A primer combination and multiplex PCR method for detecting common pneumonia pathogenic bacteria in fur animals

Abstract

The present invention discloses a primer combination and a multiplex PCR method for detecting common pneumonia pathogenic bacteria in fur animals, belonging to the technical field of medical detection. By using the specific genes (uidA, khe, LasI, and atpD) of the pathogenic bacteria, the present invention differentiates different pneumonia pathogenic bacteria in the diseased materials. Through the multiplex PCR technology, 4 pairs of primers with nucleotide sequences shown in SEQ ID NO.1 - SEQ ID NO.8 are added into the same reaction system, and the final concentration of the primers in the multiplex PCR reaction system and the annealing temperature in the reaction procedure are optimized, achieving the technical effect of amplifying multiple target DNA fragments in the same reaction system, and solving the technical problems of time-consuming and laborious single PCR and the inability to eliminate the sample loading differences between different sample wells. The multiplex PCR detection method constructed by the present invention has high sensitivity and specificity and has broad application prospects.

Description

Technical Field

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

[0002] The pathogenic bacteria of bacterial respiratory diseases in fur animals are complex and diverse, causing huge economic losses to the breeding industry. Common molecular biology methods are increasingly widely used in the detection of animal diseases, and the most prominent one is the polymerase chain reaction (PCR). The 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 pathogenic bacteria causing pneumonia in fur animals, which is both time-consuming and laborious. Therefore, there is an urgent need to develop a multiplex PCR method for quickly, accurately, sensitively, and efficiently detecting multiple common pneumonia pathogenic bacteria 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 pathogenic bacteria in fur animals to solve the problems existing in the above-mentioned prior art. The present invention introduces multiple pairs of primers into the same reaction system, solving the problems of time-consuming and laborious single PCR and the inability to eliminate the pipetting differences between different sample wells.

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

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

[0006] uidA-F with the nucleotide sequence shown in SEQ ID NO.1 and uidA-R with the nucleotide sequence shown in SEQ ID NO.2;

[0007] khe-F with the nucleotide sequence shown in SEQ ID NO.3 and khe-R with the nucleotide sequence shown in SEQ ID NO.4;

[0008] LasI-F with the nucleotide sequence shown in SEQ ID NO.5 and LasI-R with the nucleotide sequence shown in SEQ ID NO.6;

[0009] atpD-F with the nucleotide sequence shown in SEQ ID NO.7 and atpD-R with the nucleotide sequence shown in SEQ ID NO.8.

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

[0011] The amplified fragment length 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 amplified fragment length of the primer atpD-F and the primer atpD-R is 756 bp.

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

[0015] The present invention provides the application of the primer combination in the preparation of products for detecting common pneumonia pathogenic bacteria in fur animals.

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

[0017] The present invention also provides a kit for detecting common pneumonia pathogenic bacteria in fur animals, including the primer combination.

[0018] The present invention also provides the application of the primer combination or the kit in the detection of common pneumonia pathogenic bacteria in fur animals for non-diagnostic or therapeutic purposes.

[0019] The present invention also provides a method for detecting common pneumonia pathogenic bacteria in fur animals for non-diagnostic or therapeutic purposes, including the step of performing multiplex 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; 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 differentiates different pneumonia pathogens in diseased materials by using specific genes of pathogenic bacteria. Through multiplex PCR technology, more than one pair of primers are added to the same reaction system. If there are templates complementary to each pair of primers, they will respectively bind to the corresponding positions on the templates, achieving the technical effect of amplifying more than one target DNA fragment in the same reaction system, and solving the technical problems of time-consuming and laborious single PCR and the inability to eliminate the sample loading differences 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 technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0025] Figure 1 It is the electrophoresis result diagram of the annealing temperature gradient of single PCR for uidA; where M is DNA standard DL2000, the annealing temperature gradient of 1 - 12 is 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°C, and 13 is the negative control ddH 2 O;

[0026] Figure 2 It is the electrophoresis result diagram of the annealing temperature gradient of single PCR for khe; where M is DNA standard DL2000, the annealing temperature gradient of 1 - 12 is 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°C, and 13 is the negative control ddH 2 O;

[0027] Figure 3 It is the electrophoresis result diagram of the annealing temperature gradient of single PCR for LasI; where M is DNA standard DL2000, the annealing temperature gradient of 1 - 12 is 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°C, and 13 is the negative control ddH 2 O;

[0028] Figure 4It is the electrophoretogram of the annealing temperature gradient of atpD single PCR; among them, M is the DNA standard DL2000, and the annealing temperature gradients of 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°C, and 13 is the negative control ddH 2 O;

[0029] Figure 5 It is the electrophoretogram of the primer concentration gradient of uidA single PCR; among them, M is the DNA standard DL2000, and the primer concentration gradients of 1 - 8 are 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;

[0030] Figure 6 It is the electrophoretogram of the primer concentration gradient of khe single PCR; among them, M is the DNA standard DL2000, and the primer concentration gradients of 1 - 8 are 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;

[0031] Figure 7 It is the electrophoretogram of the primer concentration gradient of LasI single PCR; among them, M is the DNA standard DL2000, and the primer concentration gradients of 1 - 8 are 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;

[0032] Figure 8 It is the electrophoretogram of the primer concentration gradient of atpD single PCR; among them, M is the DNA standard DL2000, and the primer concentration gradients of 1 - 8 are 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;

[0033] Figure 9 It is the electrophoretogram of the annealing temperature gradient of quadruple PCR; among them, M is the DNA standard DL2000, and the annealing temperature gradients of 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°C;

[0034] Figure 10 It is the electrophoresis result diagram of the concentration combination of quadruple PCR primers; among them, M is DNA standard DL2000, and the final concentrations of uidA, khe, LasI, and atpD primers in 1-6 are 0.4 μmol / L, 0.4 μmol / L, 0.4 μmol / L, and 32 nmol / L, 0.5 μmol / L, 0.4 μmol / L, 0.5 μmol / L, and 32 nmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.3 μmol / L, and 32 nmol / L, 0.4 μmol / L, 0.4 μmol / L, 0.4 μmol / L, and 22.4 nmol / L, 0.5 μmol / L, 0.4 μmol / L, 0.5 μmol / L, and 22.4 nmol / L, 0.3 μmol / L, 0.4 μmol / L, 0.3 μmol / L, and 22.4 nmol / L respectively;

[0035] Figure 11 It is the electrophoresis result diagram for the sensitivity test of the quadruple PCR method; among them, M is DNA standard DL2000; the dilution factors of the mixed DNA in 1-6 are 10 0 、10 1 、10 2 、10 3 、10 4 and 10 5 ;

[0036] Figure 12 It is the electrophoresis result diagram for the specific detection of the quadruple PCR method. Among them, 1-8 are Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Bacillus cereus, Enterococcus faecium, and Staphylococcus cohnii respectively, 9 is the negative control ddH 2 O, and M is DNA standard DL2000;

[0037] Figure 13 It is the electrophoresis result diagram for detecting the pathogenic bacteria DNA of 17 fur animal pneumonia samples by using the quadruple PCR method; among them, M is DNA standard DL2000. Detailed implementation manners

[0038] Now, various exemplary implementation manners of the present invention will be described in detail. This detailed description should not be regarded as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes 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), product of Qingdao Keyuan Biotechnology Co., Ltd.; DNA standard DL2000, product of Novoprotein Scientific Inc.; Bacterial genomic DNA extraction kit, product of omega company; Fecal genomic DNA extraction kit, product of biomiga company.

[0054] 1.3 Main instruments and equipment

[0055] Electrophoresis apparatus, purchased from Beijing Liuyi Biotechnology Co., Ltd.; PCR instrument, purchased from Analytik Jena AG.

[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 is: 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 sterilized ddH 2 O 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; Finally, extension at 72°C for 10 min.

[0061] The annealing temperatures of the gradient-set single PCR reaction procedure are set as 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 remain 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 primer concentrations were controlled at 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. Using the intermediate temperature within the appropriate annealing temperature range in 2.1.1 as the annealing temperature for testing, with other conditions remaining unchanged, the concentration range for each primer reaction was explored.

[0064] 2.2 Establishment of the quadruple PCR method

[0065] 2.2.1 Optimization of amplification conditions

[0066] Using the mixed bacterial solution of 4 kinds of bacteria as the template, the primer concentration, annealing temperature, etc. of the PCR reaction were optimized to determine the optimal reaction system and reaction program.

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

[0068] Reaction program: 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; finally, extension at 72°C for 10 min.

[0069] Two reaction conditions, namely 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) and the combination of final concentrations of each primer (the combinations are set as shown in Table 2), were optimized to screen out the optimal reaction conditions for quadruple PCR amplification.

[0070] Table 2 Design table of the combination of final concentrations of quadruple PCR primers

[0071]

[0072] 2.2.2 Sensitivity test

[0073] The overnight cultured Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa bacterial solutions were counted by the plate method. Then, directly take the DNA of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa, mix and perform 10-fold serial dilutions. Using the bacterial solutions at each concentration gradient as templates, reactions were carried out under the optimized quadruple PCR amplification conditions.

[0074] 2.2.3 Specificity detection of quadruple PCR

[0075] Using the optimized amplification conditions and procedures, quadruple PCR amplification reactions were carried out with the bacterial suspensions of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Bacillus cereus, Enterococcus faecium, and Staphylococcus cohnii as templates respectively.

[0076] 2.2.4 Detection of samples

[0077] DNA of the pneumonia samples from fur animals was extracted, and the quadruple PCR method was applied for the detection of pneumonia pathogenic bacteria.

[0078] 3. Experimental results

[0079] 3.1 Establishment of single PCR method

[0080] As Figures 1 - 4 shown, the results showed that there were corresponding target fragment bands for the four genes within the annealing temperature range of 50°C - 58.2°C.

[0081] As Figures 5 - 8 shown, obvious bands were observed for the four genes when the primer concentrations were in the range of 0.4 μmol / L - 0.16 μmol / L. Among them, visible bands were still present for atpD when the final primer concentration was 32 nmol / L, and visible bands were still present for LasI 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 bacterial suspensions of the four bacteria were mixed in equal proportions as templates, and different annealing temperatures (50°C - 62°C) were selected for quadruple PCR amplification. As Figure 9 shown, the results indicated that the amplification effect was better within the annealing temperature range of 50°C - 53.8°C, and 52°C was determined as the optimal annealing temperature.

[0085] 3.2.2 Optimization of primer concentration

[0086] Quadruple PCR reactions were carried out with different combinations of primer concentrations. The results were as Figure 10 shown, indicating that the amplification effect was the best when the final primer concentrations of uidA, khe, LasI, and atpD were 0.4 μmol / L, 0.4 μmol / L, 0.4 μmol / L, and 32 nmol / L respectively.

[0087] 3.2.3 Sensitivity detection

[0088] The bacterial suspensions of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa were mixed and serially diluted 10-fold as templates, and the optimized amplification conditions were used for quadruple PCR reactions. The results were as Figure 11As shown, the results show that the minimum detection limits of the Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa bacterial suspensions are 9.57×10 3 CFU / mL, 1.07×10 6 CFU / mL, 1.53×10 6 CFU / mL, and 6.5×10 5 CFU / mL.

[0089] 3.3 Specificity detection of quadruple PCR

[0090] The optimized amplification conditions were used for the specificity detection of quadruple PCR. The results are as Figure 12 shown. Specific target fragments could be amplified using the bacterial suspensions of Escherichia coli, Proteus mirabilis, Klebsiella pneumoniae, and Pseudomonas aeruginosa as templates, while no corresponding bands were amplified using the bacterial suspensions of other common bacteria and ddH 2 O negative control, indicating that the established quadruple PCR method has good specificity.

[0091] 3.4 Sample detection

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

[0093] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for detecting common pneumonia pathogens in fur animals for non-diagnostic or therapeutic purposes, characterized in that: The method comprises the steps of performing multiple PCR amplification on the sample DNA using a primer combination; The common pneumonia pathogens are Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Proteus mirabilis; The primer combination includes the following primers: 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 sequences are atpD-F as shown in SEQ ID NO.7 and atpD-R as shown in SEQ ID NO.8; The annealing temperature for the multiplex PCR amplification was 52°C.

2. The method according to claim 1, 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.

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