CN114959083B - Triple PCR detection primer set and kit - Google Patents
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Abstract
The invention discloses a triple PCR detection primer set and a kit. Belongs to the field of biotechnology. Comprising the following steps: pld-F/R, plo-F/R and nuc-F/R. The invention designs 3 pairs of primers by taking a corynebacterium pseudotuberculosis phospholipase D (pld) gene, a stellera suppuration secretor hemolysin (plo) gene and a staphylococcus aureus heat-resistant nuclease (nuc) gene as target genes respectively, and establishes a triple PCR detection method and a kit with strong specificity, high sensitivity, rapidness and accuracy. The method is used for clinical and laboratory PCR detection of corynebacterium pseudotuberculosis, stellera suppurativa and staphylococcus aureus in the sheep cheese lymphadenitis, and has wide application potential in the aspects of rapid pathogen detection, epidemiological investigation and the like of the sheep cheese lymphadenitis. Can provide a reliable method for dynamic monitoring, epidemiological investigation, laboratory detection, purification and other aspects of sheep cheese lymphadenitis.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a triple PCR detection primer set and a kit.
Background
In the existing PCR detection method for sheep cheese lymphadenitis, a detection method is established around a bacterium of pseudotuberculosis corynebacterium from the viewpoint of pathogenic bacteria, but a plurality of scholars at home and abroad and research results of the unit show that most of bacteria separated from sheep cheese lymphadenitis abscess tissues are staphylococcus aureus, and then pseudotuberculosis corynebacterium and suppuration stealth bacillus, and animal regression experiments show that 3 bacteria can cause similar characteristic clinical symptoms and pathological changes of experimental sheep, but the infection severity is different, so that the establishment detection method for 1 bacteria is not comprehensive enough.
The method also relates to the establishment of a triple PCR detection method of 3 bacteria, but is inconsistent with the target genes applied in the invention, and has the problems of insufficient specificity and the like.
Therefore, how to provide a triple PCR primer set and a detection kit with better effect is a problem to be solved by the technicians in the field.
Disclosure of Invention
In view of this, the present invention provides a triple PCR detection primer set and a kit. The invention selects the specific virulence genes of 3 bacteria, which can further improve the specificity of the detection method, reduce or eliminate the later sequencing step and save the detection period and the cost; the detection method established in the invention has successfully prepared the kit for direct application, and has convenient application capability.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a triple PCR detection primer set comprising: pld-F/R, plo-F/R and nuc-F/R, wherein the nucleotide sequence of pld-F is shown as SEQ ID No.1, and the nucleotide sequence of pld-R is shown as SEQ ID No. 2; the nucleotide sequence of plo-F is shown as SEQ ID No.3, and the nucleotide sequence of plo-R is shown as SEQ ID No. 4; the nuc-F nucleotide sequence is shown as SEQ ID No.5, and the nuc-R nucleotide sequence is shown as SEQ ID No. 6.
The invention also provides a reagent or a kit containing the primer group.
The invention also provides a method for detecting pld genes of corynebacterium pseudotuberculosis ATCC19410, plo genes of cryptobacter pyogenes ATCC19411 and nuc genes of staphylococcus aureus ATCC25923 for non-diagnostic treatment purpose, and the primers are used.
The beneficial effects are that: the invention determines that the corynebacterium pseudotuberculosis, the cryptobacter suppuration and the staphylococcus aureus are 3 bacteria with highest separation rate in the current sheep cheese lymphadenitis abscess tissues in China, and can cause sheep cheese lymphadenitis-like lesions.
The pld, plo, nuc genes selected for the 3 bacteria were 100% carried in Corynebacterium pseudotuberculosis, milkyi suppuration and Staphylococcus aureus, respectively.
Preferably: comprising an amplification system: 2X Taq PCR MasterMix. Mu.L, 0.4. Mu. Mol/L of each of the upstream and downstream primers, 0.6. Mu.L of template, 3. Mu.L, and the remainder was made up to 30. Mu.L with sterile DEPC water.
Preferably, the amplification procedure is included: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 54℃for 30s, extension at 72℃for 40s for 35 cycles; final extension at 72℃for 10min.
Compared with the prior art, the invention discloses a triple PCR detection primer set and a kit, and the technical effects obtained are that the triple PCR detection primer set and the detection kit for 3 common pathogenic bacteria in sheep cheese lymphadenitis (pseudotuberculosis) are provided. The invention designs 3 pairs of primers by taking a corynebacterium pseudotuberculosis phospholipase D (pld) gene, a stellera suppuration secretor hemolysin (plo) gene and a staphylococcus aureus heat-resistant nuclease (nuc) gene as target genes respectively, and establishes a triple PCR detection method and a kit with strong specificity, high sensitivity, rapidness and accuracy. The method is used for clinical and laboratory PCR detection of corynebacterium pseudotuberculosis, stellera suppurativa and staphylococcus aureus in the sheep cheese lymphadenitis, and has wide application potential in the aspects of rapid pathogen detection, epidemiological investigation and the like of the sheep cheese lymphadenitis. In addition, the method can be used for single and combined detection of corynebacterium pseudotuberculosis, cryptococcus suppurative and staphylococcus aureus except for sheep cheese lymphadenitis.
The kit provides positive control strain DNA and various reagents required by PCR reaction. The detection method and the detection kit provided by the invention can provide a reliable method for dynamic monitoring, epidemiological investigation, clinical and laboratory detection, comprehensive control, purification and other aspects of sheep cheese lymphadenitis.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a diagram showing a primer specificity evaluation scheme provided by the invention, wherein M: DL2000DNA markers; 1: corynebacterium pseudotuberculosis; 2: a stellera suppurati; 3: staphylococcus aureus; 4: klebsiella pneumoniae; 5: coli; 6: pseudomonas aeruginosa; 7: clostridium perfringens; 8: streptococcus; 9: negative control.
FIG. 2 is a graph showing the reaction results of different annealing temperatures provided by the present invention, wherein M: DL2000DNA markers; 1:54 ℃;2:53 ℃;3:55 ℃;4:56 ℃;5:57 ℃.
FIG. 3 is a graph showing the results of reactions at different primer concentrations according to the present invention, wherein M: DL2000DNA markers; 1: 0.2. Mu. Mol/L;2: 0.4. Mu. Mol/L;3:0.6 mu mol/L;4:0.8 mu mol/L;5: 1.0. Mu. Mol/L.
FIG. 4 is a diagram showing the detection sensitivity of the triple PCR for detecting the number of different bacteria.
FIG. 5 is a graph showing the sensitivity of triple PCR provided by the invention to DNA concentrations of different bacteria.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a triple PCR detection primer set and a kit.
Example 1
Primer design
The PCR primers for amplifying the corresponding genes were synthesized with the pld gene of Corynebacterium pseudotuberculosis ATCC19410, plo gene of Mikrypton suppurative ATCC19411 and nuc gene of Staphylococcus aureus ATCC25923 as target genes, respectively (Table 1):
TABLE 1 synthetic primer information
Example 2
Primer specificity evaluation
Extracting DNA from isolated strains of corynebacterium pseudotuberculosis, stellera suppuration, staphylococcus aureus, klebsiella pneumoniae, escherichia coli, pseudomonas aeruginosa, clostridium perfringens and streptococcus, respectively, taking the extracted DNA as a template for PCR, and checking whether the designed primer can specifically amplify target genes of target bacteria.
The reaction system was 30. Mu.L, to which 15. Mu.L of 2X Taq PCR MasterMix was added, 0.6. Mu.L (10. Mu. Mol/L) of each primer, 3. Mu.L of template content, and the remainder was made up to 30. Mu.L with sterile DEPC water. The reaction procedure is 94 ℃ pre-denaturation for 5min; denaturation at 94℃for 30s, annealing at 55℃for 30s, extension at 72℃for 40s for 35 cycles; final extension at 72℃for 10min. The PCR products are subjected to 1.2% agarose gel nucleic acid electrophoresis, and the result shows that in the detected common bacterial DNA, each designed pair of primers only specifically amplify a single target band of target bacteria, and the primers are not combined with the DNA of other bacteria after amplification, so that the designed 3 pairs of primers have good specificity (figure 1).
Example 3
Optimum annealing temperature screening
The reaction system was 30. Mu.L, 1. Mu.L of each of DNA of Corynebacterium pseudotuberculosis, mikroorganismen suppuration and Staphylococcus aureus was used as a template, and the other components and contents were consistent with those in the specific detection.
The reaction procedure is 94 ℃ pre-denaturation for 5min; denaturation at 94 ℃ for 30s, annealing at 53-57 ℃ for 30s, extension at 72 ℃ for 40s, for 35 cycles; final extension at 72℃for 10min. The PCR products were subjected to 1.2% agarose gel nucleic acid electrophoresis, and the result showed that the amplified target band was most clear at an annealing temperature of 54 ℃ (FIG. 2).
Example 4
Optimal primer concentration screening
The primer concentrations were adjusted to 0.2. Mu. Mol/L, 0.4. Mu. Mol/L, 0.6. Mu. Mol/L, 0.8. Mu. Mol/L and 1.0. Mu. Mol/L, respectively, for the triple PCR amplification reactions, and the reaction system and reaction conditions were as described in "optimum annealing temperature screening". The results show that 3-item bands can be amplified when the primer concentration is 0.2-1.0 mu mol/L in the triple PCR reaction, but the accuracy of the reaction result can be ensured, the dilution times and the use times of the primers can be maximally increased when the primer concentration is 0.4 mu mol/L, and finally the detection cost is saved (figure 3).
Example 5
Sensitivity detection
The reaction conditions are explored and optimized, the established reaction system of the triple PCR method is finally determined to be 30 mu L, wherein 15 mu L of 2 xTaqPCRMastermix is added, 0.4 mu mol/L of each primer is 0.6 mu L, the template content is 1-3 mu L (3 mu L of tissue DNA detected by unknown pathogen and 1 mu L of each single bacterial genome DNA) and the rest is supplemented to 30 mu L by sterile DEPC water. The reaction procedure is 94 ℃ pre-denaturation for 5min; denaturation at 94℃for 30s, annealing at 54℃for 30s, extension at 72℃for 40s for 35 cycles; final extension at 72℃for 10min.
The established triple PCR detection method is utilized to detect sensitivity to the corynebacterium pseudotuberculosis, the stellera suppuration and the staphylococcus aureus, the cultured bacterial liquid is subjected to plate count and 10 times serial dilution, and the final concentration of the corynebacterium pseudotuberculosis bacterial liquid is 1.88 multiplied by 10 7 CFU/mL～1.88×10 1 CFU/mL, the concentration of the stellera suppurative bacterial liquid is 1.72X10 7 CFU/mL～1.72×10 1 CFU/mL, staphylococcus aureus bacterial liquid concentration is 2.55X10 7 CFU/mL～2.55×10 1 CFU/mL, extracting bacterial liquid DNA with different concentrations, performing triple PCR amplification, and evaluating the detection lower limit of the established triple PCR method on bacterial liquid concentration (fig. 4); meanwhile, the DNA of the 3 extracted bacteria is measured for concentration10-fold serial dilution, and finally determining the DNA concentration of the corynebacterium pseudotuberculosis to be 3.4x10 7 copies/mL～3.4×10 1 The concentration of the DNA of the cryptobacter pyogenes is 2.8X10 per mL 7 copies/mL～2.8×10 1 The concentration of staphylococcus aureus DNA is 2.0X10 per mL 7 copies/mL～2.0×10 1 Triple PCR was performed using extracted bacterial genomic DNA as a template, and the lower limit of detection of bacterial DNA content by the established triple PCR method was determined (FIG. 5).
The results show that the established triple PCR method has the minimum detection colony number and the minimum DNA detection concentration of 1.88×10 for the corynebacterium pseudotuberculosis 3 CFU/mL and 3.4X10 3 The lowest detection colony number and the lowest DNA detection concentration of the lepidobacter pyogenes are respectively 1.72X10 per mL 3 CFU/mL and 2.8X10 3 The lowest detection colony number and the lowest DNA detection concentration of staphylococcus aureus are respectively 2.55X10 per mL 2 CFU/mL and 2.0X10 2 copies/mL。
Example 6
Application of triple PCR detection method
The established triple PCR method is used for detecting the collected 50 sheep cheese lymphadenitis abscess tissues after tissue DNA is extracted, and meanwhile, bacteria separation culture and identification are carried out on each abscess, and the result shows that the detection result of the established triple PCR method is consistent with the bacteria separation identification result, and the established triple PCR method is reliable in accuracy and can be used for clinical and laboratory detection of sheep cheese lymphadenitis.
Meanwhile, 20 parts of single bacterial DNA, two bacterial mixed DNA and three bacterial mixed DNA of corynebacterium pseudotuberculosis, cryptobacter suppuration and staphylococcus aureus are used as templates, and the established triple PCR method is used for amplification respectively, and the result shows that the established triple PCR method can accurately detect single or mixed bacteria in corynebacterium pseudotuberculosis, cryptobacter suppuration and staphylococcus aureus, can be used for detecting and identifying the 3 bacteria in the sheep cheese lymph node, and can also be used for detecting the corynebacterium pseudotuberculosis, cryptobacter suppuration and staphylococcus aureus in other aspects.
The corynebacterium pseudotuberculosis, the cryptococcus suppurative and the staphylococcus aureus are 3 bacteria with highest separation rate in the tissue of the sheep cheese lymphadenitis-like abscess, and the invention can realize comprehensive detection of 3 bacteria from the joint detection angle of the 3 pathogenic bacteria.
The invention designs primer amplification by taking the specific virulence factors of 3 target bacteria as target genes, and can improve the specificity and accuracy of detection results.
In general, the detection of pathogenic bacteria in the cheesy lymphadenitis abscess requires a plurality of processes of bacterial separation culture and identification, and the growth speed of corynebacterium pseudotuberculosis, stellera suppurativa and staphylococcus aureus in the abscess tissue is slow, and the separation and purification period is long.
The corynebacterium pseudotuberculosis is most frequently infected by sheep in livestock, but staphylococcus aureus and stellera suppuration can be infected by various livestock such as cattle, pigs and part of wild animals, so the invention can be used for detecting the cheese lymphadenitis of sheep, and can also be used for detecting other livestock species of corynebacterium pseudotuberculosis, stellera suppuration and staphylococcus aureus, and has general application and popularization values.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
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Claims (4)
1. A triple PCR detection reagent or kit comprising: pld-F/R, plo-F/R and nuc-F/R, wherein the nucleotide sequence of pld-F is shown as SEQ ID No.1, and the nucleotide sequence of pld-R is shown as SEQ ID No. 2; the nucleotide sequence of plo-F is shown as SEQ ID No.3, and the nucleotide sequence of plo-R is shown as SEQ ID No. 4; the nuc-F nucleotide sequence is shown as SEQ ID No.5, and the nuc-R nucleotide sequence is shown as SEQ ID No. 6.
2. A method for detecting pld gene of corynebacterium pseudotuberculosis ATCC19410, plo gene of cryptobacter pyogenes ATCC19411, nuc gene of staphylococcus aureus ATCC25923 for the purpose of non-diagnostic treatment, characterized by using the reagent or the kit according to claim 1.
3. The method of claim 2, comprising an amplification system: 2 XTaqPCRMastermix 15. Mu.L, 0.4. Mu. Mol/L of each of the upstream and downstream primers, 3. Mu.L of template, and the remainder was made up to 30. Mu.L with sterile DEPC water.
4. A method according to claim 3, comprising an amplification procedure: pre-denaturation at 94℃for 5min; denaturation at 94℃for 30s, annealing at 54℃for 30s, extension at 72℃for 40s for 35 cycles; final extension at 72℃for 10min.
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