CN117512141A - Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis - Google Patents

Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis Download PDF

Info

Publication number
CN117512141A
CN117512141A CN202210926273.3A CN202210926273A CN117512141A CN 117512141 A CN117512141 A CN 117512141A CN 202210926273 A CN202210926273 A CN 202210926273A CN 117512141 A CN117512141 A CN 117512141A
Authority
CN
China
Prior art keywords
brucella
primer
bovis
quantitative pcr
fluorescent quantitative
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202210926273.3A
Other languages
Chinese (zh)
Inventor
赵薇
李可维
佟黎明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Changchun Chengshi Health Industry Co ltd
Original Assignee
Changchun Chengshi Health Industry Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Changchun Chengshi Health Industry Co ltd filed Critical Changchun Chengshi Health Industry Co ltd
Priority to CN202210926273.3A priority Critical patent/CN117512141A/en
Publication of CN117512141A publication Critical patent/CN117512141A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention discloses a special primer for detecting bovine brucella by double real-time fluorescent quantitative PCR, and relates to the field of molecular biology detection of bovine brucella. The special primer IS designed according to a specific conserved target sequence BCSP31 and an insertion sequence IS711 of brucella bovis, and contains 6 primers, wherein the nucleotide sequence of each primer IS shown in a specification sequence table. The invention provides a new technical method for detecting the bovine brucella, and determines an optimal reaction system for double real-time fluorescent quantitative PCR detection and a kit matched with the optimal reaction system. The method is used for screening and detecting the brucella bovis in animal husbandry production units and basic medical institutions, has wide market prospect and great economic and social benefits, and is suitable for being popularized and used in a large range.

Description

Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis
Technical Field
The invention relates to the field of molecular biology detection of bovine brucella, in particular to a dual real-time fluorescent quantitative PCR detection method of bovine brucella and a special primer and a kit thereof.
Background
Brucella (Brucella spp.) is an intracellular parasitic Sphingomonas that can cause a zoonotic disease, brucella disease (Brucella sp). The main infectious sources include sheep, cattle, pigs, etc., and are highly developed in the Mediterranean, asia, south and Central America, etc. The disease is mainly characterized by clinical manifestations such as fever, arthralgia, hepatosplenomegaly, and the like, and the more serious cases can cause damage to nervous system, skeletal muscle system and fertility, and even death. The disease has wide popularity, seriously jeopardizes the development of animal husbandry, and threatens human health, so that the laboratory diagnosis and research of the brucellosis are of great significance.
The current laboratory methods for diagnosing brucellosis mainly comprise a red-amber flat plate agglutination test (RBPT), a test tube agglutination test (SAT), a Complement Fixation Test (CFT) and the like, but the methods have the defects of low specificity, poor sensitivity, complex operation and the like. The separation culture of the brucella in the etiology detection is a gold standard for the detection and the type distinction of the brucella, but the method is long in time consumption, needs to be carried out in a biosafety three-level laboratory, and has high operation requirement and certain danger. Therefore, it is urgently required to establish a safe, rapid, highly sensitive and highly specific Brucella diagnostic test method.
The polymerase chain reaction-PCR technology is popularized and applied in Brucella detection, and the technology relies on two PCR methods of BCSP31 polymerase and AMOS polymerase to identify the genus and species (types) of suspected Brucella, so that the technology has important significance in tracing analysis of infectious agents. The BCSP31 protein exists in various biotype strains of the Brucella, and a pair of B4-B5 primers are designed according to the nucleotide sequence of the BCSP31 to carry out nucleic acid detection on suspected Brucella; AMOS-PCR is a method of PCR named Abortus, melitensis, ovis and the abbreviation for the first letter of Suis that identifies brucella bovis (type 1, 2, 4), brucella ovis (type 1, 2, 3), brucella Suis (type 1), and brucella ovis.
The PCR nucleic acid amplification technology is applied to the directions of brucellosis diagnosis, animal products, detection and typing of brucellosis in the environment and the like, can detect the brucellosis from multiple types of samples such as body fluid, tissues, serum, whole blood, milk, raw meat, cheese and the like, and can detect pathogen from serological negative samples in the early stage of disease, thereby overcoming the defects of a serological detection method. Meanwhile, the method has important significance for epidemiology, prevention and control and risk analysis in different areas. Is also a reliable method for detecting the copy number of the target nucleic acid at present, and is applied to the fields of animal medicine and molecular biology research. However, no special primers and kits for PCR detection of Brucella have been developed in the market so far.
Disclosure of Invention
The invention aims to provide a dual real-time fluorescent quantitative PCR detection method for detecting bovine brucella with strong specificity, high sensitivity and higher safety, and a special primer and a kit thereof.
The special primer for carrying out double real-time fluorescent quantitative PCR detection on bovine brucella IS designed according to a specific conserved target sequence BCSP31 and an insertion sequence IS711 of bovine brucella and IS used for qualitatively detecting bovine brucella in a sample to be detected.
The nucleotide sequence of the specific conserved target sequence BCSP31 of the Brucella bovis is GCGCCTATACGCGCGACCTGATCC
AGTCTTTTGCATCGCGGTGCCATGTGCGTCTGGTGGGGGCCGACGGGCTGGCGGCGATTGCCGAAGCGCATATTCGCGGTGAAAGTTTCGATGAGGCGCTGGTCATGGCGCAGATCGCGCCATGTTTTATCGAAAAGGATGGCAAGCGCACCGATATCGTGGTGCTTGCCTGTACGCATTATCCGTTTCTCGTCAATGTATTGCGCCGTCTGGCTCCATGGCCGGTGGATTGGCTGGACCCGGCGGAAGCCATTGCACGACGCATGAAATCGCTTTTGCCTGCGCGAAGTGACGATGATGAATTTCATTCTCAAGATGACCTAGCATTCTTCACATCCAGGAAACCCGACTATGCCATTCGCCGCCTGATGCAGGGTTTTGGGCTGCGTTTTTAATCGTTTCAGTCGGCTCTGGCGGCGCTTGTCGGCAAGCGATTGTATTCTTTGGGAAAATCCAGAATAATGGAATGCGGTGGTTGACAATCGGCCTCAAGCTTCCTATGGTTTTCGGCATAATCTATGCGGGAAGAGGACTGGTATTATGAAATTCGGAAGCAAAATCCGTCGCTTGGCTGTTGCGGCGGTGGCGGGCGCGATTGCGTTGGGAGCGAGCTTTGCGGTTGCACAGGCCCCGACATTTTTCCGTATCGGCACTGGCGGCACAGCCGGAACCTATTATCCGATTGGTGGTCTGATCGCGAACGCGATTTCCGGCGCAGGCGAAAAGGGCGTGCCGGGTCTCGTCGCGACGGCCGTTTCGTCGAATGGCTCGGTTGCCAATATCAATGCGATCAAGTCGGGCGCTCTGGAGTCCGGCTTTACGCAGTCAGACGTTGCCTATTGGGCCTATAACGGCACCGGCCTTTATGATGGCAAGGGCAAGGTGGAAGATTTGCGCCTTCTGGCGACGCTTTACCCGGAAACGATCCATATCGTTGCGCGTAAGGATGCAAACATCAAATCGGTCGCAGACCTGAAAGGCAAGCGCGTTTCGCTGGATGAGCCGGGTTCTGGCACCATCGTCGATGCGCGTATCGTTCTTGAAGCCTACGGCCTCACGGAAGACGATATCAAGGCTGAACACCTGAAGCCGGGACCGGCAGGCGAGAGGCTGAAAGATGGTGCGCTGGACGCCTATTTCTTTGTGGGCGGCTATCCGACGGGCGCAATCTCGGAACTGGCCATCTCGAACGGTATTTCGCTCGTTCCGATCTCCGGGCCGGAAGCGGACAAGATTCTGGAGAAATATTCCTTCTTCTCGAAGGATGTGGTTCCTGCCGGAGCCTATAAGGACGTGGCGGAAACACCGACCCTTGCCGTTGCCGCACAGTGGGTGACGAGCGCCAAGCAGCCGGACGACCTCATCTATAACATCACCAAGGTTCTCTGGAACGAGGATACACGCAAGGCACTCGATGCGGGCCATGCGAAGGGCAAGCTCATCAAGCTCGATAGTGCGACGAGCAGCCTCGGTATTCCGCTGCATCCCGGCGCAGAACGCTTTTACAAGGAAGCGGGCGTGCTGAAATAATCCCTCAATGATCGGTTCCTGATATCTTATTCCGAATTGAAGGGTGACATTGCGGCAGCTCGTTATGCGCGCTGCTGCGCTCCCGTTTTCCAGAGCGGTTCCGGTTAGAACGGAATCGTTGGAACCGCTCTATCTCTTTGTTTTTACGCATTATCCGACGCAAAACCGTTTCACGCTTTTGCTGGAAATGCTCTAGCCTATTGAAATGCACGACCGGCAAAGTGGAGTTGGCCCGCATGACAGAAGAACAAAATGCAAAGCTT。
The Brucella bovis insert IS711 nucleotide sequence IS GCTTGTCTGCATTCAAGGATTCCCTTTTGCATGAAATTCTGATTCAAGGTTGTTGAAGGAGAACAGCCTTGAGCAGACGAAGCCTTACAGATGAGCAATGGAACCGGATCGAAGCATATCTTCCGGGGCGAGTTGGTACGCCCGGCCGCAGTGGCGTTGATAACCGATTATTTGTCGATGCCATCTTGTGGATGGCTGCCAATGCAGCGCACTGGCGCGATCTGCCTGCGACCTTCGGCAAATGGACAGCGGTTCATGCCCGCTTTCGGCGCTGGTCGCACGCCGGTGTATGGGAAAGGCTTTTCCATGCCCTGGCTGATACGCCGGACTTTGAATATGTCCTCATTGACAGCACCATATCGAAAGTCCACGCAGATGCGGCGGGCGCAAAAGGGGGGCTGAAGCTGCCTGCATCGGTCGCTCGCGCGGTGGATTGACGACCAAGCTGCATGCTGTTGTCGATGCTATCGGCCTACCGCTGCGAATAAAGCCAACACCCGGCCATTATGGTGACTGTCCGCAAGCTTCAAGCCTTCTATCCGGCTTGAAGGGTGTGGGGCATGTCATTGCTGATGCAGCCTATGATGCCGATCACTTAAGGGCCTTCATTGCCAGCGATCTCAAGGCAACGGCTCAGATCAAGGTCAATCCAACACGTTCCAGTGCCCCAACAATCGACTGGAGGCTGTACAAGGAACGCCATCAGATTGAATGCTTTTTTAACAAGTTGAAACGCTATCGTCGTATTGCGCTGCGATGCGAGAAAACATTGACCGCATTCATGGGTTTCGTCCATCTCGCATGCGCTATGATCTGGTTACGTTAAATGCAGACACGCCC.
The first content of the invention is that the special primer for detecting the Brucella bovis by double real-time fluorescent quantitative PCR comprises 6 primers,
the nucleotide sequence of the primer 1 (B-F) is shown as <210>1 in the sequence table,
the nucleotide sequence of the primer 2 (B-R) is shown as <210>2 in the sequence table,
the nucleotide sequence of the primer 3 (B-P) is shown as <210>3 in the sequence table,
the nucleotide sequence of the primer 4 (B.a-F) is shown as <210>4 in the sequence table,
the nucleotide sequence of the primer 5 (B.a-R) is shown as <210>5 in the sequence table,
the nucleotide sequence of primer 6 (B.a-P) is shown as <210>6 in the sequence table.
The second object of the invention is to provide a method for detecting Brucella bovis by dual real-time fluorescent quantitative PCR.
The method comprises the following steps:
1) And (3) using the DNA of the sample to be detected as a template, and carrying out nucleic acid amplification under the guidance of the special primer.
2) The real-time fluorescent quantitative PCR reagent was 2X TaqMan Fast qPCR Master Mix (B639274, BBI); the quantitative PCR instrument was a LightCycler480 type II fluorescent quantitative PCR instrument (Roche, rotkreuz, switzerland).
3) The PCR reaction steps are as follows:
(1) preparing a reaction mixture as shown in Table 1
Table 1: b and Ba reaction systems (note: 1 μl DNA template is added per 10 μl system)
Reaction Component Concentration Volume(µl)
TaqMan Fast qPCR Master Mix 2X 5
Primer F/R (10 [ mu ] M) (B) 10 µM 0.25+0.25
Primer F/R (10 [ mu ] M) (Ba) 10 µM 0.25+0.25
Probe P (10 mu M) (Ba) 10 µM 0.15
Probe P (10 mu M) (B) 10 µM 0.15
ddH 2 0 2.7
Template (DNA) 1
Total 10
(2) PCR cycling conditions, as in Table 2
Table 2: PCR cycle conditions
Thermal Cycler Times and Temperatures
Initial Steps Each of 45 cycles
Melt Anneal Extend
LightCycler480 II HOLD CYCLE
4 min 95°C 7 s 95°C 35s 60℃ 2s 72℃
(3) Operation of the instrument
After the above steps are completed, the 96/384 well plate with the added sample is placed in LightCycler480II (Roche) for reaction.
4) Result determination
Positive: the Ct value of the sample detection result is less than or equal to 35.00 or a typical S-shaped amplification curve exists;
negative: the Ct value of the sample detection result is more than 35.00 or no Ct value or no typical S-type amplification curve.
The third content of the invention is to provide a dual real-time fluorescent quantitative PCR detection kit for Brucella of cattle. The kit component comprises the special primer for double real-time fluorescent quantitative PCR detection of the bovine brucella.
The invention has the following advantages:
(1) The double real-time fluorescent quantitative PCR detection is a multiplex fluorescent quantitative detection method based on the PCR technology. Compared with the common PCR, the fluorescent quantitative PCR detection technology has the characteristics of quantification, specificity, rapidness, sensitivity, safety and the like, can realize rapid and efficient detection of the Brucella bovis and improve the diagnosis level of the Brucella bovis.
(2) When the serum antibody level or the bacterial load level of the bovine brucella in the sample is low, false negative results exist in serology and common PCR detection, but the detection method can still detect bacterial nucleic acid, and can carry out qualitative and quantitative analysis on a large number of samples. The probability of contacting with pathogens is reduced in experimental operation, and the biosafety risk is reduced, so that the method has good potential application value.
(3) High specificity: the identification of the specific region of the target sequence of brucella bovis by the 6 primers ensures the high specificity of the dual fluorescent quantitative PCR detection.
(4) The operation is simple and convenient: the technology is simple and convenient to operate, low in cost and low in sample processing requirement, and the whole amplification process and the amplification efficiency can be observed to directly obtain a standard curve.
Drawings
FIG. 1 shows the amplification of target genes B and Ba (B) in gradient dilution samples.
FIG. 2 shows the results of amplification standard curves of gradient diluted samples of the target genes B and Ba (B).
FIG. 3 shows the amplification of target genes B and Ba (Ba) in gradient diluted samples.
FIG. 4 shows the results of amplification standard curves of gradient diluted samples of the target genes B and Ba (Ba).
FIG. 5 shows amplification curves of target genes B and B a (B).
FIG. 6 shows amplification curves of the target genes B and Ba (Ba).
Detailed Description
The embodiments and specific operation procedures are given by taking the technical scheme of the present invention as a premise, and the detailed embodiments and specific operation procedures are given in combination with fig. 1, fig. 2, fig. 3, fig. 4, fig. 5 and fig. 6, but the protection scope of the present invention is not limited to the following embodiments.
The methods used in the examples described below are conventional methods unless otherwise specified.
Example 1 primer design for Dual real-time fluorescent quantitative PCR detection of Brucella bovis
The Brucella gene sequence is obtained by searching from a American gene database, homology analysis is carried out through BLAST software, the species and bovine species specific conserved target sequence of the Brucella is obtained, and then a Primer for carrying out fluorescent quantitative PCR detection on the Brucella is designed by using software Primer design 5.0 according to the conserved target DNA sequence, and 1 set of Primer sequences are designed as shown in Table 3.
TABLE 3 primer sequence listing for double fluorescent quantitative PCR detection of Brucella bovis
Primer name Sequence (5 '-3')
B-F GCTCGGTTGCCAATATCAATGC
B-R GGGTAAAGCGTCGCCAGAAG 151bp 60
B-P 5' ATAGGCAACGTCTGACT 3' 66.0 5'FAM 3'MGB
B.a-F CGGTATTCTACCATTGAAGTCTGGC
B.a-R GACCGCATTCATGGGTTTCGTC 150bp 56
B.a-P 5' CTTAATCTGACCTTCCG 3' 67.0 5'HEX 3'MGB
Example 2 establishment of the method for detecting Brucella bovis by double real-time fluorescent quantitative PCR of the present invention
The six primer combinations obtained in the example 1 and used for carrying out double fluorescence quantitative PCR detection on the bovine brucella are used for carrying out real-time fluorescence quantitative PCR detection on the bovine brucella, and the specific method is as follows:
under the same reaction conditions (constant temperature of 63 ℃ for 50 min), the optimal reaction system was determined as follows:
the PCR was performed under the guidance of the six primers obtained in example 1 using a sample containing Brucella genomic DNA diluted 6-fold as the template for the detection of the upper machine.
The quantitative PCR reagent was 2X TaqMan Fast qPCR Master Mix (B639274, BBI).
The quantitative PCR instrument was a LightCycler480 type II fluorescent quantitative PCR instrument (Roche, rotkreuz, switzerland).
The Ba and B reaction systems include: genomic DNA containing Brucella 1 [ mu ] l,5 [ mu ] l lTaqMan Fast qPCR Master Mix,0.25+0.25 [ mu ] l primer F/R (10 [ mu ] M) (B), 0.25+0.25 [ mu ] l primer F/R (10 [ mu ] M) (Ba), 0.15 [ mu ] l probe P (10 [ mu ] M) (B), 2.7 [ mu ] lddH20; the reaction conditions are that the mixture is placed in a water bath kettle with constant temperature of 63 ℃ for incubation for 50min.
The ordinate in the amplification curves of the attached figures 1 and 2 is CT value; the abscissa is LogCO, which refers to Log concentration, i.e., log concentration. Slope= -3.068; amplification efficiency: e=10 -1/slope -1=10 -1/-3.068 -1 = 111.8%; correlation coefficient: r2=0.9995 YIntercept: 36.42 Error: 0.0276.
The ordinate in the amplification curves of the figure 3 and the figure 4 is CT value; the abscissa is LogCO, which refers to Log concentration, i.e., log concentration. Slope= -3.059; amplification efficiency: e=10 -1/slope -1=10 -1/-3.059 -1 = 112.3%; correlation coefficient: r2= 0.9991 yontercept: 36.62 Error: 0.0435.
Example 3 preparation of Dual real-time fluorescent quantitative PCR detection kit for Brucella bovis
The reagent kit for dual real-time fluorescence quantitative PCR detection of the bovine brucella is obtained by packaging bovine brucella fluorescent PCR reaction liquid, bovine brucella primer liquid, bovine brucella probe, bovine brucella negative control substance and bovine brucella positive control substance together.
Sequence listing
<110> vinca health and practice Co.Ltd
<120> A specific primer for double real-time fluorescent quantitative PCR detection of Brucella bovis
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (sequence name) (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 1
gctcggttgc caatatcaat gc 22
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence (sequence name) (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 2
gggtaaagcg tcgccagaag 20
<210> 3
<211> 17
<212> DNA
<213> Artificial sequence (sequence name) (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 3
ataggcaacg tctgact 17
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence (sequence name) (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 4
cggtattcta ccattgaagt ctggc 25
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence (sequence name) (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 5
gaccgcattc atgggtttcg tc 22
<210> 6
<211> 17
<212> DNA
<213> Artificial sequence (sequence name) (2 Ambystoma laterale x Ambystoma jeffersonianum)
<400> 6
cttaatctga ccttccg 17。

Claims (5)

1. The special primer and the kit for detecting the brucella bovis comprise six primers, and are characterized by comprising brucella primers and brucella bovis primers.
2. The brucella primer according to claim 1, comprising a brucella forward primer B-F and a brucella reverse primer B-R and a brucella probe B-P.
3. The brucella bovis primer according to claim 1, comprising brucella bovis forward primers b.a-F and brucella bovis reverse primers b.a-R and brucella bovis probe b.a-P.
4. The brucella primer according to claim 2, wherein the nucleotide sequence of the brucella forward primer B-F is GCTCGGTTGCCAATATCAATGC, the nucleotide sequence of the brucella reverse primer B-R is GGGTAAAGCGTCGCCAGAAG, and the nucleotide sequence of the brucella probe B-P is ATAGGCAACGTCTGACT.
5. A brucella primer according to claim 3, wherein the nucleotide sequence of the b.a-F forward primer of brucella bovis is CGGTATTCTACCATTGAAGTCTGGC, the nucleotide sequence of the b.a-R reverse primer of brucella bovis is GACCGCATTCATGGGTTTCGTC and the nucleotide sequence of the b.a-P probe of brucella bovis is CTTAATCTGACCTTCCG.
CN202210926273.3A 2022-08-04 2022-08-04 Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis Pending CN117512141A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210926273.3A CN117512141A (en) 2022-08-04 2022-08-04 Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210926273.3A CN117512141A (en) 2022-08-04 2022-08-04 Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis

Publications (1)

Publication Number Publication Date
CN117512141A true CN117512141A (en) 2024-02-06

Family

ID=89748299

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210926273.3A Pending CN117512141A (en) 2022-08-04 2022-08-04 Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis

Country Status (1)

Country Link
CN (1) CN117512141A (en)

Similar Documents

Publication Publication Date Title
CN112063756B (en) Method and kit for multiple detection of respiratory virus nucleic acid
CN113817868A (en) Primer, probe composition and kit for detecting novel coronavirus and variant thereof
CN105219772B (en) One group of nucleotide sequence and the application in detection of Salmonella and shigella dysenteriae detection
CN106520923B (en) Kit and method for simultaneously detecting staphylococcus aureus and 5 enterotoxins thereof
CN107083446B (en) Diarrhea pathogenic bacteria multiple gene detection system and kit and application thereof
CN111763767A (en) Central nervous system infection pathogen detection kit and application thereof
CN111910017A (en) Multiplex-time PCR (polymerase chain reaction) kit for detecting respiratory pathogens, method and application
CN115466800A (en) Multiplex fluorescence quantitative PCR kit for detecting monkeypox virus and subtype thereof
CN112725475A (en) Mycobacterium tuberculosis detection primer, probe composition, kit and application
WO2009087685A2 (en) Method for detection of hepatitis nucleic acid and uses thereof
CN112921126A (en) Human respiratory syncytial virus typing detection multiplex RT-qPCR kit, primer probe composition and use method thereof
CN110607381B (en) Mycobacterium tuberculosis detection kit and method
CN116814859A (en) Primer probe composition, kit and method for identifying African swine fever virus genes I and II
CN116219071B (en) Multiplex qPCR kit for simultaneously detecting and identifying type I, type IIa and type IIb monkey poxviruses
CN113564271B (en) Primer probe set for pathogenic bacterium typing quantitative detection, kit and sample purification method
CN117512141A (en) Special primer for dual real-time fluorescent quantitative PCR detection of Brucella bovis
CN112592965B (en) E.coli host DNA residue detection kit adopting TaqMan probe method
CN113755618A (en) Method for detecting brucellosis of animals with high sensitivity
CN113817849A (en) Primer group for detecting mycobacteria based on nucleic acid mass spectrometry technology and application thereof
CN116622872B (en) Salmonella gallinarum molecular detection kit and non-diagnostic detection method thereof
RU2809735C1 (en) Method for identifying mycobacterium tuberculosis bacteria using loop isothermal amplification (lamp)
CN111197094B (en) Compositions, kits and methods for genotyping vibrio parahaemolyticus
CN114703306B (en) Detection method and kit for mycoplasma genitalium parC gene mutation type
CN113736894B (en) Detection method of four intestinal tract protection bacteria and nucleic acid detection kit
WO2009087688A2 (en) Method for detection of nucleic acid of mycobacterium and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination