CN110760601A - Primer group and kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens and application of primer group and kit - Google Patents
Primer group and kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens and application of primer group and kit Download PDFInfo
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Abstract
The invention provides a primer group, a kit and an application for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens, belonging to the technical field of molecular biology detection, wherein the primer group comprises Br No1, Br No2, Ch No1, Ch No2, Cl No1 and Cl No 2; the nucleotide sequences of the Br No1, the Br No2, the Ch No1, the Ch No2, the Cl No1 and the Cl No2 are shown as SEQ ID NO: 1 to SEQ ID NO: and 6. The primer group and the kit have the advantages of strong specificity and high sensitivity.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a primer group, a kit and application for simultaneously detecting brucella, Chlamydia abortus and Clostridium perfringens.
Background
Brucella melitensis (Brucella melitensis), Chlamydia abortus (Chlamydia abortus) and clostridium perfringens (clostridium perfringens) are common bacterial pathogens in sheep flocks and are very popular. Brucella can cause mastitis, bronchitis, bursitis, arthritis and orchitis in sheep; abortion chlamydia can cause local abortion in sheep, manifested as abortion, stillbirth and weak lambs, and can also cause endometritis, conjunctivitis and arthritis; clostridium perfringens can cause acute diseases such as sheep plague, sudden gangrene of sheep, enterotoxemia of sheep, lamb dysentery and the like, can cause acute death of sheep, and bacteria widely exist in sheep flocks, thereby bringing great threat to the production of sheep industry. Brucella, Chlamydia abortus and Clostridium perfringens are zoonotic pathogens and pose risks to human health, and Clostridium perfringens can produce 12 toxins and has potential risks to public health safety.
Brucella and Chlamydia abortus are 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 cure; clostridium perfringens produces lethal toxins which can cause animals to die in a short time, so that rapid and accurate diagnosis of disease pathogens is crucial.
At present, a plurality of PCR detection methods aiming at Brucella, Chlamydia abortus and Clostridium perfringens exist, including a multiple PCR method aiming at simultaneous detection of a single genotype and a plurality of genotypes, but a multiple PCR system for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens is not reported.
Disclosure of Invention
The invention aims to provide a primer group, a kit and an application for simultaneously detecting brucella, Chlamydia abortus and Clostridium perfringens, wherein the primer group and the kit can simultaneously detect the brucella, the Chlamydia abortus and the Clostridium perfringens and have the advantages of high detection sensitivity and strong specificity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a primer group for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens, which comprises BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No 2;
the nucleotide sequence of BrNo1 is shown in SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of BrNo2 is shown in SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of ChNo1 is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of ChNo2 is shown as SEQ ID NO: 4 is shown in the specification;
the nucleotide sequence of Cl No1 is shown in SEQ ID NO: 5 is shown in the specification;
the nucleotide sequence of Cl No2 is shown in SEQ ID NO: and 6.
The invention provides a kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens, which comprises the primer group in the scheme.
Preferably, the kit further comprises 2 × Taq DNA polymerase Mix, 5 × PCR Buffer and sterile double distilled water; the 5 XPCR Buffer comprises dNTP and MgCl2(ii) a The concentration of the dNTP is 2.5 mmol/L; said MgCl2The concentration of (2) was 25 mmol/L.
Preferably, the kit further comprises a positive control and a negative control.
Preferably, the positive control comprises brucella genomic DNA, chlamydia abortus genomic DNA and clostridium perfringens genomic DNA; the negative control substance is sterile double distilled water.
The invention also provides application of the primer group or the kit in the scheme in multiple PCR detection of Brucella, Chlamydia abortus and Clostridium perfringens for non-diagnosis purposes, which comprises the following steps:
1) extracting the genome DNA of a sample to be detected;
2) taking the extracted genome DNA of a sample to be detected as a template, and carrying out multiple PCR amplification reaction by using the primer group in the scheme to obtain multiple PCR amplification products;
3) detecting the multiple PCR amplification product by agarose gel electrophoresis;
if the specific fragment with the length of 731bp is amplified, the detection result is positive for the Brucella, and if the specific fragment with the length of 731bp is not amplified, the detection result is negative for the Brucella;
if the specific fragment with the length of 345bp is amplified, the detection result is positive for the Chlamydia abortus; if the specific fragment with the length of 345bp is not amplified, the detection result is negative to the Chlamydia abortus;
if a specific fragment with the length of 228bp is amplified, the detection result is positive clostridium perfringens; if no specific fragment with the length of 228bp is amplified, the detection result is negative for clostridium perfringens.
Preferably, the reaction system used in the multiplex PCR amplification reaction in step 2) comprises, in 25 μ L:
0.5 mu L of each of BrNo1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo2, 12.5 mu L of 2 xTaq DNA polymerase Mix, 5 xPCR Buffer5 mu L, 3 mu L of genome DNA of a sample to be detected and 1.5 mu L of sterile double distilled water;
the primer group consists of the following components in final concentration: the concentrations of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and ClNo2 were independently 10. mu. mol/L.
Preferably, the procedure of the multiplex PCR amplification reaction is: 5min at 95 ℃; 30S at 95 ℃, 90S at 55 ℃ and 40S at 72 ℃ for 35 cycles; 10min at 72 ℃.
The invention has the beneficial effects that: the invention provides a primer group for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens, which comprises BrNo1, BrNo2, Ch No1, Ch No2, Cl No1 and ClNo 2. The primer group of the invention can simultaneously amplify three specific fragments of 731bp, 345bp and 228 bp. The sheep tissue DNA is taken as a template, the primer group is utilized to carry out specificity verification amplification, and a PCR amplification product is not found, so that the primer group has strong specificity; in the multiplex PCR system, the number of DNA templates is 102During copying, a visible band can be amplified, which shows that the primer group of the invention has high sensitivity; the three pairs of primers in the primer group are used in a PCR reaction system without mutual interference, which shows that the primer group has the advantages of high efficiency, systematicness, economy, simplicity and the like, and is suitable for rapid and accurate etiology identification in clinics and laboratories.
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FIG. 1 shows the electrophoresis results of PCR amplification of Brucella melitensis, Chlamydia abortus and Clostridium perfringens genome DNA templates respectively by the single-specificity detection primers for Brucella melitensis, Chlamydia abortus and Clostridium perfringens of the present invention, wherein: lanes 1 and 2 are PCR result electrophoretograms of BrNo1 and BrNo2 amplified Brucella melitensis genomic DNA template and negative control, respectively; 3,4 are the electrophoresis images of the PCR results of the specific primers ChNo1 and ChNo2 amplified Chlamydia ovis genome DNA template and the negative control, respectively; 5,6 are PCR result electrophoresis charts of specific primers Cl No1 and Cl No2 for amplifying DNA template of Clostridium perfringens genome of sheep and negative control respectively; m is DNA molecular weight standard DL 2000;
FIG. 2 shows the electrophoresis results of PCR specific detection and amplification of primers for single-specificity detection of Brucella melitensis, Chlamydia abortus and Clostridium perfringens in the invention with genomic DNA templates of Chlamydia abortus, Brucella melitensis and Clostridium perfringens, respectively, wherein: lanes 1,2 and 3 are the electrophoresis charts of the PCR amplification results of specific primers Ch No1 and Ch No2 for producing genomic DNA templates of Chlamydia abortus, Brucella melitensis and Clostridium perfringens; 4,5 and 6 are the electrophoresis images of the PCR amplification results of specific primers Br No1 and BrNo2 with Brucella melitensis, Chlamydia abortus and Clostridium perfringens genomic DNA as the template; 7,8 and 9 are electrophoresis charts of PCR amplification results with specific primers Cl No1 and Cl No2 as templates and genomic DNAs of Clostridium perfringens, Brucella melitensis and Chlamydia abortus as templates, respectively; m is DNA molecular weight standard DL 2000;
FIG. 3 shows specific detection primers for Brucella melitensis, Chlamydia abortus and Clostridium perfringens of the present invention and plasmids for Brucella melitensis, Clostridium perfringens and Chlamydia abortusThe result of the multiple PCR sensitivity detection amplification of the DNA single template is as follows: brucella melitensis (10) was amplified with primers Br No1, Br No2, Ch No1, Ch No2, Cl No1 and Cl No2 specific to 1,2 and 3, respectively3Copy), Chlamydia abortus of sheep (10)3Copies) and Clostridium perfringens ovine (10)2Copy) a multiplex PCR result electropherogram with the DNA plasmid template; lane 4 is a negative control when the specific primers Br No1, Br No2, Ch No1, Ch No2, ClNo1 and Cl No2 were amplified; m is DNA molecular weight standard DL 2000;
FIG. 4 shows the results of the primers for detecting Brucella melitensis, Clostridium perfringens and Chlamydia abortus of the present invention amplified by multiplex PCR using DNA composite templates of Brucella melitensis, Clostridium perfringens and Chlamydia abortus, wherein: 1 is a multiple PCR amplification result when a mixed primer of Br No1, Br No2, Ch No1, Ch No2, Cl No1 and Cl No2 is used for amplifying a mixed template of genome DNA of Brucella melitensis, Clostridium perfringens ovis and Chlamydia abortus; 2 is BrNo1, BrNo2, Ch No1, Ch No2, Cl No1 and Cl No2, and the DNA mixed template of Brucella melitensis and Chlamydia abortus is amplified by the multiplex PCR; 3 is a multiple PCR amplification result when a mixed primer of BrNo1, BrNo2, Ch No1, ChNo2, Cl No1 and Cl No2 is used for amplifying a genome DNA mixed template of Brucella melitensis and Clostridium perfringens; 4 is the multiple PCR amplification result when the mixed primer of BrNo1, BrNo2, Ch No1, Ch No2, Cl No1 and Cl No2 is used for amplifying the mixed template of the genome DNA of the clostridium perfringens ovis and the chlamydia abortus ovis; 5 is the multiple PCR amplification result when the mixed primer of BrNo1, BrNo2, Ch No1, Ch No2, ClNo1 and Cl No2 is used for amplifying the sheep tissue genome DNA template; m is DNA molecular weight standard DL 2000;
FIG. 5 shows the results of multiplex PCR amplification of specific detection primers for Brucella melitensis, Chlamydia abortus and Clostridium perfringens according to the present invention using sheep manure whole genome DNA suspected of having three clinical pathogen infections as a template, wherein: 1,2,3,4,5,6,7 and 8 are the multiple PCR amplification results when the mixed primers of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No2 are used for amplifying the sheep manure whole genome DNA template; m is DNA molecular weight standard DL 2000.
Detailed Description
The invention provides a primer group for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens, which comprises BrNo1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo 2;
the nucleotide sequence of the Br No1 is shown as SEQ ID NO: 1, specifically: 5'-CACTCGTGCTAGCAATTTTCTCGC-3', respectively;
the nucleotide sequence of the Br No2 is shown as SEQ ID NO: 2, specifically: 5'-CGACATTGACCGATACGTTATAGC-3', respectively;
the Ch No1 nucleotide sequence is shown as SEQ ID NO: 3, specifically: 5'-GGGCTAGACACGTGAAACCTAG-3', respectively;
the Ch No2 nucleotide sequence is shown as SEQ ID NO: 4, specifically: 5'-CCTGGTCATAAATAGCTCAC-3', respectively;
the nucleotide sequence of Cl No1 is shown in SEQ ID NO: 5, specifically: 5'-CAGCAGGTTGCAAAACTAATG-3', respectively;
the nucleotide sequence of Cl No2 is shown in SEQ ID NO: 6, specifically: 5'-CGTGTAAGAATCTATAAATATATCC-3' are provided.
In the invention, the BrNo1 and BrNo2 are a pair of primers; the BrNo1 and BrNo2 are designed aiming at brucella bp26 gene; the nucleotide sequence of the Brucella bp26 gene is shown as SEQ ID NO: 7, specifically: CACTCGTGCTAGCAATTTTCTCGCAGCCTCATTTTCCACAATCATGCTC GTCGGCGCTTTCAGCCTGCCCGCTTTCGCACAGGAGAATCAGATGACG ACGCAGCCCGCGCGCATCGCCGTCACCGGGGAAGGCATGATGACGGC CTCGCCCGATATGGCCATTCTCAATCTCTCGGTGCTACGCCAGGCAAAG ACCGCGCGCGAAGCCATGACCGCGAATAATGAAGCCATGACAAAAGT GCTCGATGCCATGAAGAAGGCCGGCATCGAAGATCGCGATCTCCAGAC AGGCGGCATCAATATCCAGCCGATTTATGTCTATCCTGACGACAAGAAC AACCTGAAAGAGCCTACCATCACCGGCTATTCTGTATCCACCAGTCTCA CGGTTCGCGTGCGCGAACTGGCCAATGTTGGAAAAATTTTGGATGAAT CCGTCACGCTCGGTGTTAATCAGGGCGGTGATTTGAACCTGGTCAATG ATAATCCCTCCGCCGTGATCAACGAGGCGCGCAAGCGCGCAGTGGCCA ATGCCATTGCCAAGGCGAAGACGCTTGCCGACGCTGCAGGCGTGGGG CTTGGCCGTGTGGTGGAAATCAGTGAACTGAGCCGCCCGCCCATGCCG ATGCCAATTGCGCGCGGACAGTTCAGAACCATGCTAGCAGCCGCACCG GACAATTCCGTGCCGATTGCCGCAGGCGAAAACAGCTATAACGTATCG GTCAATGTCG are provided.
In the invention, ChNo1 and ChNo2 are a pair of primers; the ChNo1 and ChNo2 are designed against Chlamydia abortus 23S gene; the nucleotide sequence of the Chlamydia abortus 23S gene is shown as SEQ ID NO: 8, specifically: GGGCTAGACACGTGAAACCTAGTCTGAATCTGGGGAGACCACTCTCCA AGTCTAAATACTAGTCAATGACCTATAGTGAACCAGTACTGTGAAGGAA AGGTGAAAAGAACCCTTGTTAAGGGAGTGAAATAGAACCTGAAACCA GTAGCTTATAAGCGGTCGAAGACCTATAACTTCTTCGGAAGTCATGGTT GACGGCGTGCCTTTTGCATGATGAGCCAGGGAGTTAAGTTAAACGGCG AGGTTAAGGGATCTACATTCCGGAGCCGAAGCGAAAGCGAGTTTTAAA AGAGCGTTTAGTCGTTTGATTTAGACACGAAACCAAGTGAGCTATTTAT GACCAGG are provided.
The invention discloses a method for detecting toxin in clostridium perfringens, which comprises the steps of preparing a primer pair of Cl No1 and Cl No2, designing Cl No1 and Cl No2 aiming at a clostridium perfringens α toxin gene, and designing a clostridium perfringens α toxin gene with a nucleotide sequence shown as SEQ ID NO 9, specifically CAGCAGGTTGCAAAACTAATGAGGATTTTTATGCTGATATCTTAAAAAACAAAGATTTTAATGCATGGTCAAAAGAATATGCAAGAGGTTTTGCTAAA ACAGGGAAATCAATATACTATAGTCATGCTAGCATGAGTCATAGTTGGG ATGATTGGGATTATGCAGCAAAGGTAACTCTAGCTAACTCTCAAAAAG GAACAGCGGGATATATTTATAGATTCTTACACG.
In the specific implementation process of the invention, the primer group is synthesized by biological engineering (Shanghai) corporation.
The invention provides a kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens, which comprises the primer group in the scheme; the concentration of the BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and ClNo2 is independently and preferably 10 mu mol/L; the kit preferably further comprises 2 × Taq DNA polymerase Mix (QIAGEN, Germany), 5 × PCR Buffer (QIAGEN, Germany) and sterile double distilled water; the 5 XPCR Buffer preferably comprises dNTP and MgCl2(ii) a The concentration of the dNTP is preferably 2.5 mmol/L; said MgCl2The concentration of (B) is preferably 25 mmol/L.
In the invention, the kit also comprises a positive control substance and a negative control substance; the positive control preferably comprises brucella genomic DNA, Chlamydia abortus genomic DNA and Clostridium perfringens genomic DNA; the negative control substance is preferably sterile double distilled water.
The invention also provides application of the primer group or the kit in the scheme in multiple PCR detection of Brucella, Chlamydia abortus and Clostridium perfringens for non-diagnosis purposes, which comprises the following steps:
1) extracting the genome DNA of a sample to be detected;
2) taking the extracted genome DNA of a sample to be detected as a template, and carrying out multiple PCR amplification reaction by using the primer group in the scheme to obtain multiple PCR amplification products;
3) detecting the multiple PCR amplification product by agarose gel electrophoresis;
if the specific fragment with the length of 731bp is amplified, the detection result is positive for the Brucella, and if the specific fragment with the length of 731bp is not amplified, the detection result is negative for the Brucella;
if the specific fragment with the length of 345bp is amplified, the detection result is positive for the Chlamydia abortus; if the specific fragment with the length of 345bp is not amplified, the detection result is negative to the Chlamydia abortus;
if a specific fragment with the length of 228bp is amplified, the detection result is positive clostridium perfringens; if no specific fragment with the length of 228bp is amplified, the detection result is negative for clostridium perfringens.
Firstly, extracting genome DNA of a sample to be detected; the sample to be detected comprises animal tissues, secretions and excretions; the method for extracting the genome DNA of the sample to be detected is not particularly limited, and the conventional method for extracting the genome DNA in the field can be adopted.
After extracting the genome DNA of the sample to be detected, the invention takes the extracted genome DNA of the sample to be detected as a template and utilizes the primer group of the scheme to carry out multiple PCR amplification reaction to obtain multiple PCR amplification products.
In the present invention, the multiplex PCR amplification reaction uses a reaction system comprising, in 25. mu.L: 0.5 muL (10 mumol/L) of each of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No2, 12.5 muL of 2 xTaq DNA polymerase Mix, 5 xPCR Buffer5 muL, 3 muL of genome DNA of a sample to be detected and 1.5 muL of sterile double distilled water; the concentration of the genomic DNA of the sample to be tested is preferably 100 ng/3. mu.L.
In the present invention, the procedure of the multiplex PCR amplification reaction is: 5min at 95 ℃; 30S at 95 ℃, 90S at 55 ℃ and 40S at 72 ℃ for 35 cycles; 10min at 72 ℃.
After obtaining the multiple PCR amplification products, the multiple PCR amplification products are detected by agarose gel electrophoresis;
if the specific fragment with the length of 731bp is amplified, the detection result is positive for the Brucella, and if the specific fragment with the length of 731bp is not amplified, the detection result is negative for the Brucella;
if the specific fragment with the length of 345bp is amplified, the detection result is positive for the Chlamydia abortus; if the specific fragment with the length of 345bp is not amplified, the detection result is negative to the Chlamydia abortus;
if a specific fragment with the length of 228bp is amplified, the detection result is positive clostridium perfringens; if no specific fragment with the length of 228bp is amplified, the detection result is negative for clostridium perfringens.
In the invention, the agarose gel is prepared from agarose and TAE buffer solution, and the mass-volume ratio of the agarose to the TAE buffer solution is 1.5g:100 mL.
The techniques provided by the present invention will be described in detail with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1. Respectively taking sterile double distilled water and brucella melitensis genome DNA as templates, and carrying out PCR amplification by using the following reaction systems; the reaction system was measured at 25. mu.L: 0.5 mu L of each of BrNo1 and BrNo2, 12.5 mu L of 2 XTaq DNA polymerase Mix, 5 mu L of 5 XPCRBuffer, 1 mu L of template and 5.5 mu L of sterile double distilled water;
respectively taking sterile double distilled water and sheep abortion chlamydia genome DNA as templates, and carrying out PCR amplification by using the following reaction systems; the reaction system was measured at 25. mu.L: 0.5 mu L of each of ChNo1 and ChNo2, 12.5 mu L of 2 XTaq DNA polymerase Mix, 5 mu L of 5 XPCRBuffer, 1 mu L of template and 5.5 mu L of sterile double distilled water;
respectively taking sterile double distilled water and clostridium perfringens genome DNA as templates, and carrying out PCR amplification by using the following reaction systems; the reaction system was measured at 25. mu.L: ClNo1 and Cl No2 each 0.5 μ L, 2 XTaq DNA polymerase Mix 12.5 μ L, 5 XPCRBuffer 5 μ L, template 1 μ L, sterile double distilled water 5.5 μ L;
reaction procedure: 5min at 95 ℃; 30S at 95 ℃, 90S at 55 ℃ and 40S at 72 ℃ for 35 cycles; 10min at 72 ℃.
2. The product was detected by electrophoresis on 1.5% agarose gel (agarose 1.5g was weighed, dissolved in 100mL TAE buffer, heated and dissolved to make 1.5% agarose gel), and the detection results are shown in FIG. 1, wherein: lanes 1 and 2 are PCR result electropherograms of BrNo1 and BrNo2 amplified Brucella melitensis genomic DNA template and negative control, respectively; 3,4 are the electrophoresis images of the PCR results of the specific primers ChNo1 and ChNo2 amplified Chlamydia ovis genome DNA template and the negative control, respectively; 5,6 are PCR result electrophoresis charts of specific primers Cl No1 and Cl No2 for amplifying DNA template of Clostridium perfringens genome of sheep and negative control respectively; m is DNA molecular weight standard DL 2000.
The results show that: the single detection primers are used for amplifying the DNA templates of the genomes of the Brucella melitensis, the Chlamydia abortus and the Clostridium perfringens respectively, clear target bands with the sizes of 731bp, 345bp and 228bp appear in lanes 1, 3 and 5 respectively 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 the pathogen genomes can efficiently carry out PCR amplification on target genes, and the blank control without the DNA templates does not have bands.
Example 2
1. Respectively taking sterile double distilled water, brucella melitensis genome DNA, chlamydia abortus genome DNA and clostridium perfringens genome DNA as templates, and carrying out PCR amplification by using the following reaction systems; the reaction system was measured at 25. mu.L: 0.5 mu L of each of BrNo1 and BrNo2, 12.5 mu L of 2 XTaq DNA polymerase Mix, 5 mu L of 5 XPCRBuffer, 1 mu L of template and 5.5 mu L of sterile double distilled water;
respectively taking sterile double distilled water, brucella melitensis genome DNA, chlamydia abortus genome DNA and clostridium perfringens genome DNA as templates, and carrying out PCR amplification by using the following reaction systems; the reaction system was measured at 25. mu.L: 0.5 mu L of each of ChNo1 and ChNo2, 12.5 mu L of 2 XTaq DNA polymerase Mix, 5 mu L of 5 XPCRBuffer, 1 mu L of template and 5.5 mu L of sterile double distilled water;
respectively taking sterile double distilled water, brucella melitensis genome DNA, chlamydia abortus genome DNA and clostridium perfringens genome DNA as templates, and carrying out PCR amplification by using the following reaction systems; the reaction system was measured at 25. mu.L: ClNo1 and ClNo2 each 0.5 μ L, 2 XTaq DNA polymerase Mix 12.5 μ L, 5 XPCRBuffer 5 μ L, template 1 μ L, sterile double distilled water 5.5 μ L;
the rest of the procedure is the same as in example 1;
the detection results are shown in fig. 2, wherein: lanes 1,2 and 3 are the electrophoresis charts of PCR amplification results of specific primers Ch No1 and Ch No2 for producing genomic DNA templates of Chlamydia abortus, Brucella melitensis and Clostridium perfringens; 4,5 and 6 are the electrophoresis images of the PCR amplification results of specific primers BrNo1 and BrNo2 using Brucella melitensis, Chlamydia abortus and Clostridium perfringens genomic DNA as templates; 7,8 and 9 are electrophoresis charts of PCR amplification results of specific primers ClNo1 and ClNo2 using genomic DNA of Clostridium perfringens, Brucella melitensis and Chlamydia abortus as templates, respectively; m is DNA molecular weight standard DL 2000.
The results show that: the single detection primers are used for amplifying by using DNA templates of genomes of chlamydia abortus, brucella ovis and clostridium perfringens, clear target bands with the sizes of 345bp, 731bp and 228bp respectively appear in lanes 1,4 and 7 of a PCR result electrophoresis picture, and PCR amplification products do not appear when the same primer takes genomes of other two pathogens as templates, which shows that the specificity of three pairs of primers is high, and the PCR amplification can not be performed on the DNA templates of other two pathogens.
Example 3
Performing PCR amplification by respectively using double distilled water, brucella melitensis plasmid DNA, chlamydia abortus plasmid DNA and clostridium perfringens plasmid DNA as templates; the reaction system included, in 25. mu.L: 0.5 mu L (10 mu mol/L) of Br No1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo2 respectively, 12.5 mu L of 2 xTaq DNA polymerase Mix, 5 xPCRbuffer 5 mu L, 3 mu L of genome DNA of a sample to be tested and 1.5 mu L of sterile double distilled water;
the rest of the procedure is the same as in example 1;
the results are shown in FIG. 3, in which: BrNo1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo2 as specific primers 1,2 and 3 respectively amplify Brucella melitensis (10)3Copy), Chlamydia abortus of sheep (10)3Copies) and Clostridium perfringens ovine (10)2Copy) a multiplex PCR result electropherogram with a plasmid DNA template; lane 4 is a negative control when specific primers BrNo1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo2 were amplified; m is DNA molecular weight standard DL 2000.
The results show that: the mixed detection primers are used for carrying out sensitivity detection amplification on plasmid DNA templates of Brucella melitensis, Chlamydia abortus and Clostridium perfringens respectively, clear and single-purpose bands with the sizes of 731bp, 504bp and 345bp appear in lanes 1,2 and 3 respectively in an electrophoresis chart of a PCR result, and a negative control lane 4 has no band, so that the single and low-copy DNA templates of three pathogens in a multiplex PCR system of the composite primers can be efficiently amplified, non-specific amplification bands or non-amplification bands caused by the composite primers cannot occur, and the negative control without the templates has no amplification products.
Example 4
Respectively carrying out multiple PCR amplification on (Brucella melitensis, Clostridium perfringens and Chlamydia abortus genome DNA mixed template), (Brucella melitensis and Clostridium perfringens genome DNA mixed template), (Clostridium perfringens and Chlamydia abortus genome DNA mixed template) and a sheep tissue genome DNA template by using the following reaction system; the reaction system included, in 25. mu.L: 0.5 muL (10 mumol/L) of each of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No2, 12.5 muL of 2 xTaq DNA polymerase Mix, 5 muL of 5 xPCRbuffer, 3 muL of genome DNA of a sample to be detected and 1.5 muL of sterile double distilled water;
the rest of the procedure is the same as in example 1;
the results are shown in FIG. 4, in which: 1 is the multiple PCR amplification result when the mixed primer of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No2 is used for amplifying the mixed template of the genomic DNAs of Brucella melitensis, Clostridium perfringens and Chlamydia abortus; 2 is BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No2, and the DNA mixed template of Brucella melitensis and Chlamydia abortus is amplified by the multiplex PCR; 3 is the multiple PCR amplification result when Brucella melitensis and Clostridium perfringens genome DNA mixed templates are amplified by the mixed primers of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No 2; 4 is the multiple PCR amplification result when the mixed primer of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No2 is used for amplifying the mixed template of the genomic DNA of the clostridium perfringens ovis and the chlamydia abortus; 5 is the multiple PCR amplification result when the mixed primer of BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and Cl No2 is used for amplifying the sheep tissue genome DNA template; m is DNA molecular weight standard DL 2000.
The results show that: the mixed detection primer is used for amplifying three or two mixed DNA templates of Brucella melitensis, Chlamydia abortus and Clostridium perfringens genomes and a sheep tissue DNA template, clear target bands with the sizes of 731bp, 345bp and 228bp appear in a lane 1 in a PCR result electrophoresis chart, and the fact that the DNA templates of the three pathogens are efficiently amplified and non-specific amplification does not appear in a multiplex PCR system is shown; target bands with the sizes of 731bp and 345bp appear in a lane 2, which shows that the genes of the Brucella melitensis and the Chlamydia abortus in the multiple PCR reaction system are efficiently amplified; target bands with the sizes of 731bp and 228bp appear in a lane 3, which shows that the genes of the Brucella melitensis and the Clostridium perfringens are efficiently amplified in the multiplex PCR system; target bands with the sizes of 345bp and 228bp appear in a lane 4, which shows that the genes of the sheep Chlamydia abortus and the sheep Clostridium perfringens in the multiplex PCR system are efficiently amplified; no amplification products were present in lane 5, indicating that no PCR amplification was performed using sheep tissue DNA as a template.
Example 5
1. Sheep faeces were collected from a sheep farm flock suspected of having brucella, chlamydia abortus and clostridium perfringens infections.
2. Extraction of sheep excrement whole genome DNA
1) The obtained feces were heated at 70 ℃ for 30 min.
2) Preparation of fecal DNA: the fecal DNA was extracted according to the instructions of a fecal DNA extraction kit (TIANAmp Stool, centrifugal column type, available from Tiangen Biochemical technology (Beijing) Ltd.), and the concentration of the extracted DNA was determined for use.
3. Establishment of PCR amplification System
A multiplex PCR reaction solution was prepared in a PCR reaction tube, and 5. mu.L of 5 XPCR buffer, 12.5. mu.L of 2 XPAQQ DNA polymerase Mix, 0.5. mu.L each of primers BrNo1, BrNo2, ChNo1, ChNo2, Cl No1 and ClNo2 (10. mu. mol/L) were added to a 25. mu.L reaction system, the amount of the whole genome DNA template of sheep feces was 100ng, and 25. mu.L was made up with water.
4. PCR amplification conditions
The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, [ 30S denaturation at 95 ℃, 90S annealing at 55 ℃ and 40S extension at 72 ℃ for 35 cycles ], lengthening and extending at 72 ℃ for 10min, and storing at 4 ℃ after the reaction is finished.
5. Observation and judgment of PCR amplification result
And (3) carrying out electrophoresis on the PCR reaction product in 1.5% (w/v) agarose gel, observing the amplification result by an ultraviolet gel imaging system, and judging the type of the infected pathogen according to the size of a target band.
The results of the assay are shown in FIG. 5, in which: 1,2,3,4,5,6,7 and 8 are the multiple PCR amplification results when the mixed primers of BrNo1, BrNo2, Ch No1, Ch No2, ClNo1 and ClNo2 are used for amplifying the sheep manure whole genome DNA template; m is DNA molecular weight standard DL 2000.
The results show that: the mixed detection primer is used for amplifying by taking sheep manure whole genome DNA containing Brucella, Chlamydia abortus or Clostridium perfringens as a template, clear target bands with the sizes of 345bp and 228bp appear in lanes 1 and 4 in a PCR result electrophoresis chart, and the sheep manure is proved to have the Chlamydia abortus and Clostridium perfringens; amplified bands with the sizes of 731bp and 345bp appear in lanes 2 and 5, which indicates that brucella and Chlamydia abortus exist in sheep manure; the amplified band of 228bp in size appears in lanes 3 and 7, indicating the presence of clostridium perfringens in sheep manure; an amplified band of 345bp in size appears in lanes 6 and 8, indicating the presence of Chlamydia abortus in sheep manure.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> primer group and kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens and application
<160>9
<170>SIPOSequenceListing 1.0
<210>1
<211>24
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>1
cactcgtgct agcaattttc tcgc 24
<210>2
<211>24
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>2
cgacattgac cgatacgtta tagc 24
<210>3
<211>22
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>3
gggctagaca cgtgaaacct ag 22
<210>4
<211>20
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>4
cctggtcata aatagctcac 20
<210>5
<211>21
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>5
cagcaggttg caaaactaat g 21
<210>6
<211>25
<212>DNA
<213> Artificial sequence (Artificial sequence)
<400>6
cgtgtaagaa tctataaata tatcc 25
<210>7
<211>731
<212>DNA
<213> Brucella melitensis
<400>7
cactcgtgct agcaattttc tcgcagcctc attttccaca atcatgctcg tcggcgcttt 60
cagcctgccc gctttcgcac aggagaatca gatgacgacg cagcccgcgc gcatcgccgt 120
caccggggaa ggcatgatga cggcctcgcc cgatatggcc attctcaatc tctcggtgct 180
acgccaggca aagaccgcgc gcgaagccat gaccgcgaat aatgaagcca tgacaaaagt 240
gctcgatgcc atgaagaagg ccggcatcga agatcgcgat ctccagacag gcggcatcaa 300
tatccagccg atttatgtct atcctgacga caagaacaac ctgaaagagc ctaccatcac 360
cggctattct gtatccacca gtctcacggt tcgcgtgcgc gaactggcca atgttggaaa 420
aattttggat gaatccgtca cgctcggtgt taatcagggc ggtgatttga acctggtcaa 480
tgataatccc tccgccgtga tcaacgaggc gcgcaagcgc gcagtggcca atgccattgc 540
caaggcgaag acgcttgccg acgctgcagg cgtggggctt ggccgtgtgg tggaaatcag 600
tgaactgagc cgcccgccca tgccgatgcc aattgcgcgc ggacagttca gaaccatgct 660
agcagccgca ccggacaatt ccgtgccgat tgccgcaggc gaaaacagct ataacgtatc 720
ggtcaatgtc g 731
<210>8
<211>345
<212>DNA
<213> Chlamydia abortus (Chlamydia abortus)
<400>8
gggctagaca cgtgaaacct agtctgaatc tggggagacc actctccaag tctaaatact 60
agtcaatgac ctatagtgaa ccagtactgt gaaggaaagg tgaaaagaac ccttgttaag 120
ggagtgaaat agaacctgaa accagtagct tataagcggt cgaagaccta taacttcttc 180
ggaagtcatg gttgacggcg tgccttttgc atgatgagcc agggagttaa gttaaacggc 240
gaggttaagg gatctacatt ccggagccga agcgaaagcg agttttaaaa gagcgtttag 300
tcgtttgatt tagacacgaa accaagtgag ctatttatga ccagg 345
<210>9
<211>228
<212>DNA
<213> Clostridium perfringens (Clostridium perfringens)
<400>9
cagcaggttg caaaactaat gaggattttt atgctgatat cttaaaaaac aaagatttta 60
atgcatggtc aaaagaatat gcaagaggtt ttgctaaaac agggaaatca atatactata 120
gtcatgctag catgagtcat agttgggatg attgggatta tgcagcaaag gtaactctag 180
ctaactctca aaaaggaaca gcgggatata tttatagatt cttacacg 228
Claims (8)
1. A primer set for simultaneously detecting brucella, chlamydia abortus and clostridium perfringens, wherein the primer set comprises BrNo1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo 2;
the nucleotide sequence of BrNo1 is shown in SEQ ID NO: 1 is shown in the specification;
the nucleotide sequence of BrNo2 is shown in SEQ ID NO: 2 is shown in the specification;
the nucleotide sequence of ChNo1 is shown as SEQ ID NO: 3 is shown in the specification;
the nucleotide sequence of ChNo2 is shown as SEQ ID NO: 4 is shown in the specification;
the nucleotide sequence of the ClNo1 is shown as SEQ ID NO: 5 is shown in the specification;
the nucleotide sequence of the ClNo2 is shown as SEQ ID NO: and 6.
2. A kit for simultaneously detecting Brucella, Chlamydia abortus and Clostridium perfringens, wherein the kit comprises the primer set according to claim 1.
3. The kit of claim 2, wherein the kit further comprises 2 x Taq DNA polymerase Mix, 5 x PCR Buffer and sterile double distilled water; the 5 XPCR Buffer comprises dNTP and MgCl2(ii) a The concentration of the dNTP is 2.5 mmol/L; said MgCl2The concentration of (2) was 25 mmol/L.
4. The kit of claim 2, further comprising a positive control and a negative control.
5. The kit of claim 4, wherein the positive control comprises Brucella genomic DNA, Chlamydia abortus genomic DNA, and Clostridium perfringens genomic DNA; the negative control substance is sterile double distilled water.
6. Use of the primer set of claim 1 or the kit of any one of claims 2 to 5 for multiplex PCR detection of Brucella, Chlamydia abortus and Clostridium perfringens for non-diagnostic purposes, comprising the steps of:
1) extracting the genome DNA of a sample to be detected;
2) performing a multiplex PCR amplification reaction by using the genomic DNA of the sample to be detected as a template and the primer set of claim 1 to obtain a multiplex PCR amplification product;
3) detecting the multiple PCR amplification product by agarose gel electrophoresis;
if the specific fragment with the length of 731bp is amplified, the detection result is positive for the Brucella, and if the specific fragment with the length of 731bp is not amplified, the detection result is negative for the Brucella;
if the specific fragment with the length of 345bp is amplified, the detection result is positive for the Chlamydia abortus; if the specific fragment with the length of 345bp is not amplified, the detection result is negative to the Chlamydia abortus;
if a specific fragment with the length of 228bp is amplified, the detection result is positive clostridium perfringens; if no specific fragment with the length of 228bp is amplified, the detection result is negative for clostridium perfringens.
7. The use according to claim 6, wherein the reaction system used in the multiplex PCR amplification reaction in step 2) comprises, in 25 μ L:
0.5 mu L of each of BrNo1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo2, 12.5 mu L of 2 xTaq DNA polymerase Mix, 5 xPCR Buffer5 mu L, 3 mu L of genome DNA of a sample to be detected and 1.5 mu L of sterile double distilled water;
the primer group consists of the following components in final concentration: the concentrations of BrNo1, BrNo2, ChNo1, ChNo2, ClNo1 and ClNo2 were independently 10. mu. mol/L.
8. The use of claim 7, wherein the multiplex PCR amplification reaction is performed by: 5min at 95 ℃; 30S at 95 ℃, 90S at 55 ℃ and 40S at 72 ℃ for 35 cycles; 10min at 72 ℃.
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| CN118064612A (en) * | 2024-02-20 | 2024-05-24 | 沈阳农业大学 | Primer pair of brucella 23S rRNA genes and application thereof |
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