KR20170019925A - Primer set for diagnosing Brucella bacteria and uses thereof - Google Patents
Primer set for diagnosing Brucella bacteria and uses thereof Download PDFInfo
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Abstract
Description
The present invention relates to a primer set for diagnosing Brucellosis, and more particularly to a primer set for brucellosis diagnosis, a kit for diagnosing brucellosis comprising the primer set and a reagent for performing an amplification reaction, 3, and a method for diagnosing Brucella bacterium using the primer set. 2. Description of the Related Art
Brucellosis is one of the common infectious diseases of humans and animals infected by bacteria in the genus Brucella and has been designated as a statutory infectious disease. Brucella is well airborne and it is estimated that by inhaling 10 to 100 cells can cause disease in humans. Infected animals are infected by people through infestation, milk, and tissue, and livestock such as cows, pigs, and dogs are the major infectious agents and are spread to people exposed to these tissues. It can also be infected with non-pasteurized milk or dairy products, or ingested contaminated meat. Humans are infected by the treatment of infected carcasses, other than oral infections, such as raw milk and dairy products, and they show characteristic fever. When a person is infected with this bacterium, it becomes a malt fever or a mediterranean fever after a latency of about 3 weeks, resulting in irregular fever, fatigue, malaise, and headache. . This disease has also occurred in Korea and has spread mainly to oral and contact infections and has also spread to people who have eaten unsterilized dairy products. Brucellosis is a nonspecific febrile illness similar to influenza. Common symptoms are fever, headache, muscle aches, joint pain, back pain, sweating, chills, general weakness and fatigue. Digestive symptoms such as loss of appetite, nausea, vomiting, diarrhea and constipation are present in 70% of adults and less frequently in children. Lumbago and tenderness of the lumbar spine can occur in 60% of the spine due to multiple osteoarthritis of the spine. When a sacr oiliac joint is infected on one side or both sides, a physical examination shows stress in the cephalad joints, which causes pain in the lower back and hips. About half of the patients have hepatomegaly and spleen enlargement.
There is no effective cure for this disease, but vaccination is possible in livestock, but once an infected livestock is found, it must be slaughtered by law. In the case of humans, tetracycline, streptomycin, chloramphenicol and the like are used for the treatment, but when the administration of the drug is discontinued, recurrence often occurs and resistance to treatment is difficult. The mortality rate is less than 2%, but when left untreated, it causes spondylitis and osteomyelitis.
Korean Patent No. 1288035 discloses a 'primer set for distinguishing brucella species', Korean Patent Laid-Open Publication No. 2009-0100950 discloses 'a method for detecting a strain of Brucella genus using real-time PCR' As in the case of the present invention, the 'Brucella spp. Diagnostic primer set and its use' has not been disclosed at all.
SUMMARY OF THE INVENTION The present invention has been made in view of the above circumstances and provides a rapid and sensitive bioterrorism pathogen detection And tried to develop a kit. As a result, brucellosis-causing pathogens such as Brucella abortus abortus , Brucella suis ), Brucella melitensis melitensis ) and Brucella pinnipedalis ( Brucella Based on the specific gene sequence, pinnipedialis was selected for the detection site, and three primer sets were constructed based on this region. The three primer sets thus constructed were used to detect the infection of the brucellosis pathogen The present inventors completed the present invention by developing a primer set capable of diagnosing Brucella bacteria more accurately and quickly than other primers as a result of real-time PCR.
In order to solve the above problems, the present invention provides a primer set for diagnosing Brucellosis comprising oligonucleotide primer sets of SEQ ID NOS: 1 and 2.
Further, the present invention provides a primer set comprising: the primer set; And a reagent for carrying out an amplification reaction.
In addition, the present invention provides a composition for real-time PCR for diagnosing Brucellosis comprising a set of oligonucleotide primers of SEQ ID NOS: 1 and 2 and a nucleotide sequence of SEQ ID NO: 3.
In addition,
Isolating the DNA from the suspected Brucella infection sample;
Performing the real-time PCR using the separated genomic DNA as a template and adding a DNA polymerase to the primer set and the oligonucleotide probe for detecting brucellosis comprising the nucleotide sequence of SEQ ID NO: 3 to amplify the target sequence ; And
And detecting the amplification product. BRIEF DESCRIPTION OF THE DRAWINGS Fig.
Through the present invention, the pathogenic brucellosis-causing Brucella abortus abortus , Brucella suis ), Brucella melitensis melitensis ) and Brucella pinnipedialis were selected for the diagnostic test. Through this, it is expected that it will be possible to quickly and accurately apply the diagnosis system of high-risk pathogens in the country and prepare and respond to the biological warfare (terror).
Figure 1 shows the results of confirming the reactivity of three primer sets for the detection of Brucella bacterium developed in the present invention using SYBR Green reagent.
FIG. 2 is a result of a real-time PCR test using three primer sets for detecting Brucella bacterium developed in the present invention to select an optimal primer set.
FIG. 3 is a result of examining the minimum detection limit concentration, the Ct value, the PCR efficiency and the reactivity of three primer sets for the detection of brucellosis developed in the present invention to select the optimal primer set.
FIG. 4 shows the result of measuring the minimum detection limit concentration of Brucella using a final primer set selected in the present invention.
FIG. 5 shows the results of the test for the specificity of Brucella using a final primer set selected in the present invention. B. anthracis : Anthrax, F. tularensis : Yato germ, Smallpox: Two germs, Y. pestis :
FIG. 6 shows the final test results of application of the field diagnostic equipment using the primer set developed in the present invention.
In order to achieve the object of the present invention, the present invention provides a primer set for Brucellosis diagnosis comprising the oligonucleotide primer set of SEQ ID NOS: 1 and 2.
The oligonucleotide may preferably be an oligonucleotide consisting of fragments of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22 consecutive nucleotides in the sequence of SEQ ID NO: 1 have. In addition, the oligonucleotide may preferably be an oligonucleotide consisting of fragments of 16 or more, 17 or more, 18 or more, or 19 or more consecutive nucleotides in the sequence of SEQ ID NO: 2.
In a primer set for Brucellosis detection according to an embodiment of the present invention, the Brucella bacterium is Brucella abortus abortus), Brucella can devices (Brucella suis), Brucella mellitic X cis (Brucella melitensis , and Brucella pinnipedialis , but are not limited thereto.
In the present invention, a "primer" refers to a single strand oligonucleotide sequence complementary to a nucleic acid strand to be copied, and may serve as a starting point for synthesis of a primer extension product. The length and sequence of the primer should allow the synthesis of the extension product to begin. The specific length and sequence of the primer will depend on the primer usage conditions such as temperature and ionic strength, as well as the complexity of the desired DNA or RNA target.
In the present invention, the oligonucleotide used as a primer may also include a nucleotide analogue such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or And may include an intercalating agent.
Further, the present invention provides a primer set comprising: the primer set; And a reagent for carrying out an amplification reaction. In the kit of the present invention, the reagent for carrying out the amplification reaction may include DNA polymerase, dNTPs, buffer and the like. In the present invention, the brucellosis is Brucella abortus abortus , Brucella suis ), Brucella melitensis melitensis , and Brucella pinnipedialis , but are not limited thereto.
The brucellosis diagnosis kit according to an embodiment of the present invention may further comprise an oligonucleotide probe for detecting brucellosis comprising the nucleotide sequence of SEQ ID NO:
As used herein, "probe" refers to a single-stranded nucleic acid sequence that hybridizes with a complementary single-stranded target sequence to form a double-stranded molecule (hybrid).
As used herein, an oligonucleotide used as a probe may also include a nucleotide analogue, such as phosphorothioate, alkylphosphorothioate, or peptide nucleic acid, or alternatively, And may include an intercalating agent.
In addition, the present invention provides a composition for real-time PCR for diagnosing Brucellosis comprising a set of oligonucleotide primers of SEQ ID NOS: 1 and 2 and a nucleotide sequence of SEQ ID NO: 3.
In addition,
Isolating the DNA from the suspected Brucella infection sample;
Performing the real-time PCR using the separated genomic DNA as a template and adding a DNA polymerase to the primer set and the oligonucleotide probe for detecting brucellosis comprising the nucleotide sequence of SEQ ID NO: 3 to amplify the target sequence ; And
And detecting the amplification product. BRIEF DESCRIPTION OF THE DRAWINGS Fig.
The method of the present invention comprises isolating DNA in a suspect sample of Bursella infection. Methods for isolating genomic DNA from the sample can be performed by a method known in the art, and phenol: chloroform extraction followed by ethanol precipitation may be used. Using the separated genomic DNA as a template, an oligonucleotide primer set according to an embodiment of the present invention and a DNA polymerase using an oligonucleotide probe for detecting brucellosis comprising the nucleotide sequence of SEQ ID NO: 3 were subjected to real-time PCR To amplify the target sequence.
The method of the present invention comprises detecting said amplification product. The detection of the amplification product can be performed through capillary electrophoresis, DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement or phosphorescence measurement, but is not limited thereto.
Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.
1. Pathogenicity Brucella For screening primer making
1) Detection site selection and lift design ( oligo design )
Brucella brucella, Brucella abortus , Brucella suis ), Brucella melitensis melitensis ) and Brucella pinnipedialis (Table 1). Gene sequencing was performed on a genbank based on reference sequence data. For homology analysis, MEGA (MOLECULAR EVOLUTIONARY GENETICS ANALYSIS, http://www.megasoftware.net/ ) 5.1 program was used, and DNA fragment of brucella strain DNA was prepared and used.
The selected primers and probe sequences were identified by sequence specificity using Basic Local Alignment Search Tool (BALST) (http://blast.ncbi.nlm.nih.gov/Blast ). Tm analysis of selected primers and probes was performed by IDT The oligonucleotide was analyzed using OligoAnalyzer 3.1 web-based program ( http://eu.idtdna.com/analyzer/Applications/OligoAnalyzer/ ) with oligonucleotide concentration of 0.25 uM and Na + concentration of 50 mM. Respectively.
size(
bp
)
2) Real - time PCR optimization
The template DNA of Brucella was synthesized by DNA fragmentation from the sequence obtained by homology analysis, amplified by PCR reaction and purified. For the detection of brucellosis, the primer reactivity was checked by SYBR Green test for each primer set, and the optimal set was selected by carrying out condition tests such as the limitation of detection using a probe and PCR efficiency. After performing Master mixture condition test (enzyme test, buffer test) and PCR condition test using selected primer set, LOD test, dynamic range test and specificity test were performed under optimal conditions. Genes that determine the virulence of brucellosis were screened and subjected to condition tests after different fluorescent labels of each gene probe for real-time PCR. The experiments were performed using Roche's LC480 Real-time PCR instrument. Table 3 shows the PCR protocol performed.
Example 1. Pathogenicity Brucella For screening primer selection( SYBR Green test )
SYBR Green reagent was used to confirm the reactivity of three kinds of primer sets of Brucella (Fig. 1). Each experiment was repeated three or more times.
Example 2. Real - time PCR test ( Probe Application test)
After confirming the reactivity and amplification efficiency of the primer set through the SYBR Green test, real-time PCR test was performed to confirm the reactivity of three primer sets of Brucella, and then, an optimal primer set was selected. Real-time PCR was performed on the brucella strain DNA at a concentration of 10 5 and 10 3 copies / rxn. As a result, the PCR efficiency and reactivity of the primer set 1 (SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) (Fig. 2). Each experiment was repeated three or more times.
Example 3. Optimal primer Set screening test
The template DNA was serially diluted at a concentration of 10 7 to 10 1 copies / rxn, and then primer set 1 (see Table 4) was selected considering the minimum detection limit concentration, Ct value, PCR efficiency and reactivity of each candidate primer set SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3) were screened with an optimal primer set (Fig. 3). Each experiment was repeated three or more times.
Ct
(Mean)
copy
copy
copy
Example 4. Detection minimum threshold concentration ( LOD ) Measure
The template DNA was serially diluted at a concentration of 10 7 to 10 1 copies / rxn and then the detection limit threshold (Limit of Detection, LOD) was measured using a selected primer set. Each experiment was repeated three or more times. As a result of measuring the minimum detection limit concentration of Brucella by using the finally selected primer set, it was confirmed that it was effectively detected up to 10 copies / rxn (FIG. 4).
Example 5. Brucella Specificity ( Specificity ) Test
The specificity test of Brucella bacterium was carried out using the final screened primer set. As a result, anthrax (DNA fragment, genomic DNA), yato (DNA fragment, genomic DNA), DNA fragment, Gene) target, and it was confirmed that only brucellosis was detected specifically (Fig. 5).
Example 6. In the present invention, primer The final field diagnostic equipment application test using set
In order to confirm the infection in a short time in the future, many molecular diagnostic fields are being developed for the field diagnosis. In the present invention, the method of field diagnosis was studied by applying to the UltraFast LabChip Real-time PCR instrument of NanoBio CIS. The instrument is an ultra-small device with a weight of about 5.5kg. It can test 30 cycles of real-time PCR within about 15 minutes and is a chip-based PCR device that is different from conventional real-time PCR.
After dilution of the brucella strain DNA by concentration and using an UltraFast LabChip Real-time PCR instrument, it was confirmed that up to 10 2 copies / rxn was detected within 15 minutes of reaction time (FIG. 6). Each experiment was repeated three or more times.
<110> The Armed Forces Medical Command <120> Primer set for diagnosing Brucella bacteria and uses thereof <130> PN15228 <160> 9 <170> KoPatentin <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cctcacggaa gacgatatca ag 22 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 agccgcccac aaagaaata 19 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aggcgagagg ctgaaagatg gtg 23 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aagggcaagg tggaagattt 20 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctgcgaccga tttgatgttt g 21 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 acgctttacc cggaaacgat ccat 24 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tataaggacg tggcggaaac 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ttccagagaa ccttggtgat g 21 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agcagccgga cgacctcatc tata 24
Claims (6)
Using the separated genomic DNA as a template and adding a DNA polymerase, real-time PCR was performed using the primer set according to item 1 or 2 and the oligonucleotide probe for brucellosis detection comprising the nucleotide sequence shown in SEQ ID NO: Amplifying the target sequence; And
And detecting the amplification product.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113755618A (en) * | 2021-10-14 | 2021-12-07 | 中国动物卫生与流行病学中心 | Method for detecting brucellosis of animals with high sensitivity |
| KR20220095680A (en) * | 2020-12-30 | 2022-07-07 | 대한민국(농림축산식품부 농림축산검역본부장) | Single nucleotide polymorphism marker for identification of Brucella abortus and primer set for detection |
| KR20220095679A (en) * | 2020-12-30 | 2022-07-07 | 대한민국(농림축산식품부 농림축산검역본부장) | Single nucleotide polymorphism marker for identification of Brucella and primer set for detection |
| KR20220095681A (en) * | 2020-12-30 | 2022-07-07 | 대한민국(농림축산식품부 농림축산검역본부장) | Single nucleotide polymorphism marker for identification of Brucella canis and primer set for detection |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220095680A (en) * | 2020-12-30 | 2022-07-07 | 대한민국(농림축산식품부 농림축산검역본부장) | Single nucleotide polymorphism marker for identification of Brucella abortus and primer set for detection |
| KR20220095679A (en) * | 2020-12-30 | 2022-07-07 | 대한민국(농림축산식품부 농림축산검역본부장) | Single nucleotide polymorphism marker for identification of Brucella and primer set for detection |
| KR20220095681A (en) * | 2020-12-30 | 2022-07-07 | 대한민국(농림축산식품부 농림축산검역본부장) | Single nucleotide polymorphism marker for identification of Brucella canis and primer set for detection |
| CN113755618A (en) * | 2021-10-14 | 2021-12-07 | 中国动物卫生与流行病学中心 | Method for detecting brucellosis of animals with high sensitivity |
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