CN111500754A - Primer and kit for identifying four Brucella in dog and application of primer and kit - Google Patents

Primer and kit for identifying four Brucella in dog and application of primer and kit Download PDF

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CN111500754A
CN111500754A CN202010473544.5A CN202010473544A CN111500754A CN 111500754 A CN111500754 A CN 111500754A CN 202010473544 A CN202010473544 A CN 202010473544A CN 111500754 A CN111500754 A CN 111500754A
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brucella
primer
sample
pcr reaction
kit
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徐怀英
秦卓明
黄迪海
刘星丽
仉伟
郝文娟
盛晓丹
郭卉
刘霞
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Shandong Province Jianmu Biopharmaceuticals Co ltd
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • C12Q1/686—Polymerase chain reaction [PCR]

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Abstract

The invention belongs to the technical field of molecular biology, and relates to a single PCR detection primer, a single PCR detection kit and a single PCR detection method for four kinds of brucella capable of infecting dogs (Brucella melitensis, Brucella nivora, Brucella suis and Brucella canis). Only 1 pair of primers can realize the respective amplification of 352bp and 709bp of rough brucella (357 bp deletion of Brucella canis) and smooth Brucella (bovine species, ovine species and porcine species), and can carry out effective identification; and the primer can effectively avoid generating primer dimer and hairpin structure, ensure the efficient amplification of PCR and enhance the specificity and sensitivity of the detection technology.

Description

Primer and kit for identifying four Brucella in dog and application of primer and kit
Technical Field
The invention belongs to the technical field of molecular biology, and relates to a single PCR detection primer, a single PCR detection kit and a single PCR detection method for four kinds of brucella capable of infecting dogs (Brucella melitensis, Brucella nivora, Brucella suis and Brucella canis).
Background
In recent years, the hidden danger of pet source zoonosis caused by the rapid increase of the feeding quantity of pet dogs in China has been increased day by day. Strengthening the prevention consciousness of the zoonosis of the pet source, adopting effective measures to control the occurrence of the epidemic diseases and having very important practical significance for guaranteeing the public health safety. Brucellosis is a zoonosis, is caused by invasion of brucella bacteria into organisms, has the clinical morbidity characteristics of long-term fever, hyperhidrosis, arthralgia, hepatosplenomegaly and the like, can cause reproductive system diseases of human and animals, and has great public health significance. The brucella is an intracellular parasite, so that even sensitive drugs are difficult to enter cells to play a therapeutic role, and once infection occurs, the infection is difficult to cure. The 4 Brucella species include Brucella melitensis, Brucella bovis, Brucella suis, and Brucella canicola. The brucellosis in dogs caused by brucella of dogs is called the most parasitic type, and the brucellosis in dogs caused by other 3 kinds of brucella is called the metastatic type. The Brucella canicola growth colonies are rough, while the Brucella bovis, sheep and pig Brucella growth colonies are smooth. In a pastoral area, as dogs are mostly raised scattered, the phenomenon of living, eating, sickness and death of poultry and livestock generally exists, and the positive rate of the brucella antibody in the serum of the dogs can reach 41 percent; the positive rate of the smooth antibody in a pet dog in a city in 2016-2017 is 8.76%, and the positive rate of the rough antibody is 1.99%, which indicates that the brucella infection rate in the dog is high and the potential transmission risk is high.
The clinical manifestations of brucellosis in dogs are various, but the common symptoms are abortion and sterility of female dogs and orchitis, epididymitis and scrotitis of male dogs. The brucellosis of the dogs has another important characteristic that the bacteremia period is long, and some canis bacteremia periods can last for 1-2 years. Bacteria take approximately 3 weeks from infection to colonisation in reproductive tissues, then start to multiply and excrete continuously, which can last for months to years, and once a brucella infection is diagnosed, typically a killing measure is taken. Brucella isolated from blood or aborted fetuses, vaginal secretions, and other materials is the "gold standard" for diagnosis, but because of the infectivity of humans by this pathogen, isolation in laboratories with higher biosafety is generally recommended. Polymerase Chain Reaction (PCR) is a rapid, good specificity and high sensitivity detection method, so that a primer is established, different types of brucella infected with dogs can be identified and distinguished at the same time, and the method is suitable for basic detection mechanisms and scientific research units with common PCR instruments. And the cost of the single PCR reaction reagent can be reduced by more than 1/3 of the cost of the reagent for detecting different types of Brucella by adopting the multiple PCR reaction, the nonspecific problem of the multiple PCR reaction is reduced, and the method is more economical.
Disclosure of Invention
The invention aims to provide a primer, a kit and a method which can be used for simultaneously diagnosing four types of brucella capable of infecting dogs (such as canine, bovine, ovine and porcine species) and identifying smooth type brucella (such as bovine, ovine and porcine species) and rough type brucella of dogs. The primer combination has high specificity and stability, and can effectively amplify four target fragments of brucella infecting dogs in a PCR reaction system of 1 group of primers simultaneously, thereby achieving the capability of detecting the four bacteria simultaneously in the same PCR reaction system, and leading the detection process to be simpler, more convenient, time-saving, labor-saving and economical; only 1 pair of primers can realize the respective amplification of 352bp and 709bp of rough brucella (357 bp deletion of Brucella canis) and smooth Brucella (bovine species, ovine species and porcine species), and can carry out effective identification; and the primer can effectively avoid generating primer dimer and hairpin structure, ensure the efficient amplification of PCR and enhance the specificity and sensitivity of the detection technology.
A primer for identifying four Brucella in a dog is characterized in that:
primer F sequence SEQ ID NO. 1: ttattacgga cggctggctg ag 22
Primer M sequence SEQ ID NO. 2: ggtggcccat gtggcgcttt 20
The method for detecting the Brucella by using the primers comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) establishing a PCR reaction system, and carrying out PCR reaction by using a primer M and a primer F;
(3) detecting the PCR reaction product through 1.5% agarose gel electrophoresis, and when a 352bp band is amplified in a sample, indicating that the sample contains rough brucella, namely the brucella canicola; when 709bp of the sample is amplified, the sample contains smooth Brucella, namely Brucella melitensis of sheep species, cattle species or pig species.
The PCR reaction system in the step (2) is 2 × PCR Mix, a primer F and a primer M, a sample DNA template to be detected and DEPC water without RNA enzyme.
The PCR reaction conditions in the step (2) are as follows: pre-denaturation at 95 ℃ for 4 min; performing denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 40s, and extending at 72 ℃ for 40s for 35 cycles; extension was carried out at 72 ℃ for 10min and the reaction was stopped at 4 ℃.
The kit prepared by the detection method.
The kit is applied to detecting brucella canis.
Drawings
FIG. 1 shows PCR primer sites and Brucella canis deletion positions; 1430-1460 with quoted region represents primer F binding site, 2120-2150 with shaded region represents primer R binding site, gray region represents Brucella canis deletion 357 bp;
FIG. 2 shows the specificity test of four Brucella PCR methods; wherein M: DNA Marker; 2: brucella melitensis; 3: brucella melitensis; 4: brucella in swine; 5: brucella canicola;
FIG. 3 shows the sensitivity test of four Brucella PCR methods; wherein M: DNA Marker; the four bands are, in order from left to right: brucella melitensis is a strain of bovine, ovine, porcine and canine species.
The invention has the advantages of
Strong detection specificity and high sensitivity
Specific primers aiming at gene segments of Brucella canis, bovines, ovine and porcine Brucella on the 3' end of wbkF and wbkD genes are designed, and the PCR method capable of detecting Brucella canis is finally established by optimizing the concentration of the primers and the annealing temperature. The method has the advantages of strong specificity, high sensitivity, rapidness, convenience and the like.
The detection cost is low
The primer combination has high specificity and stability, and can effectively amplify four target fragments of brucella infecting dogs in a PCR reaction system of 1 group of primers simultaneously, thereby achieving the capability of detecting the four bacteria simultaneously in the same PCR reaction system, and leading the detection process to be simpler, more convenient, time-saving, labor-saving and economical; is suitable for being applied and popularized in basic units and scientific research institutions, and provides technical support for epidemiological investigation and prevention and control of brucellosis pathogens in dogs.
Detailed Description
Example 1
Establishing and optimizing a PCR reaction system and performing specificity test:
1. the strain is as follows: the inactivated bacterial liquid of the Brucella melitensis, Brucella suis and Brucella canicola is presented by the China institute of veterinary medicine supervision, and the Escherichia coli, Salmonella and Listeria are preserved by the poultry research institute of the Shandong academy of agricultural sciences.
2. Reagents D L1000 DNA Marker 1000bp, 2 × PCR Mix were purchased from Takara Shuzo (Beijing) Co., Ltd., and other reagents were purchased from commercial Co., Ltd.
3. Extraction of bacterial genome: extracting the genomic DNA of the bacterial strain according to the instructions of the TIANGEN bacterial genome extraction kit, and storing at-20 ℃ for later use.
4. Establishing a 25 mu L reaction system:
1) annealing temperature 2 × PCR Mix 12.5 mu L, DNA template 2 mu L to be detected, 0.5 mu L of each of primer F and primer R (25 mmol/L), complementing to 25 mu L with RNA-free, pre-denaturing at 95 ℃ for 4min, denaturing at 94 ℃ for 30s, annealing at 7 annealing temperatures (50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃ and 56 ℃) for 40s, extending at 72 ℃ for 40s, performing 35 cycles, extending at 72 ℃ for 10min, and finishing the reaction at 4 ℃.
2) The concentration of the primers is 2 × PCR Mix 12.5 mu L, the DNA template to be detected is 2 mu L, the primers F and the primers R (25 mmol/L) are respectively added into 0.2-1 mu L, 5 test gradients are set in total, each gradient is increased by 0.2 mu L, finally, RNA-free is used for complementing to 25 mu L, meanwhile, the concentration of annealing temperature and reaction time are optimized according to the effect of concentration test, and finally the final reaction system and conditions of the method are determined.
Finally, the optimal system of the PCR reaction is determined as 2 × PCR Mix 12.5 mu L, the DNA template to be detected is 2 mu L, the adding amount of each primer F and R (25 mmol/L) is 0.6 mu L, the RNA-free is used for complementing to 25 mu L, the optimal reaction conditions are pre-denaturation at 95 ℃ for 4min, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 40s, extension at 72 ℃ for 40s, 35 cycles are carried out, extension at 72 ℃ for 10min and reaction at 4 ℃ is ended.
Example 2
Verification of specificity of established methods
The PCR reaction product is subjected to agarose gel electrophoresis, the electrophoresis picture is shown in figure 2, and each lane in figure 2 is respectively M, D L1000 DNAmarker, 1: negative control (a template is DEPC), 2: bovine Brucella, 3: ovine Brucella, 4: porcine Brucella, 5: canine Brucella, 6: Salmonella and 7: Escherichia coli.
As can be seen from FIG. 2, the established Brucella canis PCR method is used for detecting a specific band of 352bp from a Brucella canis sample; 709bp of specific bands are detected for the Brucella melitensis, Brucella melitensis and Brucella suis; and no specific band is amplified for escherichia coli, salmonella and negative control, and the result shows that the method has good specificity.
Example 3
Sensitivity test
1. Four genomic DNA (canine, ovine, bovine and porcine) samples were subjected to content and purity determination using a NanoDrop. One/OneC ultramicro UV spectrophotometer, respectively, and then diluted 10-fold into six gradients of 10.7 ng/. mu. L, 1.07 ng/. mu. L, 107 pg/. mu. L, 10.7 pg/. mu. L, 1.07 pg/. mu. L, and 107 fg/. mu. L, respectively.
2. The sensitivity of the detection method was verified by PCR amplification using 2. mu. L different amounts of template with diagnostic primers F and R, and the PCR reaction products were subjected to agarose electrophoresis, the electrophoretogram being shown in FIG. 3.
In FIG. 3, each lane is respectively M. DNA D L Marker, 1-6 corresponding to Brucella gene concentrations of 1, 50.7 ng/mu L, 2, 5.07 ng/mu L, 3, 507 pg/mu L, 4, 50.7 pg/mu L, 5, 5.07 pg/mu L, 6, 507 fg/mu L, 4.7-12 corresponding to Brucella gene concentrations of 7, 62 ng/mu L, 8, 6.2 ng/mu L, 9, 620 pg/mu L, 10, 62/mu L, 11, 6.2 pg/mu L, 12, 620 pg/mu L, 13-18 corresponding to Brucella gene concentrations of 13, 55 ng/mu L, 14, 5.5 ng/mu L, 15 pg/mu L, 620 pg/mu L, 13-18 corresponding to Brucella gene concentrations of 867, 27, 3.
As can be seen from FIG. 3, the method has obvious specific amplification bands on genomic DNA samples with Brucella canicola genomic DNA concentration of 50.7 pg/mu L-50.7 ng/mu L, the canine DNA fragment size is 352bp, no amplification band on genomic DNA samples with concentration of 5.07 pg/mu L or less, the method has obvious specific amplification bands on genomic DNA samples with Brucella ovis genomic DNA concentration of 62 pg/mu L-62 ng/mu L, the sheep DNA fragment size is 709bp, no amplification band on genomic DNA samples with concentration of 6.2 pg/mu L or less, the method has obvious specific amplification bands on genomic DNA samples with Brucella bovis pgg genomic DNA concentration of 55 pg/mu L-55 ng/mu L, the method has obvious specific amplification bands on genomic DNA samples with concentration of 709bp, the genomic DNA amplification bands on Brucella bovis pgg/mu L-55 ng/mu L or less than 5 pg/mu 4678, and no specific amplification bands on genomic DNA sample with concentration of pig genome DNA concentration of 5 pg/mu 468. 10. mu. and no amplification bands on Brucella porcine genomic DNA sample with concentration of 5. wt.% or less than 35. 387.
Therefore, the DNA sample with the Brucella genome DNA concentration of more than 50.7 pg/mu L can be detected at the lowest energy by the method, and the detection sensitivity of the method is high.
Example 4
Clinical sample testing
1. Detecting a sample: bacterial genomic DNA was extracted from 502 pet dog blood samples from a city.
2. PCR reaction system and reaction conditions:
the reaction system is 2 × PCR Mix 12.5 mu L, the DNA template to be detected 2 mu L, the adding amount of each primer F and R (25 mmol/L) is 0.6 mu L, and the mixture is made up to 25 mu L by RNA-free.
The reaction conditions are as follows: 5 min at 95 ℃; the temperature is 94 ℃ for 30s, 52 ℃ for 45s and 72 ℃ for 50s, and 35 cycles are carried out; the reaction was terminated at 72 ℃ for 10min and 4 ℃.
3. The PCR reaction products are detected by agarose gel electrophoresis, and the detection result shows that 2 Brucella canicola in 502 pet dogs are positive, and the others are negative, which indicates that the PCR method can be used for clinical examination.
4. The detection result is positive by 2 parts of Bruce-L adder in GB/T18646 animal brucellosis diagnosis technology, and 500 parts of negative (not infected with any brucella) by using the detection method of 4.13 parts of Bruce-L adder, which is consistent with the detection result of the invention.
Sequence listing
<110> Jianmu biological pharmaceutical Co., Ltd, Shandong province
<120> primer and kit for identifying four Brucella in dog and application thereof
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>22
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>1
ttattacgga cggctggctg ag 22
<210>2
<211>20
<212>DNA
<213> Artificial sequence (artiartiartifical sequence)
<400>2
ggtggcccat gtggcgcttt 20

Claims (6)

1. A primer for identifying four Brucella in a dog is characterized by comprising the following components:
primer F sequence SEQ ID NO. 1: ttattacgga cggctggctg ag 22
Primer M sequence SEQ ID NO. 2: ggtggcccat gtggcgcttt 20, respectively.
2. The method for detecting Brucella by using the primer as claimed in claim 1, which comprises the following steps:
(1) extracting DNA of a sample to be detected;
(2) establishing a PCR reaction system, and carrying out PCR reaction by using a primer M and a primer F;
(3) detecting the PCR reaction product through 1.5% agarose gel electrophoresis, and when a 352bp band is amplified in a sample, indicating that the sample contains rough brucella, namely the brucella canicola; when 709bp of the sample is amplified, the sample contains smooth Brucella, namely Brucella melitensis of sheep species, cattle species or pig species.
3. The detection method according to claim 3, wherein the PCR reaction system in step (2) is 2 × PCR Mix, primer F and primer M, DNA template of the sample to be detected, and DEPC water without RNase.
4. The detection method according to claim 3, wherein the PCR reaction conditions in the step (2) are as follows: pre-denaturation at 95 ℃ for 4 min; performing denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 40s, and extending at 72 ℃ for 40s for 35 cycles; extension was carried out at 72 ℃ for 10min and the reaction was stopped at 4 ℃.
5. A kit prepared by the detection method of claim 2.
6. Use of the kit of claim 5 for detecting Brucella canis.
CN202010473544.5A 2020-05-29 2020-05-29 Primer and kit for identifying four Brucella in dog and application of primer and kit Pending CN111500754A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113528685A (en) * 2021-08-09 2021-10-22 沈阳农业大学 PCR primer for distinguishing Brucella canis from other Brucella and detection method
CN116218707A (en) * 2022-12-14 2023-06-06 北京科牧丰生物制药有限公司 Rough sheep brucella attenuated strain, vaccine and application thereof

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CN103409520A (en) * 2013-08-06 2013-11-27 中国人民解放军军事医学科学院军事兽医研究所 PCR kit for simultaneously detecting Brucella abortus, Brucella melitensis, Brucella suis and Brucella canis as well as preparation method and using method thereof

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113528685A (en) * 2021-08-09 2021-10-22 沈阳农业大学 PCR primer for distinguishing Brucella canis from other Brucella and detection method
CN116218707A (en) * 2022-12-14 2023-06-06 北京科牧丰生物制药有限公司 Rough sheep brucella attenuated strain, vaccine and application thereof
CN116218707B (en) * 2022-12-14 2023-10-31 北京科牧丰生物制药有限公司 Rough sheep brucella attenuated strain, vaccine and application thereof

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