Double-target primer probe composition, application and kit

Abstract

The invention discloses a double-target primer probe composition, application and a kit. The double-target primer probe composition comprises amplification primers and fluorescent probes of a salmonella invasion related gene InvA and an avian pathogenic escherichia coli aerobacin receptor gene IutA. The composition can be used for detecting pathogenic bacteria in poultry farms, and is high in sensitivity, good in specificity, reliable in result and free of cross interference among primers; the kit is suitable for a recombinase polymerase amplification technology, can be used for clinical diagnosis and epidemic condition monitoring of avian pathogenic escherichia coli and salmonella, and has excellent industrial application value.

Description

Technical Field

[0001] The present invention relates to isothermal amplification technology, and particularly to a dual-target primer-probe composition, applications, and kits. Background Art

[0002] In the poultry farming industry, Avian Pathogenic Escherichia coli (APEC) and Salmonella sp. are two core pathogenic bacteria that seriously threaten the health of poultry. Avian Pathogenic Escherichia coli causes diseases such as septicemia, airsacculitis, and pericarditis, leading to increased poultry mortality and decreased growth performance, resulting in huge economic losses every year. Salmonella not only induces diseases such as avian enteritis and hepatitis, reducing egg production rate and feed conversion rate, but can also enter the human food chain through contaminated poultry meat or egg products, causing foodborne diseases such as fever, diarrhea, and even septicemia, becoming a major hidden danger to public health safety.

[0003] Traditional pathogenic bacteria detection methods mainly include bacterial isolation and culture, biochemical identification, serological detection, and conventional PCR technology. These methods generally have problems such as long detection cycles, complex operations, high costs, or low throughput, and are difficult to meet the needs of real-time monitoring and early warning in large-scale farming.

[0004] In recent years, isothermal amplification technology has become a research hotspot due to its lack of thermal cycling, rapid reaction, and high sensitivity. This technology achieves exponential amplification of nucleic acids at a constant temperature through specific primer design, and is particularly suitable for on-site point-of-care testing. However, the primer design for dual-target co-detection faces multiple technical challenges: First, the risk of cross-reaction between primers increases sharply. The primers corresponding to the two sets of targets not only need to avoid the formation of primer dimers within the same target, but also need to avoid non-specific binding between primers of different targets; Second, it is difficult to synchronously optimize the amplification efficiency of the two targets. Differences in GC content and secondary structure in different target regions may lead to unbalanced amplification kinetics, and balanced amplification needs to be achieved through fine adjustment of primer length, T m value, and concentration ratio; In addition, the problem of cross-interference of amplification products is particularly prominent. The amplified strands may compete for enzyme binding sites or form heteroduplexes due to structural similarity, affecting the specificity of the detection signal. Currently, there is no dual-target primer-probe composition for Avian Pathogenic Escherichia coli and Salmonella. Summary of the Invention

[0005] Objectives of the Invention: The objective of the present invention is to provide a dual-target primer-probe composition that can be used to detect pathogenic bacteria in poultry farms; the second objective is to provide applications of the dual-target primer-probe composition; the third objective is to provide a kit containing the dual-target primer-probe composition.

[0006] Technical solution: The dual-target primer-probe composition of the present invention includes:

[0007] The InvA upstream primer has the nucleotide sequence shown in SEQ ID NO: 1;

[0008] The InvA downstream primer has the nucleotide sequence shown in SEQ ID NO: 2;

[0009] The InvA fluorescent probe has the nucleotide sequence shown in SEQ ID NO: 3 and is modified with a fluorescent group, a quenching group, and a 3'-terminal spacer arm;

[0010] The IutA upstream primer has the nucleotide sequence shown in SEQ ID NO: 4;

[0011] The IutA downstream primer has the nucleotide sequence shown in SEQ ID NO: 5;

[0012] The IutA fluorescent probe has the nucleotide sequence shown in SEQ ID NO: 6 and is modified with a fluorescent group, a quenching group, and a 3'-terminal spacer arm.

[0013] Preferably, the molar ratios of the InvA upstream primer, InvA downstream primer, InvA fluorescent probe and the IutA upstream primer, IutA downstream primer, IutA fluorescent probe are all 1:1:0.2 - 0.5.

[0014] Preferably, the fluorescent groups in the InvA fluorescent probe and the IutA fluorescent probe are selected from any two of HEX, FAM, Cy5, Cy3, ROX, and TAMRA.

[0015] Preferably, the quenching groups in the InvA fluorescent probe and the IutA fluorescent probe are selected from any one or more of BHQ1, BHQ2, and Dabcyl.

[0016] Preferably, the 3'-terminal spacer arms in the InvA fluorescent probe and the IutA fluorescent probe are selected from any one or more of C3 spacer arm and C6 spacer arm.

[0017] Application of the dual-target primer-probe composition of the present invention in detecting pathogenic bacteria in poultry farms.

[0018] Preferably, the pathogenic bacteria include Salmonella and avian pathogenic Escherichia coli.

[0019] Preferably, the steps of the application include:

[0020] (1) Collect and extract DNA from poultry samples;

[0021] (2) Add the dual-target primer-probe composition and isothermal amplification enzyme composition to the sample DNA obtained in step 1, and add dNTPs and magnesium acetate solution.

[0022] (3) After pre-reaction, analyze using a real-time fluorescence quantitative PCR analyzer.

[0023] Preferably, the reaction temperature in step 3 is not higher than 40 °C.

[0024] The dual-target detection kit of the present invention includes:

[0025] (a) The aforementioned dual-target primer-probe composition;

[0026] (b) Isothermal amplification enzyme composition, containing recombinant enzyme T4 UvsX, auxiliary protein UvsY, DNA polymerase, single-stranded DNA binding protein and exonuclease III;

[0027] (c) Reaction buffer, containing 480 - 520 mM NaCl, 95 - 105 mM Tris-HCl, 95 - 105 mM MgCl2, 9 - 11 mM dithiothreitol;

[0028] (d) dNTPs solution;

[0029] (e) Magnesium acetate solution, with a concentration of 220 - 240 mM.

[0030] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. The dual-target primer-probe composition has high sensitivity, good specificity and reliable results for detecting pathogenic bacteria in poultry farms, and there is no cross-interference between primers; 2. The dual-target primer-probe composition is applicable to the recombinase polymerase amplification technology (RPA), and can be used for the clinical diagnosis and epidemic situation monitoring of avian pathogenic Escherichia coli and Salmonella, and has excellent industrial application value. Description of the Drawings

[0031] Figure 1 It is the sensitivity evaluation result of the Salmonella invasion-related gene InvA primer-probe composition for detecting Salmonella;

[0032] Figure 2 It is the sensitivity evaluation result of the aerobactin receptor gene IutA primer-probe composition of avian pathogenic Escherichia coli for detecting avian pathogenic Escherichia coli;

[0033] Figure 3 It is the specificity evaluation result of the Salmonella invasion-related gene InvA primer-probe composition for detecting Salmonella;

[0034] Figure 4Specificity evaluation results of primers and fluorescent probes of aerobactin receptor gene IutA of avian pathogenic Escherichia coli for detecting avian pathogenic Escherichia coli

[0035] Figure 5 Results of the dual-target primer-probe composition for detecting avian pathogenic Escherichia coli and Salmonella. Among them, FAM, APEC and Salmonela sp. are the amplification results of DNA samples in the FAM channel, FAM, negative control is the amplification result of sterile water in the FAM channel, Hex, APEC and Salmonella sp. are the amplification results of DNA samples in the HEX channel, and Hex, negative conirol is the amplification result of sterile water in the HEX channel. Detailed implementation mode

[0036] The technical solution of the present invention will be further described below.

[0037] Example 1: Preparation of amplification primers and fluorescent probes

[0038] (1) Entrust GenScript Biotech Corporation to synthesize the upstream primer S1 of the Salmonella invasion-related gene InvA, the downstream primer S2 of InvA, the fluorescent probe S3 of InvA, the upstream primer E1 of the aerobactin receptor gene IutA of avian pathogenic Escherichia coli, the downstream primer E2 of IutA, and the fluorescent probe E3 of IutA. The specific sequences are shown in Table 1:

[0039] Table 1 Nucleotide sequence table of synthesized amplification primers and fluorescent probes

[0040]

[0041]

[0042] Among them, as shown in Table 1, the 31st position of S3 is deoxythymidine modified with a HEX fluorescent group, the 32nd position is a vacancy, the 33rd position is deoxythymidine modified with a BHQ1 quenching group, and the 3' end is modified with a C3 spacer (C3 Spacer); the 30th position of E3 is deoxythymidine modified with a 6-FAM fluorescent group, the 31st position is a vacancy, the 32nd position is deoxythymidine modified with a BHQ1 quenching group, and the 3' end is modified with a C3 spacer (C3 Spacer).

[0043] (2) Mix S1, S2, and S3 to obtain a primer-probe composition for the Salmonella invasion-related gene InvA, where the final concentrations of S1 and S2 are both 10 μΜ, and the final concentration of S3 is 3 μΜ;

[0044] Mix E1, E2, and E3 to obtain the primer-probe composition of the aerobactin receptor gene IutA of avian pathogenic Escherichia coli, where the final concentrations of E1 and E2 are both 10 μΜ, and the final concentration of E3 is 3 μΜ;

[0045] Mix S1, S2, S3, E1, E2, and E3 to obtain the dual-target primer-probe composition, where the final concentrations of S1, S2, E1, and E2 are all 10 μΜ, and the final concentrations of S3 and E3 are both 3 μΜ.

[0046] Example 2: Sensitivity evaluation of Salmonella detection using the amplification primer and fluorescent probe of the Salmonella invasion-related gene InvA

[0047] (1) Prepare plasmid DNA samples of the Salmonella invasion-related gene InvA, and dilute the concentrations to 8×105 Copies / μL, 8×104 Copies / μL, 8×10 3 Copies / μL, 8×10 2 Copies / μL, 8×101 Copies / μL, 8×10 0 Copies / μL respectively, and use sterile water as the negative control.

[0048] (2) Construct a 50 μL reaction system. Select the enzyme composition lyophilized powder in the DNA constant temperature nucleic acid amplification kit of Wuxi Leshang Biotechnology Co., Ltd., add 2 μL of the plasmid DNA sample or sterile water obtained in step 1, 2 μL of the Salmonella invasion-related gene InvA amplification primer-probe composition obtained in Example 1, 2 μL of dNTPs, 5 μL of reaction buffer, and make up the volume to 47 μL with sterile water. Finally, add 3 μL of magnesium acetate solution to activate the reaction system and mix well. Set 3 replicates for each sample.

[0049] Among them, the reaction buffer contains 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol. The final concentrations of S1 and S2 primers are both 400 nM, the final concentration of S3 probe is 120 nM, the final concentration of dNTPs is 100 μΜ, and the final concentration of magnesium acetate solution is 14 mM;

[0050] (3) After pre-reacting at 39 °C for 4 min, use the Bori FQD-16A real-time fluorescence quantitative PCR analyzer for analysis. React at 39 °C for 20 min and read the fluorescence value every minute.

[0051] The sensitivity evaluation results of Salmonella detection are as Figure 1 shown, at 8×10 5 -8×10 1Within the range of copies / μL concentration, as the concentration of the DNA sample decreased, the fluorescence signal intensity gradually weakened. However, obvious fluorescence signals and amplification curves could be collected after the reaction ended, which was determined as a positive result. That is, the detection limit of the amplification primer and fluorescence probe for the Salmonella invasion-related gene InvA was 8×101 / reaction, with high sensitivity.

[0052] Example 3: Sensitivity evaluation of amplification primer and fluorescence probe for the aerobactin receptor gene IutA of avian pathogenic Escherichia coli in detecting avian pathogenic Escherichia coli

[0053] (1) Prepare plasmid DNA samples of avian pathogenic Escherichia coli IutA, with concentrations diluted to 5×10 5 Copies / μL, 5×10 4 Copies / μL, 5×10 3 Copies / μL, 5×10 2 Copies / μL, 5×10 1 Copies / μL, 5×10 0 Copies / μL, and use sterile water as a negative control.

[0054] (2) Construct a 50 μL reaction system. Select the enzyme composition lyophilized powder in the DNA constant temperature nucleic acid amplification kit of Wuxi Leshang Biotechnology Co., Ltd., add 2 μL of the plasmid DNA sample or sterile water obtained in step 1, 2 μL of the amplification primer-probe composition of the aerobactin receptor gene IutA of avian pathogenic Escherichia coli obtained in Example 1, 2 μL of dNTPs, 5 μL of reaction buffer, and make up the volume to 47 μL with sterile water. Finally, add 3 μL of magnesium acetate solution to activate the reaction system, and mix well. Set 3 replicates for each sample.

[0055] Among them, the reaction buffer contains 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol. The final concentrations of primers E1 and E2 are both 400 nM, the final concentration of probe E3 is 120 nM, the final concentration of dNTPs is 100 μM, and the final concentration of magnesium acetate solution is 14 mM.

[0056] ((3) After pre-reacting at 39 °C for 4 min, use the Bori FQD-16A real-time fluorescence quantitative PCR analyzer for analysis. React at 39 °C for 20 min and read the fluorescence value every minute.

[0057] The results of the sensitivity evaluation for detecting avian pathogenic Escherichia coli are as Figure 2 shown. At 5×10 5 -5×10 1In the range of Copies / μL concentration, as the concentration of the DNA sample decreases, the fluorescence signal intensity gradually weakens. However, obvious fluorescence signals and amplification curves can be collected after the reaction ends, which are determined as positive results. That is, the detection limit of the amplification primer and fluorescence probe for avian pathogenic Escherichia coli IutA is 5×101 / reaction, with high sensitivity.

[0058] Example 4: Specificity evaluation of Salmonella invasion-related gene InvA amplification primer and fluorescence probe for detecting Salmonella

[0059] (1) Prepare the strain DNA samples as shown in Table 2, with a concentration of 50 ng / μL. Among them, the DNA sample of Salmonella gallinarum ATCC70062 is used as a positive control, and sterile water is used as a negative control.

[0060] Table 2 Strains used in the specificity evaluation experiment for Salmonella detection

[0061] Strain Name Latin Name Strain Number Bacillus cereus Bacillus Cereus CMCC(B)63303 Vibrio parahaemolyticus Vibrio Prholyticus CGMCC 1.1615 Candida albicans Candida Albicans ATCC 1.2258 Klebsiella pneumoniae Corynebacterium Glutamicum CMCC(B)46117 Micrococcus lysodeikticus Micrococcus Lysodeikticus CGMCC 1.634 Bacillus subtilis Bacillus Subtilis CGMCC 1.1630 Escherichia coli Escherichia Coli ATCC 35218 Salmonella gallinarum Salmonella Sp. ATCC 70062

[0062] (2) Construct a 50 μL reaction system. Select the enzyme composition lyophilized powder in the DNA constant temperature nucleic acid amplification kit of Wuxi Leshang Biotechnology Co., Ltd. Add 2 μL of the strain DNA sample or sterile water in step 1, 2 μL of the Salmonella invasion-related gene InvA amplification primer-probe composition obtained in Example 1, 2 μL of dNTPs, 5 μL of reaction buffer, and make up the volume to 47 μL with sterile water. Finally, add 3 μL of magnesium acetate solution to activate the reaction system and mix well. Set 3 replicates for each sample.

[0063] Among them, the reaction buffer contains 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol. The final concentrations of primers S1 and S2 are both 400 nM, the final concentration of probe S3 is 120 nM, the final concentration of dNTPs is 100 μM, and the final concentration of magnesium acetate solution is 14 mM.

[0064] (3) After pre-reacting at 39 °C for 4 min, use the Bori FQD-16A real-time fluorescence quantitative PCR analyzer for analysis. React at 39 °C for 20 min and read the fluorescence value every minute.

[0065] The results of the specificity evaluation for detecting Salmonella are as Figure 3 shown. The positive control sample tube of Salmonella gallinarum produces obvious fluorescence signals and amplification curves, which are determined as positive. No fluorescence signals and amplification curves are detected in all negative control sample tubes of non-Salmonella strains, which are determined as negative. That is, the amplification primer and fluorescence probe for Salmonella invasion-related gene InvA have good specificity and can accurately distinguish Salmonella from other non-target bacteria.

[0066] Example 5: Specificity Evaluation of Amplification Primers and Fluorescent Probes of Aerobactin Receptor Gene IutA of Avian Pathogenic Escherichia coli for Detecting Avian Pathogenic Escherichia coli

[0067] (1) Prepare the strain DNA samples with a concentration of 50 ng / μL as described in step 1 of Example 4, where the DNA sample of Escherichia coli ATCC 35218 is used as a positive control and sterile water is used as a negative control;

[0068] (2) Construct a 50-μL reaction system. Select the lyophilized enzyme composition in the DNA constant-temperature nucleic acid amplification kit of Wuxi Leshang Biotechnology Co., Ltd. Add 2 μL of the strain DNA sample or sterile water in step 1, 2 μL of the amplification primer-probe composition of the aerobactin receptor gene IutA of avian pathogenic Escherichia coli obtained in Example 1, 2 μL of dNTPs, 5 μL of reaction buffer, and make up the volume to 47 μL with sterile water. Finally, add 3 μL of magnesium acetate solution to activate the reaction system and mix well. Set 3 replicates for each sample.

[0069] Among them, the reaction buffer contains 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol. The final concentrations of primers E1 and E2 are both 400 nM, the final concentration of probe E3 is 120 nM, the final concentration of dNTPs is 100 μM, and the final concentration of magnesium acetate solution is 14 mM;

[0070] (3) After pre-reacting at 39 °C for 4 min, use the Bori FQD-16A real-time fluorescence quantitative PCR analyzer for analysis. React at 39 °C for 20 min and read the fluorescence value every minute.

[0071] The results of the specificity evaluation for detecting avian pathogenic Escherichia coli are as Figure 4 shown. The positive control sample tube of avian pathogenic Escherichia coli produced obvious fluorescence signals and amplification curves and was judged as positive. No fluorescence signals and amplification curves were detected in all negative control sample tubes of non-avian pathogenic Escherichia coli, which were judged as negative. That is, the amplification primers and fluorescent probes of the aerobactin receptor gene IutA of avian pathogenic Escherichia coli have good specificity and can accurately distinguish avian pathogenic Escherichia coli from other non-target bacteria.

[0072] Example 6: Dual-Target Primer-Probe Composition for Detecting Avian Pathogenic Escherichia coli and Salmonella

[0073] (1) Obtain dead chickens from a chicken farm from a veterinary pharmacy:

[0074] For one dead chicken showing typical symptoms of Escherichia coli triple inflammation and suspected of being infected with Escherichia coli, under aseptic conditions, approximately 0.5 g of liver tissue was transferred to a test tube containing 10 mL of nutrient broth, mashed with a sterile inoculation loop to ensure sufficient release of bacteria, and subjected to enrichment culture in a constant temperature incubator at 37 °C for 18 h. Subsequently, the broth after enrichment was taken with a sterile inoculation loop and streaked on MacConkey agar medium. After culturing at 37 °C for 18 h, single colonies were screened out and identified as avian pathogenic Escherichia coli by 16S rRNA sequencing.

[0075] For another dead chicken whose autopsy showed enlarged and bronze-colored liver and was suspected of being infected with Salmonella, liver tissue was also obtained under aseptic operation for enrichment culture in nutrient broth. Then, the cultured bacterial liquid was streaked on MacConkey agar medium, and single colonies were screened out after culturing and identified as avian Salmonella by 16S rRNA sequencing.

[0076] (2) DNA of the obtained avian pathogenic Escherichia coli and avian Salmonella was extracted. Equal amounts of the DNA of the two strains were mixed and diluted to a concentration of 50 ng / μL, and sterile water was used as a negative control.

[0077] (3) A 50 μL reaction system was constructed. The lyophilized enzyme composition in the DNA isothermal nucleic acid amplification kit of Wuxi Leshang Biotechnology Co., Ltd. was selected. 2 μL of the DNA sample or sterile water obtained in step (2), 2 μL of the dual-target primer-probe composition obtained in Example 1, 2 μL of dNTPs, and 5 μL of reaction buffer were added. The volume was made up to 47 μL with sterile water, and finally 3 μL of magnesium acetate solution was added to activate the reaction system and mixed well. Three replicates were set for each sample.

[0078] Among them, the reaction buffer contained 500 mM NaCl, 100 mM Tris-HCl, 100 mM MgCl2, 10 mM dithiothreitol. The final concentrations of primers S1, S2, E1, and E2 were all 400 nM, the final concentrations of probes S3 and E3 were all 120 nM, the final concentration of dNTPs was 100 μM, and the final concentration of magnesium acetate solution was 14 mM.

[0079] (4) After pre-reaction at 39 °C for 4 min, a Bio-Rad FQD-16A real-time fluorescence quantitative PCR analyzer was used for analysis. The reaction was carried out at 39 °C for 20 min, and the fluorescence value was read every minute.

[0080] The results of the dual-target primer-probe composition for the detection of avian pathogenic Escherichia coli and Salmonella are as Figure 5 shown, and it can accurately distinguish avian pathogenic Escherichia coli and Salmonella.

Claims

1. A dual-target primer-probe composition, characterized in that, Comprising: InvA upstream primer, having the nucleotide sequence shown in SEQ ID NO: 1; InvA downstream primer, having the nucleotide sequence shown in SEQ ID NO: 2; InvA fluorescent probe, having the nucleotide sequence shown in SEQ ID NO: 3 and modified with a fluorophore, a quencher, and a 3'-terminal spacer arm; IutA upstream primer, having the nucleotide sequence shown in SEQ ID NO: 4; IutA downstream primer, having the nucleotide sequence shown in SEQ ID NO: 5; IutA fluorescent probe, having the nucleotide sequence shown in SEQ ID NO: 6 and modified with a fluorophore, a quencher, and a 3'-terminal spacer arm.

2. The dual-target primer-probe combination according to claim 1, wherein The molar ratios of the InvA upstream primer, InvA downstream primer, InvA fluorescent probe, and the molar ratios of the IutA upstream primer, IutA downstream primer, and IutA fluorescent probe are all 1:1:0.2 - 0.

5.

3. The dual-target primer-probe combination according to claim 1, wherein The fluorophores in the InvA fluorescent probe and IutA fluorescent probe are selected from any two of HEX, FAM, Cy5, Cy3, ROX, and TAMRA.

4. The dual-target primer-probe combination according to claim 1, wherein The quenchers in the InvA fluorescent probe and IutA fluorescent probe are selected from any one or more of BHQ1, BHQ2, and Dabcyl.

5. The dual-target primer-probe combination according to claim 1, wherein The 3'-terminal spacer arms in the InvA fluorescent probe and IutA fluorescent probe are selected from any one or more of C3 spacer arm and C6 spacer arm.

6. Use of the dual-target primer-probe composition according to any one of claims 1 - 5 in detecting pathogenic bacteria in a poultry farm.

7. The application according to claim 6, wherein The pathogenic bacteria include Salmonella and avian pathogenic Escherichia coli.

8. The application according to claim 6, wherein The steps of the use include: (1) Collecting and extracting poultry sample DNA; (2) Adding the dual-target primer-probe composition and an isothermal amplification enzyme composition to the sample DNA obtained in step 1, and adding a dNTPs solution, a reaction buffer, and a magnesium acetate solution; (3) Analyzing using a real-time fluorescence quantitative PCR analyzer after pre-reaction.

9. The application according to claim 8, characterized in that, The reaction temperature in step 3 is not higher than 40°C.

10. A dual-target detection kit, characterized in that, Comprising: (a) The dual-target primer-probe composition according to any one of claims 1 - 5; (b) An isothermal amplification enzyme composition containing recombinant enzyme T4 UvsX, accessory protein UvsY, DNA polymerase, single-stranded DNA binding protein, and exonuclease III; (c) A reaction buffer containing NaCl, Tris-HCl, MgCl2, and dithiothreitol. (d) A dNTPs solution; (e) A magnesium acetate solution.

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