Quadruple PCR (Polymerase Chain Reaction) detection primer combination for detecting diarrhea pathogenic bacteria of young deer and application of quadruple PCR detection primer combination

Abstract

The invention discloses a quadruple PCR (Polymerase Chain Reaction) detection primer combination for detecting diarrhea pathogenic bacteria of young deer and application, and belongs to the technical field of biology. The quadruple PCR detection primer combination for detecting the diarrhea pathogenic bacteria of the young deer comprises an escherichia coli primer pair as shown in SEQ ID NO.1-2, a klebsiella pneumoniae primer pair as shown in SEQ ID NO.3-4, a salmonella primer pair as shown in SEQ ID NO.5-6 and a proteus mirabilis primer pair as shown in SEQ ID NO.7-8. The invention further discloses a kit for detecting the diarrhea pathogenic bacteria of the young deer. The multiple PCR detection method is established by using the specific genes of the main pathogenic bacteria of the diarrhea of the baby deer, so that the pathogenesis of the diarrhea of the baby deer can be quickly and accurately detected, the detection efficiency of epidemic diseases is greatly improved, and the method has important significance in preventing and treating bacterial diarrhea of the baby deer.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a quadruple PCR detection primer combination for detecting pathogenic bacteria of fawn diarrhea and an application thereof. Background Art

[0002] Diarrhea in fawns has a high incidence rate and has become one of the main reasons that hinder the growth and development of fawns, affect production performance, and cause economic losses in large-scale sika deer farms. Common molecular biological methods are increasingly used in animal disease detection, among which the most prominent is polymerase chain reaction (PCR). PCR technology amplifies DNA or RNA fragments of pathogens, making them easier to detect in samples. This technology has high sensitivity and specificity, making it one of the preferred methods for detecting many animal diseases. However, traditional PCR can only amplify one target. It takes more time to detect the pathogenic bacteria of fawn diarrhea, which is time-consuming and laborious. Therefore, it is of great significance to provide a multiple detection method for the identification of pathogenic bacteria of fawn diarrhea. Summary of the invention

[0003] The purpose of the present invention is to provide a quadruple PCR detection primer combination and application for detecting pathogenic bacteria of fawn diarrhea, so as to solve the problems existing in the above-mentioned prior art. The present invention establishes a multiplex PCR detection method based on the specific genes of the main pathogenic bacteria of fawn diarrhea, which is convenient for quickly and accurately detecting the cause of fawn diarrhea, greatly improving the detection efficiency of the disease, and is of great significance for the prevention and treatment of bacterial diarrhea in fawns.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The invention provides a quadruple PCR detection primer combination for detecting pathogenic bacteria of fawn diarrhea, wherein the primer combination comprises: an Escherichia coli primer pair as shown in SEQ ID NO.1-2, a Klebsiella pneumoniae primer pair as shown in SEQ ID NO.3-4, a Salmonella primer pair as shown in SEQ ID NO.5-6, and a Proteus mirabilis primer pair as shown in SEQ ID NO.7-8.

[0006] The present invention also provides an application of the above-mentioned quadruple PCR detection primer combination in preparing a reagent or a kit for detecting pathogenic bacteria of fawn diarrhea.

[0007] The present invention also provides a quadruple PCR detection kit for detecting pathogenic bacteria of fawn diarrhea, wherein the kit comprises the quadruple PCR detection primer combination.

[0008] The present invention also provides a method for detecting pathogenic bacteria of diarrhea in young deer for non-disease diagnosis purposes, comprising the following steps:

[0009] (1) Extracting genomic DNA from feces of diarrhea fawns;

[0010] (2) using the above-mentioned quadruple PCR detection primer combination or the above-mentioned kit, and using the genomic DNA obtained in step (1) as a template, to perform PCR amplification; and determining the type of pathogenic bacteria causing deer diarrhea based on the band size of the PCR amplification product.

[0011] Preferably, the reaction system for PCR amplification is: 2×ES Taq Mastermix 12.5 μL, 1 μL each of 10 μmol / L Escherichia coli upstream and downstream primers, 0.4 μL each of 10 μmol / L Klebsiella pneumoniae upstream and downstream primers, 1 μL each of 10 μmol / L Salmonella upstream and downstream primers, 0.5 μL each of 10 μmol / L Proteus mirabilis upstream and downstream primers, 1 μL template, and ddH2O supplemented to 25 μL.

[0012] Preferably, the reaction procedure of the PCR amplification is: 94°C for 10 min; then 94°C for 30 s, 52.4°C for 30 s, 72°C for 1 min, 30 cycles and finally extension at 72°C for 10 min.

[0013] Preferably, the judgment method is: if the PCR amplification product contains a band of 420bp, the pathogenic bacteria of deer diarrhea include Escherichia coli; if the PCR amplification product contains a band of 214bp, the pathogenic bacteria of deer diarrhea include Klebsiella pneumoniae; if the PCR amplification product contains a band of 564bp, the pathogenic bacteria of deer diarrhea include Salmonella; if the PCR amplification product contains a band of 975bp, the pathogenic bacteria of deer diarrhea include Proteus mirabilis.

[0014] The present invention discloses the following technical effects:

[0015] The present invention uses the specific genes of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella to distinguish pathogenic bacteria in feces, and uses multiple PCR technology to add more than one pair of primers in the same reaction system, so as to achieve the technical effect of amplifying more than one target DNA fragment in the same reaction system, and solves the technical problem that single PCR is time-consuming and labor-intensive and cannot eliminate the difference in sample addition between different sample wells. The present invention scientifically optimizes the concentration of each primer, controls the band size of the amplified products of multiple pathogenic bacteria, makes the reaction system stable, and is easy to operate. It can achieve the effects of high sensitivity, strong specificity, and easy results, and provides a new technical method for the identification of pathogenic bacteria in diarrhea fawns, and lays a theoretical foundation for the treatment of diarrhea fawns. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 It is the test result of annealing temperature gradient of single PCR method; A is uidA, B is khe, C is invA, and D is atpD; lanes 1-12 are 50℃, 50.4℃, 51.2℃, 52.4℃, 53.8℃, 55.3℃, 56.7℃, 58.2℃, 59.6℃, 60.8℃, 61.6℃, and 62.0℃, respectively; lane 13 is the negative control (detected with the best single annealing temperature, A is 52.2℃, B is 50.4℃, C is 53.8℃, and D is 50℃); M is the DNA standard DL2000;

[0018] Figure 2 The final concentration gradient test results of the primers in the single PCR method; A is uidA, B is khe, C is invA, and D is atpD; lanes 1-10 are 0.4μmol / L, 0.32μmol / L, 0.24μmol / L, 0.16μmol / L, 0.08μmol / L, 0.04μmol / L, 32nmol / L, 24nmol / L, 16nmol / L, and 8nmol / L, respectively; lane 11 is a negative control (tested at the best single concentration, A, B, C, and D are all 0.4μmol / L); M is the DNA standard DL2000;

[0019] Figure 3 The results of optimization of annealing temperature for quadruple PCR method, where lanes 1-12 are 50°C, 50.4°C, 51.2°C, 52.4°C, 53.8°C, 55.3°C, 56.7°C, 58.2°C, 59.6°C, 60.8°C, 61.6°C, and 62.0°C, respectively; lane 13 is a negative control (detected at the optimal multiplex annealing temperature of 52.4°C); M is DNA standard DL2000;

[0020] Figure 4 The optimization results of the annealing temperature of the quadruple PCR method, wherein lanes 1-9 are primer final concentration combinations 1-9 in Table 1, and M is the DNA standard DL2000;

[0021] Figure 5 The sensitivity test results of the quadruple PCR method are shown in Figure 2. Lanes 1 to 6 are 10 0 , 10 1 , 10 2 , 103 , 10 4 , 10 5 Mixed bacterial solution diluted in multiple ratios, M is the DNA standard DL2000;

[0022] Figure 6 The results of the quadruple PCR specificity test are shown in Figure 1, where lanes 1-8 are Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Salmonella, Bacillus subtilis, Lactobacillus johnsonii, Enterococcus faecium, and Staphylococcus aureus, respectively; lane 9 is the negative control; and M is the DNA standard DL2000.

[0023] Figure 7 These are the test results of 20 clinical samples using the quadruple PCR method, where lanes 1-20 represent 20 clinical samples. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] 1. Experimental Materials

[0031] 1.1 Strains and primers

[0032] Escherichia coli (CICC 10003), Proteus mirabilis (CICC 20049), Klebsiella pneumoniae (CICC20051), and Salmonella (CICC 21610) were provided by the Animal Infection and Prevention Technology Laboratory, Institute of Special Products, Chinese Academy of Agricultural Sciences.

[0033] According to the gene sequence information included in GenBank and reference to relevant literature, the primer sequences of Escherichia coli uidA, Klebsiella pneumoniae hemolytic enzyme gene khe, Salmonella invA, and Proteus mirabilis atpD were cited and synthesized by Sangon Biotechnology Co., Ltd. The sequences of the primers and the length of the amplified fragments are shown in Table 1.

[0034] Table 1 Primer sequences of four bacteria

[0035]

[0036] 1.2 Main Reagents

[0037] Tryptone soy broth (TSB) is a product of Qingdao Keyuan Biotechnology Co., Ltd.; DNA standard DL2000 is a product of Jinan Protein Technology Co., Ltd.; bacterial genomic DNA extraction kit is a product of Omega Company; fecal genomic DNA extraction kit is a product of Biomiga Company.

[0038] 1.3 Main instruments and equipment

[0039] Electrophoresis instrument, a product of Beijing Liuyi Biotechnology Co., Ltd.; PCR instrument, a product of Jena Analytical Instruments AG, Germany.

[0040] 2. Experimental methods

[0041] 2.1 Establishment of single PCR method

[0042] 2.1.1 Single PCR annealing temperature gradient test

[0043] The annealing temperature of the single PCR reaction program was set to 50℃, 50.4℃, 51.2℃, 52.4℃, 53.8℃, 55.3℃, 56.7℃, 58.2℃, 59.6℃, 60.8℃, 61.6℃, and 62.0℃, and other reaction conditions remained unchanged to explore the appropriate annealing temperature range. At the same time, sterile water was used for detection at the optimal annealing temperature as a negative control.

[0044] 2.1.2 Single PCR primer concentration gradient test

[0045] The concentration of primers added to the single PCR reaction was controlled by gradient dilution to 0.4μmol / L, 0.32μmol / L, 0.24μmol / L, 0.16μmol / L, 0.08μmol / L, 0.04μmol / L, 32nmol / L, 24nmol / L, 16nmol / L, and 8nmol / L. The middle temperature of the suitable annealing temperature range obtained in 2.1.1 was used as the annealing temperature for testing to explore the concentration range of each primer. At the same time, sterile water was used for testing at the optimal concentration as a negative control.

[0046] 2.2 Establishment of quadruple PCR method

[0047] 2.2.1 Optimization of amplification conditions

[0048] A mixture of four bacteria (Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella) with a bacterial concentration of 9.4×10 8 CFU / mL, 8.7×10 9 CFU / mL, 1.13×10 8 CFU / mL, 4.8×10 9 CFU / mL) was used as a sample, and the DNA of the mixed bacterial solution was extracted using a bacterial genomic DNA extraction kit. The primer concentration, annealing temperature, etc. of the PCR reaction were optimized using the DNA as a template to determine the best reaction system and reaction procedure. The reaction system was: 2×ES TaqMastermix 12.5μL, 4 pairs of primers with different combination concentrations, 1μL of template bacterial solution mixture, and sterilized ddH2O to make the reaction volume 25μL. Reaction procedure: 94℃10min; then 94℃30s, 52℃30s, 72℃1min, 30 cycles, and finally 72℃ extension for 10min. The annealing temperature (50, 50.4, 51.2, 52.4, 53.8, 55.3, 56.7, 58.2, 59.6, 60.8, 61.6, 62.0°C, all set to 0.4 μmol / L), the final concentration combination of each primer (the initial concentration of each primer was 10 μmol / L, the combined concentration values ​​are shown in Table 2) were optimized to screen out the best reaction conditions for quadruple PCR amplification. In the process of annealing temperature optimization, sterile water was used to detect at the optimal annealing temperature as a negative control.

[0049] Table 2 Primer final concentration combination concentration values

[0050] Combination No. uidA inv khe atpD Combination 1 0.4μmol / L 0.4μmol / L 0.2μmol / L 0.12μmol / L Combination 2 0.4μmol / L 0.4μmol / L 0.2μmol / L 0.16μmol / L Combination 3 0.4μmol / L 0.4μmol / L 0.2μmol / L 0.2μmol / L Combination 4 0.4μmol / L 0.4μmol / L 0.16μmol / L 0.12μmol / L Combination 5 0.4μmol / L 0.4μmol / L 0.16μmol / L 0.16μmol / L Combination 6 0.4μmol / L 0.4μmol / L 0.16μmol / L 0.2μmol / L Combination 7 0.4μmol / L 0.4μmol / L 0.12μmol / L 0.12μmol / L Combination 8 0.4μmol / L 0.4μmol / L 0.12μmol / L 0.16μmol / L Combination 9 0.4μmol / L 0.4μmol / L 0.12μmol / L 0.2μmol / L

[0051] 2.2.2 Sensitivity test

[0052] The overnight cultured Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella were counted by plate method. Then Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella were directly mixed to obtain concentrations of 9.4×10 8 CFU / mL, 8.7×10 9 CFU / mL, 1.13×10 8 CFU / mL, 4.8×10 9 CFU / mL of mixed bacterial solution was diluted 10 times and each gradient bacterial solution was used as a sample. DNA was extracted as a template and the optimized quadruple PCR amplification conditions were used for reaction.

[0053] 2.2.3 Quadruple PCR specificity detection

[0054] The optimized amplification conditions and procedures were used to amplify the bacterial cultures of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Salmonella, Bacillus subtilis, Lactobacillus johnsonii, Enterococcus faecium and Staphylococcus aureus (10 8 CFU / mL) was used as template for quadruple PCR amplification reaction.

[0055] 2.2.4 Testing of clinical samples

[0056] Fecal DNA of diarrhea fawns was extracted using a fecal genomic DNA extraction kit, and quadruple PCR amplification reaction was performed using optimized conditions.

[0057] 3. Experimental results

[0058] 3.1 Establishment of single PCR method

[0059] 3.1.1 Single PCR annealing temperature test results

[0060] The results are as follows Figure 1 As shown, A is uidA, B is khe, C is invA, and D is atpD. The corresponding target fragment bands appeared in the PCR reaction of the four genes at the annealing temperature between 50℃ and 56.7℃.

[0061] 3.1.2 Single PCR primer concentration test results

[0062] The results are as follows Figure 2 As shown, A is uidA, B is khe, C is invA, and D is atpD. The PCR reactions of the four genes all had obvious bands at primer concentrations of 0.16μM-0.4μM, among which atpD still had visible bands at a primer concentration of 24nM, invA still had visible bands at a primer concentration of 0.04μM, and khe still had visible bands at a primer concentration of 32nM.

[0063] 3.2 Establishment of quadruple PCR method

[0064] 3.2.1 Optimization results of annealing temperature

[0065] The four bacterial cultures were mixed in equal proportions as templates, and different annealing temperatures (50°C to 62°C) were selected for quadruple PCR amplification. The results are shown in Figure 3 As shown, the amplification effect is better when the annealing temperature is 50℃~53.8℃, and 52.4℃ is determined to be the optimal annealing temperature.

[0066] 3.2.2 Optimization of primer concentration

[0067] Different concentrations of primer combinations were used for quadruple PCR reactions. The results were as follows: Figure 4 As shown, lanes 1-9 are the final concentration combinations of primers 1-9, respectively. It can be seen that the amplification effect is best when the final concentrations of uidA, invA, khe, and atpD primers are 0.4 μmol / L, 0.4 μmol / L, 0.16 μmol / L, and 0.2 μmol / L.

[0068] 3.2.3 Sensitivity testing

[0069] The mixed bacterial solution of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella was diluted 10 times as a template and the quadruple PCR reaction was performed using the optimized amplification conditions. The results are as follows: Figure 5 As shown in the figure, the minimum detection limit of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella is 9.4×10 4 CFU / mL, 8.7×10 5 CFU / mL, 1.13×10 4 CFU / mL, 4.8×10 5 CFU / mL.

[0070] 3.2.4 Specificity detection

[0071] The optimized amplification conditions were used for the specific detection of quadruple PCR. The results were as follows Figure 6 As shown in the figure, specific target fragments can be amplified using the bacterial suspensions of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella as templates, while no corresponding bands were amplified using other common bacterial DNA and ddH2O negative control, indicating that the established quadruple PCR method has good specificity.

[0072] 3.2.5 Clinical sample testing

[0073] The established quadruple PCR method was used to detect 20 feces samples of clinical deer diarrhea. The results were as follows: Figure 7As shown, the detection rates of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Salmonella using the quadruple PCR method were 95%, 40%, 20% and 20%, respectively.

[0074] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A quadruple PCR primer combination for detecting pathogenic bacteria of deer diarrhea, characterized in that: The primer combination includes: an Escherichia coli primer pair as shown in SEQ ID NO.1-2, a Klebsiella pneumoniae primer pair as shown in SEQ ID NO.3-4, a Salmonella primer pair as shown in SEQ ID NO.5-6, and a Proteus mirabilis primer pair as shown in SEQ ID NO.7-8.

2. Use of the quadruple PCR detection primer combination according to claim 1 in the preparation of a reagent or kit for detecting pathogenic bacteria of fawn diarrhea.

3. A quadruple PCR detection kit for detecting pathogenic bacteria of deer diarrhea, characterized in that: The kit comprises the quadruple PCR detection primer combination according to claim 1.

4. A method for detecting pathogenic bacteria of deer diarrhea for non-disease diagnosis purposes, characterized in that: The steps include: (1) Extracting genomic DNA from feces of diarrhea fawns; (2) Using the quadruple PCR detection primer combination described in claim 1 or the kit described in claim 3, and using the genomic DNA obtained in step (1) as a template, PCR amplification is performed; and the type of pathogenic bacteria causing deer diarrhea is determined based on the band size of the PCR amplification product.

5. The detection method according to claim 4, characterized in that: The reaction system of the PCR amplification is: 2×ESTaq Mastermix 12.5 μL, 1 μL of 10 μmol / L Escherichia coli upstream and downstream primers, 0.4 μL of 10 μmol / L Klebsiella pneumoniae upstream and downstream primers, 1 μL of 10 μmol / L Salmonella upstream and downstream primers, 0.5 μL of 10 μmol / L Proteus mirabilis upstream and downstream primers, 1 μL template, and ddH2O supplemented to 25 μL.

6. The detection method according to claim 4, characterized in that: The reaction procedure of the PCR amplification is: 94°C for 10 min; then 94°C for 30 s, 52.4°C for 30 s, 72°C for 1 min, 30 cycles and finally extension at 72°C for 10 min.

7. The detection method according to claim 4, characterized in that: The judgment method is: if the PCR amplification product contains a 420bp band, the pathogenic bacteria of deer diarrhea include Escherichia coli; if the PCR amplification product contains a 214bp band, the pathogenic bacteria of deer diarrhea include Klebsiella pneumoniae; if the PCR amplification product contains a 564bp band, the pathogenic bacteria of deer diarrhea include Salmonella; if the PCR amplification product contains a 975bp band, the pathogenic bacteria of deer diarrhea include Proteus mirabilis.

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