CN110656190A - Multiple PCR detection kit for common bacterial disease pathogens of cattle - Google Patents

Multiple PCR detection kit for common bacterial disease pathogens of cattle Download PDF

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CN110656190A
CN110656190A CN201911062733.7A CN201911062733A CN110656190A CN 110656190 A CN110656190 A CN 110656190A CN 201911062733 A CN201911062733 A CN 201911062733A CN 110656190 A CN110656190 A CN 110656190A
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abortus
brucella
chlamydia
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CN110656190B (en
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娄忠子
周继章
李兆才
刘星
谭书敏
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Lanzhou Veterinary Research Institute of CAAS
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Abstract

The invention designs three pairs of specific amplification primers for three pathogens based on genes of Brucella abortus, Mycobacterium bovis and Chlamydia abortus, establishes a multiplex PCR method for detecting and distinguishing single or mixed infection of the three common pathogens of cattle by matching PCR reaction components, and packages a detection kit for detecting and distinguishing Brucella abortus, Mycobacterium bovis and Chlamydia abortus.

Description

Multiple PCR detection kit for common bacterial disease pathogens of cattle
Technical Field
The invention relates to a kit for common cow bacterial pathogens, in particular to a detection kit for detecting Brucella, Mycobacterium bovis and Chlamydia abortus.
Background
Brucella abortus (B)Brucella abortus) Mycobacterium: (A), (B), (C)Mycobacterium bovis) And Chlamydia abortus: (Chlamydia abortus) Is the most common bacterial pathogen in cattle herd, among which brucella can cause abortion, orchitis, tenosynovitis and arthritis in cattle; mycobacterium bovis can cause nodular granuloma and caseous calcification of various tissues and organs such as lung, lymph node and colonThe necrotic focus of the cow is characterized in that the cow abortion chlamydia can cause abortion of cows, stillbirth and weak fetuses, and clinical symptoms such as endometritis, orchitis of bulls, enteritis of calves, conjunctivitis and the like appear. The three pathogens are also chronic zoonosis pathogens, can cause similar clinical symptoms and tissue and organ injuries in human bodies, are threats to human health and life safety, and cause immeasurable economic loss.
Brucella abortus, mycobacteria and Chlamydia abortus are all intracellular parasitic bacteria, the prevalence trend in the world is continuously rising in recent years, and once the disease is developed, the disease is difficult to be cured, so that the rapid and accurate detection of the disease pathogen is very critical. The diagnosis of bacterial pathogens is mainly based on methods such as bacteriology, serology and molecular biology, and various methods are currently applied to the detection of the pathogens. The bacteriological detection method mainly comprises the isolation culture and the growth biochemical identification of bacteria, is a gold standard of the bacteriological identification, is reliable, but has long required time, great danger and is inconvenient to popularize. The serological detection method comprises a plurality of test tube agglutination tests, enzyme-linked immunosorbent assays, colloidal gold reagent strips and the like, and the detection principle is antigen-antibody binding reaction. The serological detection has good specificity and sensitivity, the operation method is easy to be applied clinically, and the detection cross-reaction with other pathogenic bacteria infection occasionally occurs in the application process to present false positive results.
The molecular biology detection technology develops very rapidly due to the advantages of strong specificity, high sensitivity, high detection rate and the like. At present, common PCR, multiplex PCR, fluorescent quantitative PCR and the like are mainly used, wherein the multiplex PCR can detect various pathogens or genotypes due to the characteristic of one-time reaction, and the requirements of various aspects such as inspection and quarantine, epidemiological investigation, clinical diagnosis and the like are met. At present, a plurality of PCR detection methods aiming at the Brucella abortus, mycobacteria and Chlamydia abortus exist, including a multiple PCR method aiming at the simultaneous detection of a single genotype and a plurality of genotypes, such as a method for detecting the OMP 25kd gene segment of the Brucella abortus in milk disclosed in the Chinese invention patent 2005100079813, a PCR kit for simultaneously detecting four Brucella in cattle, sheep and pig and a preparation method thereof disclosed in the Chinese invention patent 2013103391873, a PCR rapid diagnosis kit for bovine tuberculosis disclosed in the Chinese invention patent 2011101103143 and a kit for detecting the Chlamydia abortus disclosed in the Chinese invention patent application 201810259741X.
The Brucella abortus, the mycobacteria and the Chlamydia abortus are all endoparasitic and outbreak in cattle groups which are easy to be bred in a centralized way, so the method which can simultaneously detect the Brucella abortus, the mycobacteria and the Chlamydia abortus has a very positive effect on the animal husbandry production. But is not disclosed at this stage.
Disclosure of Invention
The invention provides a kit which can solve the problems which are not solved by the prior art and can simultaneously detect three pathogens.
The multiplex PCR detection kit for detecting and distinguishing the common bovine bacterial pathogens comprises three pairs of specific primers which are respectively as follows: brucella abortus specific upstream and downstream primers SEQ ID No. 1 and SEQ ID No. 2, Mycobacterium bovis specific upstream and downstream primers SEQ ID No. 3 and SEQ ID No. 4, and Chlamydia abortus specific upstream and downstream primers SEQ ID No. 5 and SEQ ID No. 6.
Preferably, the multiplex PCR detection kit for detecting and distinguishing common bovine bacterial disease pathogens of the invention also comprises: 2 XTaq DNA polymerase Mix, 5 XPCR Buffer (containing 2.5 mmol/L dNTP, 25 mmol/L MgCl2) Sterile double distilled water.
The invention is based on the biological base of pathogenic molecules, based on the genes of Brucella abortus, Mycobacterium bovis and Chlamydia abortus, respectively designs specific amplification primers for three pathogens, and establishes a multiplex PCR method for detecting and distinguishing single or mixed infection of the three pathogens. The multiplex PCR technology established in the invention systematically designs three pairs of specific detection primers aiming at three common bovine infectious pathogens of brucella bovis, mycobacterium bovis and chlamydia abortus for the single and composite detection of the three pathogens, thereby initiating the detection method of the three pathogens. The multiplex PCR technology has all the advantages of common PCR technology, including high specificity of detection result, sensitive detection reaction, fast detection, high flux detection, etc., and also has the advantages of high efficiency, systematicness, economy, simplicity, etc. of multiplex PCR, and is suitable for clinical and laboratory to perform fast and accurate etiology identification.
The invention relates to a kit for typing detection of Brucella, Mycobacterium bovis and Chlamydia abortus infection by using a multiple PCR method, wherein three pairs of specific primers in the kit are used for carrying out PCR amplification on a detected sample, then the amplified product is subjected to electrophoresis, whether the detected sample is infected is determined according to whether a specific strip appears in an electrophoretogram, and the specific type and the number of infection pathogens are judged according to the number of amplified strips. The kit can be used for single infection or multiple infection detection in any material containing pathogenic genomic DNA. The operation process related to the invention comprises the steps of extracting whole genome DNA from suspected brucella bovis, mycobacterium bovis and chlamydia abortus infected tissues, secretions and excretions, preparing a multiple PCR reaction system containing three pairs of primers and a whole genome template, carrying out agarose gel electrophoresis after PCR reaction to observe an amplification result, and judging the type and the quantity of an infection pathogen according to the size and the number of target bands. The three pairs of specific PCR detection primers of the invention are used, bovine tissues and other two bacterial genome DNAs with inconsistent sources with the primers are used as templates for specific verification amplification, and PCR amplification products are not found, which indicates that the three pairs of specific PCR detection primers have high specificity. In the multiplex PCR system, the number of plasmid DNA templates is 102In this case, the visible band was also amplified, indicating that the method is highly sensitive. The three pairs of primers are simultaneously used in a PCR reaction system without mutual interference, which shows that the primer has the advantages of high efficiency, systematicness, economy, simplicity and the like, and is suitable for clinical and laboratory rapid and accurate etiology identification.
The multiplex PCR method for simultaneously detecting three pathogens has all the advantages of common PCR, can distinguish and detect single pathogen or mixed infection of a plurality of pathogens in one reaction, and can avoid the problems of complicated detection process and material waste when in detection. The invention fills the technical blank that three common bacterial pathogens of the cattle are simultaneously detected by one-time PCR reaction which is not appeared at present, and has good clinical application prospect.
Drawings
FIG. 1 is a PCR result electrophoresis diagram of the single specificity detection primers of Brucella abortus, Mycobacterium bovis and Chlamydia abortus of the present invention respectively amplifying the genomic DNA templates of Brucella abortus, Mycobacterium bovis and Chlamydia abortus of milk cows, wherein: 1 and 2 lanes are PCR result electrophoretograms of specific primers SEQ ID No. 1 and 2 amplified brucella abortus genome DNA template of dairy cow and negative control respectively; 3, 4 lanes of PCR result electrophoretogram of specific primers SEQ ID No. 3 and 4 amplified Mycobacterium bovis genome DNA template and negative control respectively; 5, 6 lanes are PCR result electrophoretograms of specific primers SEQ ID No. 5 and 6 amplified cow chlamydia abortus genome DNA template and negative control respectively; m is DNA molecular weight standard DL 2000.
FIG. 2 shows the PCR results of the primers for single specific detection of Brucella abortus, Mycobacterium bovis and Chlamydia abortus of the present invention amplified with the genomic DNA templates of Brucella abortus, Mycobacterium bovis and Chlamydia abortus of milk cows, respectively, wherein: lanes 1, 2 and 3 are electrophoresis charts of PCR amplification results of specific primers SEQ ID No. 1 and 2 when genomic DNA templates of mycobacterium bovis, Brucella abortus and Chlamydia abortus of cows are used; 4, 5 and 6 are electrophoresis charts of PCR amplification results of specific primers SEQID No. 3 and 4 respectively when genomic DNAs of milk cow chlamydia abortus, milk cow brucella abortus and mycobacterium bovis are taken as templates; 7, 8 and 9 are electrophoresis images of PCR amplification results of specific primers SEQ ID No. 5 and 6 respectively, when genomic DNA of Brucella abortus, Mycobacterium bovis and Chlamydia abortus of cow is used as a template; m is DNA molecular weight standard DL 2000.
FIG. 3 shows the result of multiple PCR amplification of specific primers for Brucella abortus, Mycobacterium bovis and Chlamydia abortus of milk cow using a single DNA template for Brucella abortus, Mycobacterium bovis and Chlamydia abortus of milk cow, wherein: 1, 2 and 3 are specific primers SEQ ID No. 1 and 2, and the multiple PCR result electrophoresis patterns when the DNA templates of the Brucella abortus, the Mycobacterium bovis and the Chlamydia abortus of the dairy cows are respectively amplified by the SEQ ID No. 3, 4 and the SEQ ID No. 5 and 6; lane 4 is the negative control when the specific primers SEQ ID No. 1 and 2, SEQ ID No. 3 and 4 and SEQ ID No. 5 and 6 are amplified; m is DNA molecular weight standard DL 2000.
FIG. 4 shows the result of multiple PCR amplification of specific primers for Brucella abortus, Mycobacterium bovis and Chlamydia abortus in milk cow with a DNA composite template for Brucella abortus, Mycobacterium bovis and Chlamydia abortus, wherein: 1 is a multiplex PCR amplification result when a mixed primer of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 is used for amplifying a mixed template of genome DNA of Brucella abortus, Mycobacterium bovis and Chlamydia abortus of a cow; 2 is a multiplex PCR amplification result when a mixed primer of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 is used for amplifying a mixed template of brucella abortus and mycobacterium bovis of dairy cows; 3 is a multiplex PCR amplification result when a mixed primer of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 is used for amplifying a mixed template of the genome DNA of the Brucella abortus and the Chlamydia abortus of the cow; 4 is a multiplex PCR amplification result when a mixed primer of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 is used for amplifying a mixed template of the genomic DNAs of the mycobacterium bovis and the chlamydia abortus of the dairy cow; 5 is a multiplex PCR amplification result when a mixed primer of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 is used for amplifying a milk cow tissue genome DNA template; 6 is the negative control multiple PCR amplification result of the mixed primers of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6; m is DNA molecular weight standard DL 2000.
FIG. 5 shows the results of the primers for specific detection of Brucella abortus, Mycobacterium bovis and Chlamydia abortus of the present invention when performing multiplex PCR amplification using cow dung whole genome DNA suspected to have three pathogen infections clinically as a template, wherein: 1, 2,3, 4, 5, 6, 7 and 8 are multiplex PCR amplification results when the mixed primers of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 are used for amplifying the cow dung whole genome DNA template; m is DNA molecular weight standard DL 2000.
Detailed Description
In this example, cow feces were selected for brucella, mycobacteria, and chlamydia abortus infection detection. Taking a proper amount of cow dung, inactivating bacteria at 70 ℃ for 30 minutes, extracting whole genome DNA by using a dung DNA extraction kit, and measuring the concentration of the extracted DNA for later use. Preparing multiple PCR reaction liquid in a PCR reaction tube, adopting a 25 mu L reaction system, containing 5 multiplied by PCRBuffer 5 mu L, 2 multiplied by Taq DNA polymerase Mix 12.5 mu L, the cow dung whole genome DNA template amount is 100 ng, and an upstream primer F1 used for Brucella specificity detection: 5'-CACTCGTGCTAGCAATTTTCTCGC-3' (SEQ ID No. 1). 0.5. mu.L (10. mu. mol/L) and the downstream primer R1: 5'-CGACATTGACCGATACGTTATAGC-3' (SEQ ID No. 2) 0.5 μ L (10 μmol/L), upstream primer F2: 5'-GGCCTGGGCGGCACTGTG-3' (SEQ ID No. 3) 0.5 μ L (10 μmol/L) and a downstream primer R2: 5'-CAGACTGTCGTCGGTGATAGC-3' (SEQ ID No. 4) 0.5 μ L (10 μmol/L), upstream primer F3 for Chlamydia abortus specific detection: 5'-GGGCTAGACACGTGAAACCTAG-3' (SEQ ID No. 5) 0.5 μ L (10 μmol/L) and a downstream primer R3: 5'-CCTGGTCATAAATAGCTCAC-3' (SEQ ID No. 6) 0.5 μ L (10 μmol/L), and double distilled water make-up to 25 μ L. Completing the PCR reaction process in a thermal cycler, wherein the reaction procedure is as follows: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30S, annealing at 55 ℃ for 90S, extension at 72 ℃ for 40S, 35 cycles, extension at 72 ℃ for 10 min, and storage at 4 ℃ after the reaction is finished. And (3) carrying out agarose gel electrophoresis on the PCR reaction product, observing the amplification result by a gel imaging system, judging and analyzing the result, wherein the sizes of specific amplification bands of the Brucella abortus, the Mycobacterium bovis and the Chlamydia abortus are 728 bp, 504 bp and 345 bp respectively, and carrying out nucleic acid sequencing analysis on the connection cloning vector after the target band gel is recovered to show that the three target bands belong to the Brucella abortus, the Mycobacterium bovis and the Chlamydia abortus.
The specific detection process of the invention is as follows:
1 cow dung procurement
Collecting the cow dung suspected to be infected by the Brucella abortus, the Mycobacterium bovis and the Chlamydia abortus.
2 extraction of cow dung whole genome DNA
1) The feces obtained were heated at 70 ℃ for 30 minutes.
2) Preparation of fecal DNA: and (4) extracting the excrement DNA according to the instruction of the excrement DNA extraction kit.
3 establishment of PCR amplification System
A multiplex PCR reaction solution is prepared in a PCR reaction tube, a 25-microliter reaction system is adopted, 5 microliter of 5 XPCR Buffer and 12.5 microliter of 2 XTaq DNA polymerase Mix are added, 0.5 microliter (10 micromole/liter) of each of primers Br No. 1, Br No. 2, My No. 1, My No. 2, Ch No. 1 and Ch No. 2 is added, the total genome DNA template amount of cow feces is 100 ng, and 25 microliter is supplemented with water.
4 PCR amplification conditions
The PCR reaction program is: pre-denaturation at 95 ℃ for 5 min, denaturation at 95 ℃ for 30S, annealing at 55 ℃ for 90S, extension at 72 ℃ for 40S, 35 cycles, extension at 72 ℃ for 10 min, and storage at 4 ℃ after the reaction is finished.
5 PCR amplification result observation and judgment
The PCR reaction product was electrophoresed in 1.5% (w/v) agarose gel, and the amplification result was observed by UV gel imaging system.
PCR amplification results and conclusions
1. Referring to the attached figure 1, the single detection primers of the invention are respectively used for amplifying the DNA templates of the milk cow brucella abortus, the bovine mycobacterium and the milk cow chlamydia abortus genomes, clear target bands with the sizes of 728 bp, 504 bp and 345 bp respectively appear in lanes 1, 3 and 5 in an electrophoresis chart of a PCR result, and amplification products of a negative control do not exist, so that the primers designed according to various pathogen genomes can efficiently carry out PCR amplification on target genes, and a blank control without the DNA templates does not have bands.
2. Referring to the attached figure 2, the single detection primers of the invention are amplified by using mycobacterium bovis, chlamydia abortus and brucella abortus genomic DNA templates, clear target bands with the sizes of 504 bp, 345 bp and 728 bp appear in lanes 1, 4 and 7 respectively in an electrophoresis chart of a PCR result, and PCR amplification products do not appear when the same primer uses genomes of other two pathogens as templates, which shows that the three pairs of primers have high specificity and do not perform PCR amplification on the DNA templates of other two pathogens.
3. Referring to the attached figure 3, the mixed detection primers of the invention are used for amplifying the genome DNA templates of the brucella abortus of the dairy cows, the mycobacterium bovis and the chlamydia abortus of the dairy cows respectively, clear and single-purpose bands with the sizes of 728 bp, 504 bp and 345 bp appear in lanes 1, 2 and 3 respectively in an electrophoresis chart of a PCR result, and no band exists in a lane 4 of a negative control, which shows that the single DNA templates of three pathogens can be efficiently amplified in a multiplex PCR system of the composite primers, non-specific amplification bands can not appear due to the composite primers, and no amplification product appears in the negative control without the template.
4. Referring to the attached figure 4, the mixed detection primer is used for amplifying three or two mixed DNA templates of the milk cow brucella abortus, the bovine mycobacterium and the milk cow chlamydia abortus genomes and a bovine tissue DNA template, clear target bands with the sizes of 728 bp, 504 bp and 345 bp appear in a lane 1 in a PCR result electrophoresis chart, and the fact that the DNA templates of three pathogens are efficiently amplified and non-specific amplification does not appear in a multiple PCR system is shown; target bands with the sizes of 728 bp and 504 bp appear in a lane 2, which shows that the genes of the dairy cow brucella abortus and the bovine mycobacterium in the multiple PCR system are efficiently amplified; target bands with the sizes of 728 bp and 345 bp appear in a lane 3, which shows that the genes of the dairy cow brucella abortus and the dairy cow chlamydia abortus in the multiple PCR system are efficiently amplified; target bands with the size of 504 bp and 345 bp appear in a lane 4, which shows that the genes of mycobacterium bovis and cow chlamydia abortus in the multiplex PCR system are efficiently amplified; no amplification products are present in lane 5, indicating that there is no PCR amplification using bovine tissue DNA as a template.
5. Referring to the attached figure 5, the mixed detection primer of the invention is used for amplifying by taking the cow dung whole genome DNA containing Brucella, Mycobacterium bovis or Chlamydia abortus as a template, clear target bands with sizes of 728 bp and 345 bp appear in lanes 1, 2 and 5 in an electrophoresis chart of a PCR result, and the Brucella and Chlamydia abortus exist in cow dung; amplified bands with sizes of 728 bp, 504 bp and 345 bp appear in a lane 3, which indicates that brucella, mycobacterium bovis and chlamydia abortus exist in cow dung; an amplified band with a size of 345 bp appears in lanes 4, 6 and 8, indicating the presence of Chlamydia abortus in cow dung; amplified bands of 504 bp and 345 bp appear in lane 7, indicating the presence of M.bovis and Chlamydia abortus in cow dung.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> multiplex PCR detection kit for common bacterial pathogens of cattle
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
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<212> DNA
<213> upstream primer for specific detection of Brucella (Brucella abortus 26F)
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<212> DNA
<213> downstream primer for specific detection of Brucella (Brucella abortus 26R)
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cgacattgac cgatacgtta tagc 24
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<213> upstream primer for specific detection of Mycobacterium bovis (Mycobacterium bovis 36F) Artificial sequence
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ggcctgggcg gcactgtg 18
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<212> DNA
<213> downstream primer for specific detection of Mycobacterium bovis (Mycobacterium bovis 36R) of Artificial sequence
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cagactgtcg tcggtgatag c 21
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<213> upstream primer for specific detection of induced sequence Chlamydia abortus (Chlamydia abortus 23F)
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<213> downstream primer for specific detection of induced sequence Chlamydia abortus (Chlamydia abortus 23R)
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Claims (2)

1. A multiple PCR kit for detecting and distinguishing common bovine bacterial pathogens is characterized in that the kit comprises three pairs of specific primers which are respectively as follows: brucella abortus specific upstream and downstream primers SEQ ID No. 1 and SEQ ID No. 2, Mycobacterium bovis specific upstream and downstream primers SEQ ID No. 3 and SEQ ID No. 4, and Chlamydia abortus specific upstream and downstream primers SEQ ID No. 5 and SEQ ID No. 6.
2. The multiplex PCR kit for common bacterial pathogens of cattle according to claim 1, wherein the kit further comprises: 2 XTaq DNA polymerase Mix, 5 XPCR Buffer (containing 2.5 mmol/L dNTP, 25 mmol/L MgCl2) Sterile double distilled water.
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