed with primer concentrations of 0.10. mu.M and 0.2. mu.M, respectively, and annealing temperatures of 60 ℃ and 62 ℃. The test results are shown in fig. 9 to 12.
As is clear from FIG. 9, the Cj323 primer was positive to both the PCR electrophoresis method and the paper sheet method for Campylobacter jejuni at the primer concentration of 0.2. mu.M and the annealing temperature of 60 ℃ and was weakly positive to the paper sheet detection line due to the dimer although the band of interest for electrophoresis was not present in other non-Campylobacter jejuni. Therefore, it is considered to reduce the primer concentration to 0.1. mu.M and increase the annealing temperature to 62 ℃ and the results are shown in FIG. 10.
As can be seen from FIG. 10, when the primer concentration is reduced to 0.1. mu.M and the annealing temperature is increased to 62 ℃, primer dimers are reduced on electrophoresis and a paper sheet, but the amplification bands of Campylobacter jejuni are greatly weakened, so that the PCR paper sheet detection line cannot effectively distinguish Campylobacter jejuni from non-Campylobacter jejuni. Thus, the annealing temperature was lowered to 60 ℃ and the primer concentration was kept constant, as a result, see FIG. 11.
As is clear from FIGS. 11 and 9, under the condition of an annealing temperature of 60 ℃ C, the Cj323 primer was positive in both the amplification electrophoresis method and the sheet method of Campylobacter jejuni regardless of whether the primer concentration was 0.1. mu.M or 0.2. mu.M, and the pair of primers was weakly positive in the sheet detection line for Campylobacter jejuni because the pair of primers easily forms a dimer although the band of interest for electrophoresis was not present in other Campylobacter jejuni. Therefore, this primer is not usable.
3. The designed primer of the invention is compared with other primers
Since both nonspecific amplification and primer dimer showed positive on the test strip, the primers designed in this study and the other 3 pairs of primers were subjected to PCR electrophoresis in advance to test the specificity of the primers and the dimer production (the dimer had a greater effect on the PCR paper test results). The results are shown in FIG. 12.
Therefore, primers P1(Cj161) are weak in amplification bands of campylobacter jejuni at 56-62 ℃ and poor in adaptability to annealing temperature; primer P2(hipO344) has nonspecific amplification to campylobacter coli ATCC43478 at an annealing temperature of 56-62 ℃; primer P3(mapA) has amplification on campylobacter coli ATCC43478 at an annealing temperature of 56-62 ℃, and campylobacter jejuni and campylobacter coli cannot be distinguished; the primer P4(CjlpxA) can specifically amplify campylobacter jejuni at the annealing temperature of 56-62 ℃, and dimers of campylobacter coli and sterile water are few, so that false positive cannot be shown on a test strip, and therefore repeated detection and verification finally select a primer P4(CjlpxA) gene with an amplification fragment of 335 bp.
SEQUENCE LISTING
<110> Shanghai customs animal and plant and food inspection and quarantine technology center
<120> nucleic acid immune gold-labeled test strip, kit and method for detecting campylobacter jejuni
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Claims (10)
1. A kit for detecting Campylobacter jejuni (Campylobacter jejuni) by a test strip is characterized by comprising a PCR reaction system, wherein the PCR reaction system comprises a primer pair, the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID No.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID No. 2.
2. The kit of claim 2, wherein the forward primer is digoxigenin-labeled and the reverse primer is isothiocyanate-labeled.
3. The kit of claim 1 or 2, wherein the amplification reaction system of the kit comprises: 0.25. mu.L of the upstream primer at a concentration of 10. mu.M, 0.25. mu.L of the downstream primer at a concentration of 10. mu.M, 12.5. mu.L of Premix ExTaq, and 7. mu.L of double distilled water.
4. The kit of any one of claims 1 to 3, further comprising a test strip, wherein the test strip comprises a water-absorbing filter pad, a conjugate pad, a cross-linked body attached with FITC antibody and colloidal gold particles, a lateral chromatography matrix, a detection zone, a quality control zone, a substrate and a water-absorbing pad; the detection band is attached with digoxin antibody; the quality control band is attached with a goat anti-mouse secondary antibody, and the secondary antibody comprises an anti-FITC antibody and an anti-digoxin antibody.
5. The kit of claim 4, wherein the concentration of digoxin antibody is 1 mg/mL;
the concentration of the FITC antibody is 1 mg/mL;
and/or, the concentration of the anti-digoxin antibody is 1 mg/mL.
6. The kit of claim 5, wherein the lateral chromatography matrix is a nitrocellulose membrane.
7. The kit of any one of claims 1 to 6, further comprising a positive control and a negative control;
the positive control is preferably campylobacter jejuni DNA;
the negative control is preferably double distilled water.
8. A nucleic acid immune gold-labeled test strip for detecting campylobacter jejuni comprises a water absorption filter paper pad, a conjugate pad, a cross-linked body attached with FITC antibody and colloidal gold particles, a lateral chromatography matrix, a detection band, a quality control band, a substrate and a water absorption pad, and is characterized in that the detection band is attached with digoxin antibody; the quality control band is attached with a goat anti-mouse secondary antibody, and the secondary antibody comprises an anti-FITC antibody and an anti-digoxin antibody; preferably:
the concentration of the digoxin antibody is 1 mg/mL;
and/or the concentration of the FITC antibody is 1 mg/mL;
and/or, the concentration of the anti-digoxin antibody is 1 mg/mL.
9. A primer pair for detecting campylobacter jejuni by a test strip is characterized in that the nucleotide sequence of an upstream primer of the primer pair is shown as SEQ ID NO.1, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 2.
10. A method for the non-diagnostic detection of campylobacter jejuni, comprising the steps of:
(1) extracting total DNA in a sample to be detected by using a DNA extraction reagent;
(2) taking the total DNA extracted in the step (1) as a template, and carrying out reaction by using a reaction system in the kit according to any one of claims 1-7 to obtain a PCR product;
(3) spotting the PCR product obtained in the step (2) on a test strip in the kit of any one of claims 5 to 7 for detection; and analyzing the detection result;
preferably, the reaction conditions in step (2) are: a.95 ℃ pre-denaturation for 5 min; b.95 ℃ denaturation for 30sec, 60 ℃ annealing for 30sec, 72 ℃ extension for 1 min; c. c, performing 35 cycles on the b and b, and then performing heating at 72 ℃ for 7 min; d.4 ℃ storage.
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| CN116064726A (en) * | 2022-07-19 | 2023-05-05 | 江西师范大学 | Identification method of enterotoxin-producing escherichia coli |
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