Primer combination and multiple PCR method for detecting common pneumonia pathogenic bacteria of fur-bearing animals

Abstract

The invention discloses a primer combination and multiple PCR method for detecting common pneumonia pathogenic bacteria of fur-bearing animals, and belongs to the technical field of medical detection. Specific genes (uidA, khe, LasI and atpD) of pathogenic bacteria are used for distinguishing different pneumonia pathogenic bacteria in a disease material, four pairs of primers with nucleotide sequences as shown in SEQ ID NO.1-SEQ ID NO.8 are added into the same reaction system through a multiplex PCR technology, the final concentration of the primers in the multiplex PCR reaction system and the annealing temperature in the reaction procedure are optimized, and the pneumonia pathogenic bacteria in the disease material are distinguished. The technical effect of amplifying multiple items of DNA fragments in the same reaction system is achieved, and the technical problems that time and labor are wasted by single PCR, and meanwhile sample adding differences among different sample holes cannot be eliminated are solved. The multiplex PCR detection method constructed by the invention has relatively high sensitivity and specificity, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of medical detection, and in particular to a primer combination and a multiplex PCR method for detecting common pneumonia pathogens of fur animals. Background Art

[0002] The pathogens of bacterial respiratory diseases in fur animals are complex and diverse, causing huge economic losses to the breeding industry. Common molecular biological methods are increasingly used in animal disease detection, the most prominent of which is the 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, and it takes more time to detect the pathogens of pneumonia in fur animals, which is time-consuming and laborious. Therefore, it is urgent to develop a multiplex PCR method that can quickly, accurately, sensitively and efficiently detect a variety of common pneumonia pathogens in fur animals. Summary of the invention

[0003] The purpose of the present invention is to provide a primer combination and a multiplex PCR method for detecting common pneumonia pathogens in fur animals, so as to solve the problems existing in the above-mentioned prior art. The present invention introduces multiple pairs of primers into the same reaction system, thereby solving the problems that single-plex PCR is time-consuming and labor-intensive and cannot eliminate the differences in sample addition between different sample wells.

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

[0005] The present invention provides a primer combination for detecting common pneumonia pathogens in fur animals, comprising the following primers:

[0006] The nucleotide sequences are uidA-F as shown in SEQ ID NO.1 and uidA-R as shown in SEQ ID NO.2;

[0007] The nucleotide sequences are khe-F as shown in SEQ ID NO.3 and khe-R as shown in SEQ ID NO.4;

[0008] The nucleotide sequence is LasI-F as shown in SEQ ID NO.5 and LasI-R as shown in SEQ ID NO.6;

[0009] The nucleotide sequence is atpD-F as shown in SEQ ID NO.7 and atpD-R as shown in SEQ ID NO.8.

[0010] Preferably, the length of the amplified fragment of the primer uidA-F and the primer uidA-R is 421 bp;

[0011] The length of the amplified fragment of the primer khe-F and the primer khe-R is 143 bp;

[0012] The amplified fragment length of the primer LasI-F and the primer LasI-R is 315 bp;

[0013] The length of the amplified fragment of the primer atpD-F and the primer atpD-R is 756 bp.

[0014] Preferably, the common pneumonia pathogens are Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis.

[0015] The present invention provides application of the primer combination in preparing a product for detecting common pneumonia pathogens of fur animals.

[0016] Preferably, the product is a reagent or a kit.

[0017] The present invention also provides a kit for detecting common pneumonia pathogens of fur animals, comprising the primer combination.

[0018] The present invention also provides the use of the primer combination or the kit in detecting common pneumonia pathogens of fur animals for non-diagnostic or therapeutic purposes.

[0019] The present invention also provides a method for detecting common pneumonia pathogens of fur animals for non-diagnostic or therapeutic purposes, comprising the step of performing multiple PCR amplification on sample DNA using the primer combination or the kit.

[0020] Preferably, in the reaction system of the multiplex PCR amplification, the concentrations of uidA-F and uidA-R are both 0.4 μmol / L; the concentrations of khe-F and khe-R are both 0.4 μmol / L; the concentrations of LasI-F and LasI-R are both 0.4 μmol / L; and the concentrations of atpD-F and atpD-R are both 32 nmol / L.

[0021] Preferably, the annealing temperature of the multiplex PCR amplification is 52°C.

[0022] The present invention discloses the following technical effects:

[0023] The present invention distinguishes different pneumonia pathogens in the diseased material by using the specific genes of the pathogens. By using the multiplex PCR technology, more than one pair of primers are added to the same reaction system. If there is a template complementary to each pair of primers, they are respectively combined at the corresponding part of the template, achieving the technical effect of amplifying more than one target DNA fragment in the same reaction system, solving the technical problem that single-plex PCR is time-consuming and labor-intensive and cannot eliminate the difference in sample addition between different sample wells. The multiplex PCR detection method constructed by the present invention has high sensitivity and specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] 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.

[0025] Figure 1 The figure is the result of annealing temperature gradient electrophoresis of uidA single PCR; M is the DNA standard DL2000, annealing temperature gradient 1-12 is 50℃, 50.4℃, 51.2℃, 52.4℃, 53.8℃, 55.3℃, 56.7℃, 58.2℃, 59.6℃, 60.8℃, 61.6℃ and 62℃, 13 is the negative control ddH2O;

[0026] Figure 2 This is the result of khe single PCR annealing temperature gradient electrophoresis; where M is the DNA standard DL2000, 1-12 annealing temperature gradient is 50℃, 50.4℃, 51.2℃, 52.4℃, 53.8℃, 55.3℃, 56.7℃, 58.2℃, 59.6℃, 60.8℃, 61.6℃ and 62℃, 13 is the negative control ddH2O;

[0027] Figure 3 The figure is the result of LasI single PCR annealing temperature gradient electrophoresis; wherein, M is DNA standard DL2000, 1-12 annealing temperature gradient is 50℃, 50.4℃, 51.2℃, 52.4℃, 53.8℃, 55.3℃, 56.7℃, 58.2℃, 59.6℃, 60.8℃, 61.6℃ and 62℃, 13 is negative control ddH2O;

[0028] Figure 4The figure is the result of atpD single PCR annealing temperature gradient electrophoresis; wherein, M is DNA standard DL2000, 1-12 annealing temperature gradient is 50℃, 50.4℃, 51.2℃, 52.4℃, 53.8℃, 55.3℃, 56.7℃, 58.2℃, 59.6℃, 60.8℃, 61.6℃ and 62℃, 13 is negative control ddH2O;

[0029] Figure 5 The figure is the result of single PCR primer concentration gradient electrophoresis of uidA; wherein, M is DNA standard DL2000, and the concentration gradient of primers 1-8 is 0.4μmol / L, 0.32μmol / L, 0.24μmol / L, 0.16μmol / L, 0.08μmol / L, 0.04μmol / L, 32nmol / L and 24nmol / L;

[0030] Figure 6 The figure is the result of khe single PCR primer concentration gradient electrophoresis; wherein, M is the DNA standard DL2000, and the concentration gradient of primers 1-8 is 0.4μmol / L, 0.32μmol / L, 0.24μmol / L, 0.16μmol / L, 0.08μmol / L, 0.04μmol / L, 32nmol / L and 24nmol / L;

[0031] Figure 7 The figure is the result of LasI single PCR primer concentration gradient electrophoresis; wherein, M is DNA standard DL2000, and the concentration gradient of primers 1-8 is 0.4μmol / L, 0.32μmol / L, 0.24μmol / L, 0.16μmol / L, 0.08μmol / L, 0.04μmol / L, 32nmol / L and 24nmol / L;

[0032] Figure 8 The figure is the result of atpD single PCR primer concentration gradient electrophoresis; wherein, M is DNA standard DL2000, and the concentration gradient of primers 1-8 is 0.4μmol / L, 0.32μmol / L, 0.24μmol / L, 0.16μmol / L, 0.08μmol / L, 0.04μmol / L, 32nmol / L and 24nmol / L;

[0033] Fig. 9 The figure is the result of quadruple PCR annealing temperature gradient electrophoresis; wherein, M is DNA standard DL2000, and the 1-12 annealing temperature gradient is 50℃, 50.4℃, 51.2℃, 52.4℃, 53.8℃, 55.3℃, 56.7℃, 58.2℃, 59.6℃, 60.8℃, 61.6℃ and 62℃;

[0034] Fig.10 : is the result of electrophoresis of quadruple PCR primer concentration combination; wherein, M is DNA standard DL2000, and the final concentrations of uidA, khe, LasI and atpD primers in 1-6 are 0.4μmol / L, 0.4μmol / L, 0.4μmol / L and 32nmol / L, 0.5μmol / L, 0.4μmol / L, 0.5μmol / L and 32nmol / L, 0.3μmol / L, 0.4μmol / L, 0.3μmol / L and 32nmol / L, 0.4μmol / L, 0.4μmol / L, 0.4μmol / L and 22.4nmol / L, 0.5μmol / L, 0.4μmol / L, 0.5μmol / L and 22.4nmol / L, 0.3μmol / L, 0.4μmol / L, 0.3μmol / L and 22.4nmol / L;

[0035] Fig.11 This is the electrophoresis result of the sensitivity test of the quadruple PCR method; M is the DNA standard DL2000; the dilution factor of the 1-6 mixed DNA is 10 0 , 10 1 , 10 2 , 10 3 , 10 4 and 10 5 ;

[0036] Fig.12 The electrophoresis results of the quadruple PCR method for specific detection are shown in Figure 1-8, respectively, for Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Bacillus cereus, Enterococcus faecium, and Staphylococcus cohnii; 9 is the negative control ddH2O; M is the DNA standard DL2000;

[0037] Fig.13 The figure shows the electrophoresis results of detecting DNA of pathogenic bacteria in 17 fur animal pneumonia samples using the quadruple PCR method; where M is the DNA standard DL2000. DETAILED DESCRIPTION

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] In the present invention, the final concentration of a primer refers to the concentration of the upstream primer or downstream primer used for the DNA fragment in the configured PCR reaction system.

[0044] Example 1 Establishment of multiplex PCR method for Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis

[0045] 1. Experimental Materials

[0046] 1.1 Strains and primers

[0047] Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Proteus mirabilis were isolated, identified and preserved by the Animal Infection and Prevention Technology Laboratory of the Institute of Special Products, Chinese Academy of Agricultural Sciences.

[0048] 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, Pseudomonas aeruginosa LasI, and Proteus mirabilis atpD were cited and synthesized by Sangon Biotechnology Co., Ltd. The sequences of the primers and the lengths of the amplified fragments are shown in Table 1.

[0049] Table 1 Quadruple PCR primer sequences

[0050]

[0051]

[0052] 1.2 Main Reagents

[0053] 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.

[0054] 1.3 Main instruments and equipment

[0055] The electrophoresis apparatus was purchased from Beijing Liuyi Biotechnology Co., Ltd.; the PCR apparatus was purchased from Jena Analytical Instruments AG, Germany.

[0056] 2. Experimental Methods

[0057] 2.1 Establishment of single PCR method

[0058] 2.1.1 Single PCR annealing temperature gradient test

[0059] The reaction system was: 12.5 μL of 2×ES Taq Mastermix, 1 μL of 10 μmol / L single primer solution, 1 μL of corresponding template bacterial solution, and sterile ddH2O to make up the reaction volume to 25 μL.

[0060] Reaction procedure: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 s, annealing at different temperatures for 30 s, extension at 72°C for 1 min, 30 cycles; and final extension at 72°C for 10 min.

[0061] The annealing temperatures of the single PCR reaction program were set in a gradient manner to 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, and other reaction conditions remained unchanged to explore the appropriate annealing temperature range.

[0062] 2.1.2 Single PCR primer concentration gradient test

[0063] By diluting the concentration of primers added to a single PCR reaction, the final concentration of the primers was controlled to be 0.4 μmol / L, 0.32 μmol / L, 0.24 μmol / L, 0.16 μmol / L, 0.08 μmol / L, 0.04 μmol / L, 32 nmol / L and 24 nmol / L. The middle temperature of the suitable annealing temperature range in 2.1.1 was used as the annealing temperature for testing. Other conditions remained unchanged to explore the concentration range of each primer reaction.

[0064] 2.2 Establishment of quadruple PCR method

[0065] 2.2.1 Optimization of amplification conditions

[0066] Using the mixed bacterial solution of four bacteria as template, the primer concentration, annealing temperature, etc. of the PCR reaction were optimized to determine the best reaction system and reaction procedure.

[0067] The reaction system was: 12.5 μL of 2×ES Taq Mastermix, 4 pairs of primers with different final concentrations, 1 μL of template bacterial solution mixture, and sterile ddH2O to make up the reaction volume to 25 μL.

[0068] Reaction procedure: pre-denaturation at 94°C for 10 min; denaturation at 94°C for 30 s, annealing at 52°C for 30 s, extension at 72°C for 1 min, 30 cycles; final extension at 72°C for 10 min.

[0069] The two reaction conditions of 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) and the final concentration combination of each primer (the combination setting is shown in Table 2) were optimized to screen out the optimal reaction conditions for quadruple PCR amplification.

[0070] Table 2 Quadruple PCR primer final concentration combination design table

[0071]

[0072] 2.2.2 Sensitivity test

[0073] The overnight cultured Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa were counted by the plate method. Then the DNA of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa were directly taken, mixed and diluted 10 times, and the concentration gradient bacterial liquids were used as templates to react using the optimized quadruple PCR amplification conditions.

[0074] 2.2.3 Quadruple PCR specificity detection

[0075] The optimized amplification conditions and procedures were used to carry out quadruple PCR amplification reactions with Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Bacillus cereus, Enterococcus faecium and Staphylococcus cohnii as templates.

[0076] 2.2.4 Sample testing

[0077] DNA from pneumonia samples of fur animals was extracted and the pneumonia-causing bacteria were detected using quadruple PCR method.

[0078] 3. Experimental results

[0079] 3.1 Establishment of single PCR method

[0080] like Figure 1-Figure 4 As shown, the results showed that the four genes had corresponding target fragment bands when the annealing temperature was between 50°C and 58.2°C.

[0081] like Figure 5-Figure 8 As shown, the four genes all had obvious bands when the primer concentration was 0.4 μmol / L-0.16 μmol / L, among which atpD still had a visible band when the final primer concentration was 32 nmol / L, and LasI still had a visible band when the primer concentration was 0.08 μmol / L.

[0082] 3.2 Establishment of quadruple PCR method

[0083] 3.2.1 Optimization of annealing temperature

[0084] The four bacterial cultures were mixed in equal proportions as templates, and different annealing temperatures (50°C-62°C) were selected for quadruple PCR amplification. Fig. 9 As shown, the results showed that the amplification effect was better when the annealing temperature was 50℃-53.8℃, and 52℃ was determined to be the optimal annealing temperature.

[0085] 3.2.2 Optimization of primer concentration

[0086] Different concentrations of primer combinations were used for quadruple PCR reactions. The results were as follows: Fig.10 As shown, the amplification effect was best when the final concentrations of uidA, khe, LasI and atpD primers were 0.4 μmol / L, 0.4 μmol / L, 0.4 μmol / L and 32 nmol / L.

[0087] 3.2.3 Sensitivity testing

[0088] The optimized amplification conditions were used to perform quadruple PCR reaction using a mixture of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa bacterial suspensions diluted 10 times as templates. Fig.11The results showed that the minimum detection limit of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa was 9.57×10 3 CFU / mL, 1.07×10 6 CFU / mL, 1.53×10 6 CFU / mL, 6.5×10 5 CFU / mL.

[0089] 3.3 Quadruple PCR specific detection

[0090] The optimized amplification conditions were used for the specific detection of quadruple PCR. The results were as follows Fig.12 As shown in the figure, specific target fragments can be amplified using bacterial cultures of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa as templates, while no corresponding bands were amplified using bacterial cultures of other common bacteria and ddH2O negative control, indicating that the established quadruple PCR method has good specificity.

[0091] 3.4 Sample testing

[0092] The established quadruple PCR method was used to detect the DNA of pathogenic bacteria in 16 samples of fur animal pneumonia. The results are as follows: Fig.13 As shown, the positive rates of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae and Pseudomonas aeruginosa by the quadruple PCR method were 100%, 43.75%, 100% and 43.75%, respectively.

[0093] 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 primer combination for detecting common pneumonia pathogens in fur animals, characterized in that: The following primers were included: The nucleotide sequences are uidA-F as shown in SEQ ID NO.1 and uidA-R as shown in SEQ ID NO.2; The nucleotide sequences are khe-F as shown in SEQ ID NO.3 and khe-R as shown in SEQ ID NO.4; The nucleotide sequence is LasI-F as shown in SEQ ID NO.5 and LasI-R as shown in SEQ ID NO.6; The nucleotide sequence is atpD-F as shown in SEQ ID NO.7 and atpD-R as shown in SEQ ID NO.

8.

2. The primer combination according to claim 1, characterized in that The length of the amplified fragment of the primer uidA-F and the primer uidA-R is 421 bp; The length of the amplified fragment of the primer khe-F and the primer khe-R is 143 bp; The amplified fragment length of the primer LasI-F and the primer LasI-R is 315 bp; The length of the amplified fragment of the primer atpD-F and the primer atpD-R is 756 bp.

3. The primer combination according to claim 1, characterized in that The common pneumonia pathogens are Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis.

4. Use of the primer combination as described in any one of claims 1 to 3 in the preparation of a product for detecting common pneumonia pathogens in fur animals.

5. The use according to claim 4, characterized in that The product is a reagent or a kit.

6. A kit for detecting common pneumonia pathogens in fur animals, characterized in that: The invention comprises the primer combination according to any one of claims 1 to 3.

7. Use of the primer combination according to any one of claims 1 to 3 or the kit according to claim 6 in detecting common pneumonia pathogens in fur animals for non-diagnostic or therapeutic purposes.

8. A method for detecting common pneumonia pathogens in fur animals for non-diagnostic or therapeutic purposes, characterized in that: The method comprises the step of performing multiple PCR amplification on the sample DNA using the primer combination described in any one of claims 1 to 3 or the kit described in claim 6.

9. The method according to claim 8, characterized in that In the reaction system of the multiplex PCR amplification, the concentrations of uidA-F and uidA-R are both 0.4 μmol / L; the concentrations of khe-F and khe-R are both 0.4 μmol / L; the concentrations of LasI-F and LasI-R are both 0.4 μmol / L; and the concentrations of atpD-F and atpD-R are both 32 nmol / L.

10. The method according to claim 8, characterized in that The annealing temperature for the multiplex PCR amplification was 52°C.

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